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NMR Spectroscopy of Body Fluids
A Metabolomics Approach to Inborn Errors of Metabolism
Udo Engelke
NMR Spectroscopy of Body Fluids:
A Metabolomics Approach to Inborn Errors of Metabolism
Engelke, Udo
Thesis Radboud University Nijmegen with a summary in Dutch
ISBN 978-90-9022059-8
Cover Design by Robert Engelke
Printed by Ponsen & Looijen BV, Wageningen
NMR Spectroscopy of Body Fluids
A Metabolomics Approach to Inborn Errors of Metabolism
een wetenschappelijke proeve op het gebied van de Medische Wetenschappen
Proefschrift
ter verkrijging van de graad van doctor
aan de Radboud Universiteit Nijmegen
op gezag van de Rector Magnificus prof. mr. S.C.J.J. Kortmann,
volgens besluit van het College van Decanen
in het openbaar te verdedigen op donderdag 20 september 2007
om 10.30 uur precies
door
Udo Franziskus Heinrich Engelke
geboren op 28 april 1966
te Kevelaer (Duitsland)
Promotor
Prof. dr. R.A. Wevers
Copromotor
Dr. M.A.A.P. Willemsen
Manuscriptcommissie
Prof. dr. A. Heerschap (voorzitter)
Dr. M. Spraul (Bruker BioSpin GmbH, Duitsland)
Dr. E. Morava
Publication of this thesis was financially supported by
- Department of Neurology, Radboud University Nijmegen Medical Center
- Bruker BioSpin GmbH
Voor mijn ouders
CONTENTS
6
Abbreviations and symbols
8
Chapter 1
INTRODUCTION
11
1.1
General Introduction and aim of the thesis
13
1.2
NMR basics and Methods
17
1.3
NMR spectroscopy of body fluids as a metabolomics
approach to inborn errors of metabolism.
27
N-ACETYLATED METABOLITES IN BODY FLUIDS
55
N-Acetylated metabolites in urine: proton nuclear magnetic
57
In: The Handbook of Metabonomics and Metabolomics.
2007; (14) 373 – 410. Published by Elsevier
Editors: Lindon JC, Nicholson JK, Holmes E.
Chapter 2
2.1
resonance spectroscopic study on patients with inborn
errors of metabolism
Clinical Chemistry 2004; 50(1): 58-66
Severe hypomyelination associated with increased levels of
N-acetylaspartylglutamate in CSF
77
NMR spectroscopy of aminoacylase 1 deficiency, a novel
inborn error of metabolism
91
Chapter 3
THE COMBINATION OF IN VIVO AND IN VITRO NMR
SPECTROSCOPY
109
3.1
Dimethyl sulfone in human cerebrospinal fluid and blood
plasma confirmed by one-dimensional 1H and twodimensional 1H-13C NMR
111
2.2
Neurology 2004; 62(9): 1503-1508
2.3
NMR in Biomedicine 2007; in press
NMR in Biomedicine 2005; 18(5): 331-336
3.2
Methylsulfonylmethane
(MSM)
ingestion
causes
a
significant resonance in proton magnetic resonance
spectra of brain and cerebrospinal fluid
123
NMR spectroscopic studies on the late onset form of 3methylglutaconic aciduria, type I and other defects in
leucine metabolism
129
Leukoencephalopathy in adult-onset 3-methylglutaconic
aciduria type I
145
LIPID METABOLISM
155
Neuropediatrics 2006; 37(5): 312-314
3.3
NMR in Biomedicine 2006; 19(2): 271-278
3.4
Submitted for publication
Chapter 4
4.1
Diagnosing inborn errors of lipid metabolism using
NMR spectroscopy
1H-
157
Clinical Chemistry 2006; 52(7): 1395-1405
Chapter 5
SUMMARY AND FUTURE PERSPECTIVES
177
5.1
Summary and samenvatting
179
5.2
Future Perspectives
187
Reference List
Dankwoord
Curriculum Vitae
List of Publications
195
211
213
215
Abbreviations and symbols
δ
γ
υ0
1D
2D
3HIVA
3MG
3MGA
3MGH
7DHC
8DHC
ACY1
ACY2
AUH
B0
CNS
COSY
CSF
CTX
DMS
DMSO
DMSO2
EPI
FID
GC
GC-MS
GlcNAc
HSQC
IEM
ISSD
JRES
LC-APCI-MS
MAT I/III
MRI
MRS
MRSI
MSM
NAA
NAAG
Chemical shift
Gyromagnetic ratio
Lamour precession frequency
One-dimensional
Two-dimensional
3-Hydroxyisovaleric acid
3-Methylglutaric acid
3-Methylglutaconic acid
3-Methylglutaconyl-CoA hydratase
7-Dehydrocholesterol
8-Dehydrocholesterol
Aminoacylase type 1
Aminoacylase type 2
AU-specific RNA-binding enoyl-CoA hydratase
Magnetic field strength
Central nervous system
Correlation spectroscopy
Cerebrospinal fluid
Cerebrotendinous xanthomatosis
Dimethyl sulfide
Dimethyl sulfoxide
Dimethyl sulfone
Echo planar imaging
Free induction decay
Gas chromatography
Gas chromatography-mass spectroscopy
N-acetylglucosamine
Heteronuclear Single-Quantum Correlations
Inborn error of metabolism
Infantile sialic acid storage disorder
J-resolved
Liquid chromatography-atmospheric pressure chemical-ionization mass
spectroscopy
Methionine adenosyltransferase I/III
Magnetic resonance imaging
Magnetic resonances spectroscopy
Magnetic resonance spectroscopic imaging
Methylsulfonylmethane
N-Acetylaspartic acid
N-Acetylaspartylglutamic acid
NMR
OMIM
OMS
PLP
PMD
PPM
PRESS
SLOS
T1
tCho
tCr
TGSE
TLC
TMS
TOCSY
TR
TSP
WET
Nuclear magnetic resonance
Online mendelian inheritance in man
Octamethylcyclotetrasiloxane
Proteolipid protein
Pelizaeus-Merzbacher disease
Parts per million
Point resolved spectroscopy
Smith-Lemli-Opitz syndrome
Spin-lattice or longitudinal relaxation time
Total Choline (choline, phosphocholine and glycerophosphocholine)
Total Creatine (creatine and phosphocreatine)
Turbo gradient spin echo
Thin layer chromatography
Tetramethylsilane
Total correlation spectroscopy
Repetition time
Trimethylsilyl-2,2,3,3-tetradeuteropropionic acid
Water suppression enhanced through T1 effects
ONE
INTRODUCTION
11
12
CHAPTER
1.1
General Introduction and aim of the thesis
13
14
Introduction and aim
General introduction and aim of the thesis
The precise number of different metabolites that play a role in human metabolism
is unknown; estimates ranges from 3000 to around 20000 [1]. Approximately
30000 genes and 100000 proteins are involved in human cellular metabolism and
homeostasis [1]. Inborn errors of metabolism (IEM) may occur in any enzymatic
process that interconverts two metabolites. Currently more than 500 IEM are
known [2]. These data suggest that many IEM remain as yet unknown. It seems
that the analytical techniques that are used in routine diagnostics are insufficient
to pick up a higher percentage of the estimated theoretical number of IEM. These
techniques always rely on the detection of groups of metabolic intermediates with
a specific chemical group. Because protons are available in almost any metabolite
1H-NMR spectroscopy may be able to simultaneously quantify almost all
metabolites in the micromolar and millimolar concentration range. This opens the
way towards a metabolomics view on metabolism.
The aim of the present study is to explore the diagnostic potential of body fluid
NMR spectroscopy. The outcome of the experiments can be the basis of a
successful introduction of NMR spectroscopy as a complementary analytical
technique in the metabolic laboratory of the (near) future.
OUTLINE OF THE THESIS
Chapter 1 of the thesis gives a brief introduction to the basic physical principles of
NMR spectroscopy and discusses the diagnostic NMR approach, as developed in
Nijmegen, for the diagnosis of inherited metabolic disorders (chapter 1.2). In
chapter 1.3 we provide a literature overview of current applications of NMR
spectroscopy to metabolic diseases.
Chapter 2 describes the NMR findings for a group of compounds that would
normally not be detected in a metabolic laboratory. These compounds contain an
N-acetyl group and the study, as described in chapter 2.1, investigates thirteen
known IEM involving N-acetylated metabolites. The diagnostic importance of Nacetylated metabolites is demonstrated in chapters 2.2 and 2.3: two novel
diseases with accumulation of N-acetylated compounds in body fluid are
described.
Next to metabolomics of body fluids with 1H-NMR spectroscopy it is also possible
to obtain metabolic information of tissues in humans by in vivo MR spectroscopy.
In chapter 3 we used both in vivo and in vitro NMR spectroscopic techniques for
metabolite identification. An unknown peak was identified as dimethyl sulfone
(synonym: methylsulfonylmethane) in NMR spectra in human cerebrospinal fluid
and blood plasma (chapter 3.1). The use of this compound as a dietary
15
Chapter 1.1
supplement led to the occurrence of an abnormal peak in the in vivo brain MR
spectrum (chapter 3.2). In vivo and in vitro NMR studies on a patient with 3methylglutaconic aciduria type 1 are described in chapters 3.3 and 3.4.
Chapter 4 investigates the potential use of 1H-NMR spectroscopy to identify and
quantify lipids present in the blood of patients with different inborn errors of lipid
metabolism. The technique provides a new tool in diagnosis and follow up of
patients with inborn errors in lipid metabolism.
16
CHAPTER
1.2
NMR Basics and Methods
17
18
Basic principles of NMR spectroscopy
Basic principles of NMR spectroscopy
The clinical applications of nuclear magnetic resonance (NMR) are broad and
diverse. In many hospitals, NMR is commonly used as a non-invasive technique to
obtain clinical images (in vivo MRI, magnetic resonance imaging). In more
specialized hospitals, the technique is used to obtain metabolic information of
tissues in humans (in vivo MRS, magnetic resonance spectroscopy). Also, NMR has
been used as an analytical technique to detect metabolites, drugs and toxic agents
in body fluids (in vitro NMR).
NMR EXPERIMENT
The principle of NMR is based on the behavior of atoms placed in a strong
magnetic field. Superconducting magnets provide such a strong, extremely
homogenous magnetic field (Figure 1).
Figure 1
Left: Standard NMR sample tube with 5 mm diameter. The sample volume needed is 650 µl.
Middle: NMR spectrometer (500 MHz , 11.4 Tesla) with sample changer in the Nijmegen Goudsmit NMR pavilion.
Right: NMR magnet operating at the world's highest, actively shielded field (950 MHz, 22.3 Tesla)
The nuclei of most atoms (for example protons in a water sample) possess a
property known as spin. The spinning of these charged particles generates a
magnetic moment along the axis of spin, so that these nuclei act like tiny bar
magnetics. Normally, the magnetic moments in a collection of nuclei will be
randomly oriented. However, when an external magnetic field B0 is applied, they
adopt one of a small number of allowed orientations with different energy. 1H
nuclei can adopt only two permitted orientations, either aligned with (parallel) or
against (anti-parallel) the direction of the external magnetic field. These two
energy states are unequally populated, according to the Boltzmann distribution.
The parallel orientation has a slightly lower energy state leading to more spin
19
Chapter 1.2
protons aligned parallel and leading to a net magnetization (Mz) (the vector sum of
the individual magnetic moments).
Figure 2
Figure 3
The collection of spins creating a net magnetization (Mz)
A short radio-frequent radiation rotates Mz from
(the vector sum of the individual magnetic moments).
the z-axis into the x,y-plane.
Mz will be aligned with the applied magnetic field (BO)
which is defined as the z-direction in a x,y,z plane.
This net magnetization will be aligned with the applied magnetic field (BO) which is
defined as the z-direction in a x,y,z plane (Figure 2). In this equilibrium state, the
net magnetization vector is static and does not induce a signal. To acquire an NMR
signal, a short radio-frequent radiation (a pulse) is applied to the sample (Figure
3). This causes the magnetization vector Mz to rotate from the z-axis into the x,yplane (Figure 3). After a 90° pulse, the only remaining field is the external field
(BO). Since (BO) and Mx,y are now perpendicular to each other, the magnetization
vector experiences a torque, which causes it to precess around the magnetic field
at the Larmor frequency (1):
ν 0=
ν0
B0
γ
γ
B0
2π
(1)
= Lamor precession frequency (Hz)
= Magnetic field strength
= Gyromagnetic ratio (different for each type of nuclei)
The rotating magnetization induces a small oscillating electric current in a
detection coil placed in the x,y-plane. The resulting signal, also called Free
Induction Decay (FID), is a sum of many sine functions with different frequencies.
20
Basic principles of NMR spectroscpy
Furthermore, the FID shows decay due to relaxation processes, which bring the
magnetization vector back to its equilibrium state. The FID can be converted to a
frequency-domain NMR spectrum by Fourier transformation.
PARAMETERS FROM NMR MEASUREMENTS
A 1H-NMR spectrum provides a characteristic ‘fingerprint’ of almost all protoncontaining metabolites. The spectral parameters chemical shift (δ in ppm), spinspin coupling (multiplicity) and signal intensity are important for body fluid
analysis and will be described briefly here.
Chemical Shift (Resonance position)
The Larmor frequency is determined by the gyromagnetic ratio and the strength of
the external field BO (equation 1, page 20). However, the resonance frequency also
depends on the chemical environment, since nearby nuclei and electrons slightly
alter the local magnetic field of a specific proton. This effect, called the chemical
shift, causes nuclei with different chemical environments to resonate at different
frequencies and therefore provides structural information. The position of a
frequency, in absolute frequency terms, varies with the exact field strength of the
magnet used. Therefore, a chemical shift reference is used to define the positions
of the resonances in the spectrum. These shifts are generally very small and are
defined as parts per million (ppm) fractional shift from an internal reference
compound according to equation 2.
⎛
⎞
6
δ = ⎜⎜ Shift from an internal reference in Hz ⎟⎟ ×10
⎝
Spectromet er frequency in Hz
(2)
⎠
δ = Chemical shift (ppm)
The chemical shift expressed as ppm is not dependent on the field strength of the
external magnetic field. For body fluid NMR spectroscopy, trimethylsilyl-2,2,3,3tetradeuteropropionic acid (TSP) is often used as a chemical-shift reference.
The chemical shift can be used to discriminate the 1H-NMR spectra of molecules,
even when their chemical structure is only slightly different. For example, two
molecules that have a quite similar chemical structure are lactic acid and alanine.
The only difference is that lactic acid has a hydroxyl group whereas alanine has an
amino group. As shown in Figure 3, the CH3-protons of lactic acid resonate at
1.41 ppm and the CH-proton resonates at 4.37 ppm under the conditions used.
21
Chapter 1.2
Due to the small difference in chemical structure, the resonance positions of the
CH3- and CH-protons of alanine are slightly different (the CH3-protons resonate at
1.50 ppm, the CH proton at 3.89 ppm).
Spin-spin coupling (Multiplicity)
NMR spectra furthermore provide valuable information about the magnetic
interactions between nuclei (also known as spin-spin coupling, scalar coupling or
J-coupling). These couplings cause the signal of each nucleus to split into a
distinct number of compounds with characteristic relative intensities and spacings,
which depend on the number and type of spins coupling with the nucleus of
interest. This is illustrated using the spectrum of lactic acid shown in Figure 3.
The splitting of the resonances is caused by an interaction between neighbouring
protons. Some rules governing this splitting are:
1. No splitting is caused between equivalent protons, e.g. the CH3-group protons of lactic
acid.
2. A proton that is coupled to n equivalent protons gives rise to (n + 1) lines. The relative
intensities of these lines are given by the binomial distribution. In Figure 3, the
equivalent CH3-group protons coupled to the CH-group proton give rise to two lines (a
doublet) with relative intensities of 1:1. The CH-group proton coupled to the
equivalent CH3-group protons gives rise to four lines (a quartet) with relative
intensities of 1:3:3:1. Since the hydroxyl proton in lactic acid exchanges rapidly with
water protons under the conditions used, it does not couple to any of the
nonexchangeable protons in this molecule.
22
Basic principles of NMR spectroscpy
Figure 3
1H-NMR
spectra of lactic acid (upper panel) and alanine (lower panel) dissolved in D2O measured at 500 MHz; pD = 2.5
Signal intensity
The peak area or signal intensity of a resonance in a NMR spectrum is proportional
to the number of protons contributing to the signal when appropriate
experimental conditions are used. For example in Figure 3, the doublet is assigned
to the CH3-group (3 protons contributing) and the quartet is assigned to the CHgroup (1 proton contributing). Therefore, the peak area of the doublet is three
times as large as the peak area of the quartet. Since the peak area is proportional
to the number of protons contributing to the signal, it is also proportional to the
concentration of the molecule of interest. Therefore, it is possible to use NMR
spectroscopy for metabolite quantification. The detection limit of the technique is
in the low micromolar range for most metabolites.
NMR SPECTROSCOPY OF BODY FLUIDS IN PATIENTS WITH INBORN ERRORS OF
METABOLISM
In 1992, Prof. Dr. Ron Wevers, started the work on NMR spectroscopy for
diagnosis and follow-up of patients with inborn errors of metabolism in Nijmegen.
During the subsequent years, this approach was successful and standard methods
for recording 1H-NMR spectra in cerebrospinal fluid and blood plasma have been
published [3;4].
23
Chapter 1.2
Sample preparation is limited to the removal of proteins from CSF and plasma
serum samples on a 10 kD filter and to pH standardization of all samples (pH 2.5).
Routinely we start with a 1D 1H-NMR experiment. By comparing the spectrum with
databases containing model compounds and normal body fluid NMR spectra,
assignments can be made. This may lead to the diagnosis of an IEM [5].
Sometimes the overlap of resonances is too severe and assignments are not
possible from a 1D spectrum. In such case a two-dimensional COSY NMR
experiment is used to provide additional information (Figure 3B, page 36).
Sometimes unknown resonances are not present in the database. If we can exclude
medication or food component as source of these unknown peaks additional NMR
experiments can be used to identify the structure of the unknown metabolite
(Table 1, page 34).
Which body fluid should be investigated?
It is difficult to give a general advice on the type of body fluid that is most suited
to find the diagnosis. Urine and blood are easy to collect and urine is often used
as a first approach. The kidneys rapidly excrete some metabolites but others
remain preferentially in the blood. Urine NMR spectra are very complex and many
IEM can be diagnosed in this body fluid [5]. CSF NMR investigation may be valuable
for patients suspected of suffering from neurometabolic disease. Chapter 2.2
demonstrates the presence of the dipeptide N-acetylaspartylglutamic acid (NAAG)
in CSF of two patients with a novel neurometabolic disease while NAAG could not
be demonstrated in the urine NMR spectra of the patients.
Case selection
NMR spectroscopy of body fluids may be considered when the patient is suspected
of suffering from a so far undiagnosed inborn error of metabolism or when further
investigations leading to a diagnosis are required.
Clinical indications are:
1. Two or more children in the same family have unexplained similar clinical signs and
symptoms.
2. The patient has an unusual body odor
3. An unknown metabolite is observed using in vivo NMR spectroscopy of the brain or
other organ (Chapter 3).
Biochemical indications are:
1. An abnormal unknown metabolite is repeatedly observed in body fluids with another
technique. NMR spectroscopy may provide structural information about the metabolites
involved.
2. Confirmation of a special diagnosis with an independent technique is needed (Chapter
2.3).
24
Basic principles of NMR spectroscpy
3. The need for a reliable quantification of a metabolite that is difficult to quantify
otherwise (Chapter 2 and 3).
4. Consistent abnormal results were found using conventional screening techniques for
one or more metabolites that did not lead to a classifying diagnosis. NMR spectroscopy
may provide additional information.
25
26
CHAPTER
1.3
NMR spectroscopy of body fluids
as a metabolomics approach to inborn errors of
metabolism
Udo F.H. Engelke, Marlies Oostendorp and Ron A. Wevers
In: The Handbook of Metabonomics and Metabolomics.
2007; (14) 373 – 410. Published by Elsevier
Editors: Lindon JC, Nicholson JK, Holmes E
27
28
NMR spectroscopy of body fluids
Introduction
Nuclear magnetic resonance (NMR) spectroscopy has been used to analyze
metabolites, drugs and toxic agents in body fluids and reviews on these
topics have been published [6-8]. Proton NMR (1H-NMR) spectroscopy has also
been successfully applied to the field of inborn errors of metabolism [9]. The
technique is essentially non-selective and provides an overview of protoncontaining metabolites. Moreover, simultaneous quantification of many
metabolites over large concentration ranges can be done easily in a short
time-frame. Because of these features proton NMR spectroscopy easily
picks up a metabolic derangement or an abnormality in the metabolome.
The technique either as a standalone approach or in combination with
liquid chromatography and/or mass spectroscopy is at the frontiers of
metabolomics and systems biology approaches.
THE DIAGNOSTIC APPROACH OF INBORN ERRORS OF METABOLISM
Inborn errors of metabolism (IEM) form a considerable group of genetic diseases.
The majority is due to defects of single genes coding for enzymes. Figure 1A
shows a schematic representation of a metabolic pathway. In most of the
disorders, problems arise due to accumulation of substances, which are toxic or
interfere with normal function (Figure 1B, metabolites S1 and S2). Clinical signs
may also relate to the decreased availability of metabolites behind the metabolic
block (Figure 1B, metabolites S3 and S4). Also alternative pathways may metabolize
an accumulating metabolite. Such pathways may play a minor role or no role at all
in normal metabolism. In a patient with a metabolic block alternative pathways
may result in unusual metabolites (Figure 1B, metabolites S5 and S6). These often
are characteristic for the disease and therefore diagnostically important. Also they
may play a role in the pathophysiology of the disease.
Figure 1
A; A schematic representation of a metabolic
pathway. Enzymes E1, E2 and E3 catalyze the
reactions converting the metabolite S1 into S2,
S3 and S4.
B; A defect in enzyme E2 (black square) will
give rise to the accumulation of metabolites S2
and to a lesser extent S1 and to a decreased
availability of metabolites S3 and S4. Also
unusual alternative metabolites like S5 and S6
may occur.
29
Chapter 1.3
One of the methods to diagnose IEM is the detection of abnormal metabolite
concentrations in body fluids such as urine, blood plasma, blood serum or
cerebrospinal fluid (CSF). From these concepts it is easy to understand that the
diagnosis of an IEM may rely on (1) an abnormal accumulation of a specific
metabolite, (2) the presence of metabolites that are normally not present in body
fluids or (3) a decreased concentration of a metabolite that is always present in the
body fluid. Laboratories specialized in diagnosing IEM use a variety of analytical
methods to measure various metabolic groups. These are for instance GC-MS for
the detection of organic acids, ion exchange chromatography for the detection of
amino acids and HPLC for the detection of purines and pyrimidines. These
techniques are selective and sample pretreatment such as derivatization and
extraction procedures are often necessary. Proton nuclear magnetic resonance
(1H-NMR) spectroscopy is not selective and because most metabolites contain
protons this technique may be considered as an alternative analytical approach for
diagnosing known, but also as yet unknown, IEM.
The detection limit for body fluid NMR spectroscopy depends on a number of
factors such as field strength, number of protons contributing to a resonance and
the region of the spectrum where the resonance is observed. In general, the
detection limit is in the low micromolar range in the less crowded regions of the
spectrum. For example, the detection limit at S/N = 3 for the six equivalent
protons in dimethyl sulfone (observed as a singlet resonance at 3.14 ppm in blood
plasma and CSF) was determined to be 0.5 µmol/l (500 MHz, 128 transients; using
a conventional 5 mm TXI probehead operating at ambient temperature) [10].
Another study showed detection limts between 2 and 40 µmol/l for various
metabolites in unconcentrated plasma samples measured at 600 MHz and 132
transients [4].
Metabolite quantification in plasma and CSF can be performed by adding a known
concentration of 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid (TSP) to the
sample and concentrations can be expressed as µmol/liter. For urine, the singlet
resonance of creatinine at 3.13 ppm (pH=2.50) can be used as a concentration
reference. Metabolite concentrations in urine are often expressed per mmol
creatinine. For several compounds good correlations have been obtained with
other analytical techniques. In CSF and blood plasma, quantitative data for alanine,
threonine and lactic acid correlated well with data obtained with conventional
techniques [3;4]. Excellent linearity, recovery and reproducibility have been found
for N-acetyltyrosine, N-acetyltryptophan and N-acetylaspartic acid in urine,
normally not analyzed in conventional screening methods [11].
In this review, we provide a literature overview of current applications of NMR
spectroscopy to metabolic disease.
30
NMR spectroscopy of body fluids
Methods
SAMPLE COLLECTION, PREPARATION AND CHOICE
NMR spectroscopy has been used to investigate a wide range of body fluids [7].
However, when used as a diagnostic tool for inborn errors of metabolism, NMR
spectra are mainly recorded on blood plasma, blood serum, urine or CSF samples.
Blood is collected by venipuncture into standard vials containing either lithium
heparin or ethylene diamine tetra acetate (EDTA) as anti-coagulant. Subsequently,
plasma can be separated from the cells by centrifugation. When EDTA is used to
prevent clotting, extra resonances can be observed in the NMR spectrum due to
complexes formed between EDTA and Ca2+ and Mg2+ ions present in plasma [12].
CSF can be obtained by lumbar puncture and has to be centrifuged to remove
cells. If NMR analysis cannot be performed immediately after collection of blood,
urine or CSF, the samples have to be kept frozen at -20°C or lower until further
usage.
Whereas urine is measured directly, plasma and serum are usually deproteinized.
The blood plasma protein concentration (approximately 70 g/l) results in broad
overlapping signals that obscure resonances from low- molecular mass
metabolites of interest [4]. Adsorption of the commonly used internal standard
trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP-d4) on proteins has been
shown to have a negative influence on quantification [13]. Additionally, resonances
for small, protein-bound metabolites are broadened due to T2-relaxation
processes, making accurate quantification difficult [14]. The effect of
deproteinization on the 1H NMR spectrum of a control plasma sample is illustrated
in Figure 2. Normal CSF has a rather low protein concentration (< 500 mg/l) and
NMR measurements on both native [15-20] and deproteinized samples have been
reported [3].
Various techniques are available for plasma or serum deproteinization, including
extraction with acetone or acetonitrile, perchloric acid precipitation, solid phase
extraction chromatography and ultrafiltration. Of these methods, ultrafiltration
using a 10 kDa cut-off filter (e.g. Sartorius) is best suited for metabolic studies
[21]. Before use, the filter has to be washed by centrifugation of water to remove
glycerol that is present in several commercially available filters. The main
disadvantage of deproteinization is possible loss of protein bound metabolites.
However, excellent correlation between 1H-NMR and enzymatically determined
lactate concentrations was found [4], even though lactate is assumed to interact
with plasma proteins and so become partly NMR invisible [22].
The pH of the sample has an important influence on the observed chemical shifts
and also determines whether acidic or basic protons are detectable. Unfortunately,
a large range of pH values has been used in the literature. The physiological pH
and pH of 2.5 have been used most frequently. The pH of urine varies considerably
between samples. The pH of CSF samples increases significantly upon standing. To
31
Chapter 1.3
A
B
Figure 2
500 MHz 1H-NMR spectrum of a plasma sample before (B) and after (A) deproteinization over a 10-kilodalton filter.
Assignmets: Isoleucine (Ile), leucine (Leu), valine (Val), alanine (Ala), lysine (Lys), glutamine (Glu), tyrosine (Tyr), phenylalanine
(Phe), histidine (His). HDL Chol referes to the C18 signal from cholesterol in high-density lipoprotein. CH3 and CH2 refer to the
CH3 and CH2 groups of fatty acids in lipoproteins. L1-L6 refer to proton signals of various lipids [23].
32
NMR spectroscopy of body fluids
improve intersample reproducibility and to allow comparison between different
samples, standardization of pH was initially proposed by Lehnert and Hunkler in
1986 [24]. They chose pH 2.5 because the slope of the titration curve is expected
to be minimal for all organic acids and because the proton concentration is not yet
so high as to cause hydrolysis of certain compounds. Chemical shifts in this review
are given for pH 2.5 unless mentioned otherwise.
Finally, an aliquot of TSP-d4 in 2H2O has to be added to the sample to provide a
chemical shift (δ = 0.00) and concentration reference as well as a deuterium lock
signal. Alternatively, an internal reference or the residual solvent signal can be
used as chemical shift reference. In this case addition of pure 2H2O is sufficient.
The ionic strength of body fluids varies considerably and may be high enough to
adversely affect the tuning and matching of the RF circuits of a spectrometer
probe, particularly at high field strengths. Therefore, it may be necessary to retune
the spectrometer probe for each sample. Automatic tune and match (ATM) probes
are available.
Traditionally the approach in diagnosing IEM is to make use of urine as the body
fluid of first choice. The kidneys excrete most water-soluble small molecules.
However, some are reabsorbed like for instance glucose. The urine NMR spectrum
is by far the most complex body fluid spectrum allowing the diagnosis of most
inborn errors of metabolism. However, there may be a case for separate CSF NMR
studies in patients suspected to suffer from neurometabolic disease. A paper by
Wolf et al. [25] demonstrated the presence of the dipeptide Nacetylaspartylglutamic acid (NAAG) in CSF of two patients with a novel
neurometabolic disease while NAAG could not be demonstrated in the urine NMR
spectra of the patients. Only single studies have addressed the NMR spectra of
body fluid cells. Studies are available in leucocytes and erythrocytes [26]. The
composition of the intracellular fluid differs considerably from the body fluid
composition. Several metabolites are unable to leave the cell, as they cannot pass
the outer cell membrane. Phosphorylated compounds may serve as an example.
Inborn errors where such compounds accumulate may be easily diagnosed in
homogenates of leucocytes. However, such examples have not yet been presented
in literature.
ONE AND TWO-DIMENSIONAL NMR SPECTROSCOPY TECHNIQUES
The number of observed metabolites in body fluids is largely dependent on the
magnetic field strength of the NMR spectrometer. At higher fields, the spectral
dispersion and sensitivity increase, allowing assignment of many metabolites that
cannot be detected at lower fields. Therefore, working at the highest field available
will provide the most complete metabolic information. Currently, most studies are
performed on 400, 500 or 600 MHz spectrometers, but higher field strengths up
to 800 MHz have been used [23;27;28]. Additionally, low frequency spinning of
33
Chapter 1.3
the sample can be used to improve spectral resolution. Reported measurement
temperatures are found between 20 and 37˚C.
Table 1: Overview of different experiments used in the diagnosis of inborn errors of metabolism.
Experiment
Description
1D 1H (Figure 3A)
Single pulse with solvent presaturation. Routinely used to determine
chemical shifts, J-values and metabolite quantities (only for well-resolved
peaks).
H-1H COSY (Figure 3B)
Used to establish which protons are spin-coupled. Helpful in resolving
H-1H DQF-COSY
COSY variant with greater resolution. Cross peaks near the diagonal can be
H-1H TOCSY (Figure 3C)
COSY variant displaying relayed connectivities. Used as an aid in assignment.
H J-resolved (Figure 3F)
Separates J-splitting and chemical shifts on to two orthogonal axes.
1
1
overlap and to assign crowded regions.
resolved. Sensitivity is 50% less compared to COSY.
1
1
Reduced overlap and more accurate determination of J-values and chemical
shifts.
Spectral simplification based on difference in relaxation times. Not suitable
H 1D Spin-echo
1
1D 1H-decoupled
for quantification.
C (Figure 3D) More resonances resolved due to larger spectral width than 1H. Suffers from
inherent low sensitivity and low natural abundance.
13
H-13C HSQC (Figure 3E)
1
Displays connectivities between chemically bounded
Helpful when assigning complicated spectra.
H and
1
C nuclei.
13
A wide range of one-dimensional (1D), two-dimensional (2D) and even higher
dimensional NMR experiments are available to the modern NMR operator.
Especially in the field of structural biology, multidimensional experiments are
important for successful determination of protein or nucleic acid structures
[29;30].
For studying inborn errors of metabolism, 1D 1H NMR spectra are most frequently
employed. Additionally, several homo- and heteronuclear 2D experiments can be
recorded, which are particularly helpful in assisting the assignment of resonances.
The most commonly used experiments for diagnosing metabolic diseases will now
be discussed. The information content of the spectra will be demonstrated using
spectra recorded on a sample containing pure methionine (Figure 3). A schematic
overview of the experiments is in Table 1.
ONE-DIMENSIONAL (1D) NMR SPECTROSCOPY
Since proton NMR is relatively sensitive and because protons are present in almost
every metabolite, a 1D, single pulse 1H NMR experiment with solvent presaturation
34
NMR spectroscopy of body fluids
is a good starting point when diagnosing a possible inborn error of metabolism.
When overlap is not too severe and metabolite concentrations are high enough (>
2 – 40 µmol/l), the chemical shifts and J-coupling constants can be determined
directly and subsequent quantification of these metabolites is possible. The singlet
resonance at 2.13 ppm from the ε methyl protons of methionine is most suited to
quantify this metabolite in body fluids (Figure 3A). However, assignment can be
rather difficult when high order spin coupling patterns are present or when
spectral regions are crowded. Furthermore, presaturation of the water signal can
result in loss of nearby peaks.
TWO-DIMENSIONAL (2D) NMR SPECTROSCOPY
Two-dimensional NMR experiments can provide additional information to solve
overlap problems and allow identification of metabolites that otherwise remain
undetected. They are based on the couplings between magnetic nuclei. These can
be dipolar (through space) or scalar (through bond) couplings. Here, only
experiments from the latter category will be discussed.
1H-1H
correlation spectroscopy (COSY)
The first homonuclear 2D experiment is correlation spectroscopy (COSY). The
underlying concept is coherence transfer from one spin to another via J-coupling
[31]. It reveals the network of spin-spin couplings in each molecule and can
therefore be used to aid in spectral assignment. The peaks under the diagonal of a
COSY spectrum correspond to the peaks normally observed in a 1D spectrum. The
off-diagonal peaks (cross peaks) provide the interesting information on which
protons are spin coupled. From the COSY spectrum of methionine for instance it is
clear that the α -proton is J-coupled to the β-protons (Figure 3B). Unfortunately,
there is a 90˚ phase difference between the diagonal and cross peaks, which
makes it impossible to correct the phase of a COSY spectrum in such a way that all
peaks have absorption phase. When the phase of the cross peaks is corrected to
pure absorption, the diagonal peaks display dispersive line shapes in both
dimensions and their long dispersion tails can conceal nearby cross peaks. A
solution is to record a Double Quantum Filtered COSY (DQF-COSY) experiment, in
which both diagonal and cross peaks have absorption line shapes. Consequently,
the spectral resolution is improved and cross peaks close to the diagonal can be
readily detected.
35
Chapter 1.3
Figure 3
Six different 500 MHz NMR spectra of methionine. (Legends page 37)
36
NMR spectroscopy of body fluids
Figure 3 (Page 36)
Six different 500 MHz NMR spectra of methionine.
A: 1D 1H-NMR spectrum
ε
ε
S-C H3: The methyl protons on C give a singlet resonance at 2.13 ppm. This singlet corresponds to the three
equivalent protons of the methyl group. The fact that it is a singlet illustrates that the neighboring atom (the S
atom) does not carry any proton(s). In body fluids the 2.13 ppm resonance is often used to quantify
methionine.
γ
β
γ
C H2: Due to the J-coupling with the two neighboring protons on C the protons on C give a triplet resonance
γ
at 2.63 ppm ( Jγ, β = 7.3 Hz)
β
β
α
C H2: The protons on C give two multiplets at 2.10 and 2.20 ppm. This is explained by the fact that C is an
β
asymmetrical carbon atom, and the two methylene protons on C are therefore diastereotopic; consequently
α
γ
they give separate resonances. Due to the J-coupling with the proton on C and the protons on C we find two
multiplets.
α
α
C H: Due to the J-coupling with the two diastereotopic methylene protons on Cβ, the proton on C gives a
disturbed triplet (doublet/doublet) resonance at 3.95ppm.
The peak area for the various resonances in methionine is proportional to the number of protons attached to
the various C atoms.
B: 2D 1H-1H COSY spectrum
Chemical shift and scalar coupling can be obtained simultaneously for methionine by detecting the offdiagonal cross peaks, which are symmetric with respect to the diagonal. The diagonal and cross peaks
connected by dashed lines indicate which protons of methionine have a scalar coupling. The diagonal peak of
β
the two protons on C forms a corner of two squares, as these protons are coupled both to the proton on C
α
γ
and to those on C .
C: 2D 1H-1H TOCSY spectrum
Compared with the COSY spectrum (Figure 3B) two new signals have now appeared. These provide evidence of
α
γ
a correlation between the protons on C and C .
D: 1D 1H-decoupled
13
C spectrum
In the 1D 1H-decoupled
α
β
γ
ε
C NMR spectrum, C , C , C and C from methionine give resonances at 56.7, 32.5,
13
31.7 and 16.7 ppm, respectively.
Note: This 1D 1H-decoupled
13
C spectrum was recorded at pH 7.0.
E: 2D 1H-13C HSQC spectrum
This type of NMR spectroscopy reveals which carbons and protons in methionine are chemically bonded. For
methionine, the 1H/13C shift values for the correlation peaks are 3.86 / 56.7 (α), 2.20; 2.10 / 32.5 (β), 2.65 /
34.7 (γ) and 2.13 / 16.7 (α).
Note: This HSQC spectrum of methionine was recorded at pH 7.0.
F: 2D 1H J-resolved spectrum
The J-multiplets are displayed along the F1 dimension, while the chemical shifts are shown in the F2 dimension.
This minimizes overlap and allows better determination of coupling constants, which also are detectable in 1D
γ
γ
1H-NMR. The protons on C give a triplet resonance at 2.63 ppm with a J-coupling of 7.3 Hz ( J
γ, β = 7.3 Hz)
Also, chemical shifts and coupling constants can be determined more easily.
However, a disadvantage is that the sensitivity is reduced compared to a regular
COSY, because only two out of four scans lead to cross peaks and the other two
lead to diagonal peaks [31]. Another solution to the phase problem is displaying
37
Chapter 1.3
the conventional COSY spectrum in magnitude mode. All peaks appear absorptive,
although cross peaks near the diagonal are still likely to be obscured. In literature,
COSY spectra of body fluids have been widely used in patients with IEM, e.g., in
fucosidosis [11], 3-hydroxy-3-methylglutaryl CoA lyase deficiency [32], ribose 5phosphate isomerase deficiency [33] and Salla disease [11;34].
Total Correlation Spectroscopy (TOCSY)
COSY spectra of complex mixtures like body fluids can still show a substantial
amount of overlap. Resulting ambiguities in the spectral assignment may be
resolved by detecting multiple relayed connectivities using a Total Correlation
Spectroscopy (TOCSY) experiment. For instance, in an AMX spin system cross
peaks are observed between A and X, where both spins are coupled to spin M, but
not to each other. This allows detection of long range interactions (>3 bonds), that
are usually too weak in normal COSY spectra. Other advantages are a higher
sensitivity for larger molecules and absorption line shapes for both diagonal and
cross peaks. The number of observed cross peaks depends on the length of the
applied TOCSY mixing sequence. A spectrum recorded with a short mixing period
mainly contains cross peaks of directly coupled spins, whereas long mixing gives
rise to relay cross peaks. In the case of methionine, the TOCSY spectrum (Figure
3C) for instance shows cross peaks between the α-proton and the γ-protons,
which were not present in the regular COSY (c.f. Figure 3B). 750 MHz 1D TOCSY
spectra from seminal fluid was published by Spraul et al. [35]. Furthermore, 1D
TOCSY has been used to identify 3-ureidoisobutyric acid as accumulating
compound in ureidopropionase deficiency [36]. To our knowledge there are no
examples of successful application of 2D TOCSY to the field of inborn errors of
metabolism.
J-resolved spectroscopy (JRES)
Another useful homonuclear 2D experiment is the J-resolved (JRES) experiment,
which separates the J fine structure from the chemical shifts. In the resulting
spectrum, the J-multiplets are displayed along the t1 dimension, while the
chemical shifts are shown in the t2 dimension. This minimizes overlap and allows
better determination of coupling constants. Furthermore, a proton decoupled 1D
spectrum can be obtained by projection of the 2D J-resolved spectrum onto the F2
axis. The JRES spectrum of methionine is shown in Figure 3F. 600 MHz 2D JRES
1H-NMR spectra from urine and blood plasma samples was published by Foxall et
al. [37]. In literature, JRES 1H-NMR spectra of body fluids have been used in
patients with IEM, e.g, ribose 5-phosphate isomerase deficiency [33], maple syrup
urine disease [27] and 2-methylacetoacetyl CoA thiolase deficiency [38].
38
NMR spectroscopy of body fluids
Spin-echo spectra
Proton spectra can be also simplified by making use of the differences in
relaxation behavior between different molecules. This principle forms the basis of
spin-echo experiments. In spin-echo spectra, signals arising from relatively large
molecules (i.e.protein signals in intact plasma) are attenuated due to their shorter
relaxation times. Furthermore, even-numbered multiplets are displayed as
negative peaks, whereas singlets and odd-numbered multiplets appear positive
[38;39]. Spin-echo experiments can therefore be helpful when assigning complex
spectra. However, the method is less suited for quantification due to signal loss
during long echo times. Since plasma samples are routinely deproteinized, spin
echo experiments are not recorded often nowadays when trying to diagnose an
inborn error of metabolism.
13C
NMR Spectroscopy
With the advances made in NMR spectrometer technology, 13C NMR becomes more
and more feasible. Despite its low intrinsic sensitivity (~60 times less than 1H) and
low natural abundance (1.1%), 13C NMR can provide a wealth of additional
information. This is mainly because of the large range of observed chemical shifts,
resulting in many resolved signals, and because precursors of the metabolite of
interest can be isotopically enriched. This last option enhances sensitivity and can
simplify the study of metabolic pathways [40]. Another advantage of 13C NMR
spectroscopy is the absence of the water resonance, making solvent suppression
no longer necessary.
The simplest 13C method used in the field of inborn errors of metabolism is the 1D
1H decoupled 13C experiment. Proton decoupling results in enhanced sensitivity
due to collapsing of multiplets into singlets and due to the Nuclear Overhauser
Effect (NOE; up to 4 times for 13C). By comparing the decoupled spectrum with the
corresponding 1H-coupled spectrum, structural information can be obtained about
the number of protons bound to each carbon [40]. As an example, the 1D proton
decoupled 13C spectrum of methionine is shown in Figure 3D. Although 1D 13C
spectra are not commonly recorded yet, their effective use in studying several
inborn errors of metabolism, including galactosemia [41], dimethylglycine
dehydrogenase deficiency [42], 2-hydroxyglutaric aciduria [43], Canavan disease
[44;45] and argininosuccinic aciduria [46], has been shown.
Heteronuclear Single Quantum Coherence (HSQC) spectroscopy
An example of an experiment that combines 1H and 13C spectroscopy is the
Heteronuclear Single Quantum Coherence (HSQC). Since proton detection is
employed, it is much more sensitive than a carbon detected experiment. The
resulting spectrum reveals which carbons and protons are chemically bounded
(see Figure 3E for methionine). Hence, it can be used to facilitate spectral
assignment. For instance, the assignment of 3-methylglutaconic acid, 3hydroxyisovaleric acid and 3-methylglutaric acid in urine from a patient with 3-
39
Chapter 1.3
methylglutaconic aciduria type I (OMIM: 250950) [47]. Furthermore
effectiveness in diagnosing Canavan disease has been suggested [45].
its
DATA PROCESSING AND SPECTRAL INTERPRETATION
The NMR data are typically processed by Fourier transformation following
apodization and zero filling of the free induction decay (FID). The phase is then
corrected to obtain absorption line shapes and if necessary, a subsequent baseline
correction can be performed. Metabolites can be quantified by fitting their
resonances and the internal concentration reference signal with Lorentzian line
shapes. Subsequently the concentrations are calculated from the relative integrals
of the fitted resonances and the known concentration of the internal standard. For
blood and CSF metabolite concentrations are expressed in µmol/l. For urine, the
creatinine singlet at 3.13 ppm (3 equivalent protons) is commonly used as internal
reference and metabolite concentrations are usually reported in µmol/mmol
creatinine.
The NMR spectrum of a body fluid taken from a patient with an inborn error of
metabolism can be different from a control spectrum in three ways. First, unusual
metabolites can be observed. Secondly, certain metabolites that are always present
can be totally or partially absent. Thirdly, a novel constituent of the body fluid
occurs in increased concentration compared to an age related reference range. For
relatively sparse spectral regions, this information can be directly obtained from a
1H 1D spectrum and consequently lead to the diagnosis. However, body fluid
spectra are usually very complex (especially for urine), showing hundreds to
thousands of resonances in which small biochemical changes may be easily lost.
Automatic data reduction and pattern recognition methods reduce this wealth of
information and can therefore provide an alternative way for the diagnosis of
inherited metabolic diseases [27;48]. The first step is dividing the 1D NMR
spectrum into small sections, which provide a series of descriptors for each
sample. These descriptors are then used to map the spectrum using non-linear
mapping (NLM) or principal component analysis (PCA). The resulting twodimensional map displays clusters of similar samples. A nice separation from
control urine samples was obtained for patients suffering from cystinuria, oxalic
aciduria, porphyria, Fanconi syndrome and 5-oxoprolinuria [48]. It should be
noted that different classification parameters can result in different clustering.
Therefore, several maps may be required when screening for distinct metabolic
diseases.
40
NMR spectroscopy of body fluids
NMR spectra of body fluids
HEALTHY INDIVIDUALS AND DATABASES OF MODEL COMPOUNDS
Urine
Figure 4 shows a part of the 500 MHz 1H-NMR spectra (4.4 – 0.8 ppm) of urine
from a healthy child and a healthy adult. In both spectra, the major metabolites
and the peaks with chemical shift at pH 2.5 are creatinine (singlet: 3.13 ppm;
singlet: 4.29 ppm), creatine (singlet: 3.05 ppm; singlet 4.11 ppm), citric acid ([AB]2
spin system: 2.99; 2.83 ppm), dimethyl amine (triplet: 2.71 ppm), alanine
(doublet: 1.51 ppm) and lactic acid (doublet: 1.41 ppm).
Figure 4
500 MHz 1H-NMR spectrum of urine
from a healthy adult (A) and a healthy
child (B).
High concentrations of betaine and
N,N-dimethylglycine (DMG) can be
observed in the urine from the child
(B). These concentrations are normal
for age.
Several sources with peak assignments, chemical shift and multiplicity for a wide
range of metabolites are available in literature [5;6;24;49]. For the interpretation
of urine NMR spectra the age dependency in the concentration of many
metabolites is of relevance. For instance, high concentrations of betaine (singlet:
3.27 ppm; singlet: 3.94 ppm) and N,N-dimethylglycine (DMG) (singlet 2.93 ppm)
41
Chapter 1.3
can be observed in the urine 1D 1H spectrum from the child (Figure 4B). Especially
in the first month of life values up to 1500 μmol/mmol and 550 µmol/mmol
creatinine may be found for betaine [50] and DMG respectively [42]. In adult urine,
the concentration of betaine and DMG generally is lower with a maximal normal
value of 80 µmol/mmol creatinine and 26 µmol/mmol creatinine, respectively [42].
Betaine and DMG are metabolites that will not be detected using conventional
screening techniques and NMR studies have reported age-related reference values
for these metabolites [42;50]. Beside age, other factors such as gender,
medication and diet may cause severe variations in body fluid NMR spectra. These
factors will be discussed on page 52 (Drugs and food components in body fluids).
Plasma/serum
In deproteinised blood plasma, the major metabolites and the peaks with chemical
shift at pH 2.5 are lactic acid (doublet: 1.41 ppm, quartet: 4.36 ppm), citric acid
([AB]2 spin system: 2.99; 2.83 ppm), glucose (doublet of doublets: 3.23 ppm,
various: 3.30 – 3.95 ppm, doublet: 4.63 ppm, doublet: 5.22 ppm), acetic acid
(singlet 2.08 ppm) (Figure 2A). Due to the high concentration of glucose (5
mmol/l), identification of metabolites in the region between 3.30 and 3.95 ppm is
difficult with 1D 1H NMR spectroscopy. For instance, the singlet resonance of
glycine at 3.72 ppm, caused by the 2 protons of the methylene group, is hard to
identify at physiological concentration (concentration range: 100 – 384 µmol/l).
NMR data of 38 identified metabolites and 14 unidentified peaks measured in
deproteinesed blood plasma at pH 2.5 have been published [4]. Recently, the
singlet resonance at 3.14 ppm that was described as unknown in this blood
plasma study has been identified as dimethyl sulfone (DMSO2) [10]. Several other
reports about assignments in human blood plasma have been published [23;51].
Cerebrospinal fluid
In literature, there are several 1H-NMR studies of CSF [3;17;19;20;52]. Sweatman
et al. [17] reported assignments of 138 resonances in 1H-NMR spectra of human
CSF. These derived from 46 metabolites and the assignments were achieved by the
measurement of a combination of 2D experiments (JRES and COSY spectra). A
standardized method for recording 1D 1H-NMR spectra from CSF was published by
Wevers et al. [3]. Quantitative analysis by NMR vs. conventional techniques showed
good correlation for alanine, threonine, valine and lactic acid. Resonances from 50
metabolites in 40 CSF were found in this study. Furthermore, 16 unidentified
resonances were reported. Myo-inositol (triplet: 3.27 ppm; triplet: 4.05 ppm) in
CSF is strongly age-dependent. The concentration of myo-inositol may be as high
as 700 µmol/l in the CSF of young children (0 – 3 months) while in adults the
upper reference range limit is estimated to be < 30 µmol/l.
42
NMR spectroscopy of body fluids
NMR SPECTRA OF KNOWN INBORN ERRORS OF METABOLISM
Figure 5 shows an example of a 1D 1H-NMR spectrum of a urine sample from a
patient with an IEM. The patient has alkaptonuria (OMIM: 203500). The disease is
caused by a deficiency of the enzyme homogentisic acid oxidase in tyrosine
metabolism (Figure 6, enzyme H). The characteristic metabolite for this disease is
homogentisic acid. Normally this compound cannot be detected in body fluids.
The concentration in this patient’s urine amounted to 2500 μmol/mmol creatinine.
Figure 5
500 MHz
1H-NMR
spectrum of urine from a patient with
Figure 6
Metabolic pathway of tyrosine.
alcaptonuria. The spectrum shows a high concentration of
Homogentisic acid oxidase deficiency
detected in urine.
homogentisic
homogentisic acid (2500 μmol/mmol creatinine) normally not
(black square) results in accumulation of
acid
in
patients
with
alkaptonuria.
T: Tyrosine transaminase
P: 4-Hydroxyphenylpyruvate dioxygenase
H: Homogentisic acid oxidase
Table 2 provides a list of available papers on metabolic diseases studied with NMR
spectroscopy. In many studies, peak assignments were achieved by the
measurement of two-dimensional experiments, measurement of the pure model
compound, adding the pure model compound to the body fluid or specific
resonance shifts upon pH changes. In the next pages we provide an overview of
some key publications on several groups of inherited metabolic disorders studied
with NMR spectroscopy. The available literature indicates the power of NMR
spectroscopy for the field of inborn errors of metabolism. This review shows that
at least 100 different IEM can be diagnosed by NMR spectroscopy of urine. For
most of these diseases the handbook of NMR spectroscopy in inborn errors of
metabolism shows the characteristic parts of the NMR spectrum [5].
43
Chapter 1.3
Table 2: Inborn errors of metabolism studied using body fluid NMR spectroscopy.
Inborn error of metabolism
References
Amino acid disorders
Aminoacylase deficiency, Type I
[5;53-56]
Argininosuccinic acid lyase deficiency
[5;46;57;58]
Citrullinaemia
[5;11;57]
Cystathionine beta synthase deficiency
[5]
Cystinuria
[48;59;60]
Hawkinsinuria
[5;61]
Histidinaemia
[3;5]
Homocystinuria
[62-64]
Hyperlysinaemia II
[5]
Lysinuric protein intolerance
[5]
Methionine adenosyl transferase deficiency
[5;10]
Non-ketotic hyperglycinaemia
[5;24]
Ornithine carbamoyl transferase deficiency
[5;57]
Phenylketonuria
[5;24;65]
Prolidase deficiency
[5;66]
Prolinaemia II
[5;67]
Sarcosinaemia
[5]
Tyrosinaemia I
[5;11]
Carbohydrate and related disorders
Congenital Defect of Glycosylation type IIb
[5;68]
Galactosaemia
[5;33;41]
L-Arabinosuria
[5;69]
Ribose 5-phosphate isomerase deficiency
[5;33;70]
Lysosomal diseases
β- Mannosidosis
[5;11]
α-Mannosidosis
[5;11]
Aspartylglucosaminuria
[5;11]
Fucosidosis
[5;11]
GM1-gangliosidosis
[5;11]
GM2-gangliosidosis (Sandhoff Disease)
[5;11]
Salla disease
[5;11;34;71]
Sialidosis
[5;11]
Organic acidurias
β-Ketothiolase deficiency
[5;24]
2-Aminoadipic aciduria
[5]
2-Hydroxyglutaric aciduria
[5;24;27;43;72]
2-Methyl-3-hydroxybutyryl CoA dehydrogenase deficiency
[5]
2-Methylacetoacetyl CoA thiolase deficiency
[38]
3-Hydroxy-3-methylglutaryl CoA lyase deficiency
[5;24;32;47;73]
3-Methylcrotonyl CoA carboxylase deficiency, isolated
[3;5;24;47;74]
3-Methylglutaconic aciduria, Type I
[5;47]
44
NMR spectroscopy of body fluids
Table 2: Inborn errors of metabolism studied using body fluid NMR spectroscopy. (Continued)
4-Hydroxybutyric aciduria
[5]
5-Oxoprolinuria
[4;5;48;72;73;75]
Alkaptonuria
[5;59;76-78]
Canavan disease
[3;5;11;44;45;79]
Cytochrome c oxidase deficiency
[80]
Glutaric aciduria type I
[5;39]
Glutaric aciduria type II
[5;24;81]
Isovaleric acidaemia
[5;24;39;47;82;83]
Lactic acidosis
[5;24]
Malonic aciduria
[5]
Maple syrup urine disease (=branched-chain ketoaciduria)
[5;24;27;39;47;73]
Methylmalonic aciduria
[5;24;39;82;84-87]
Mevalonic aciduria
[5]
Oxalic aciduria
[48;73]
Propionic acidaemia
[5;24;39;85;86;88]
Pyruvate dehydrogenase E1 deficiency
[80]
Pyruvate dehydrogenase E3 deficiency
[80]
Lipid metabolism
Cerebrotendinous xanthomatosis (CTX)
[5;89]
Glycerol kinase deficiency
[5;59]
Medium-chain acyl CoA dehydrogenase (MCAD) deficiency
[90]
Refsum disease
[89]
Short-chain acyl CoA dehydrogenase (SCAD) deficiency
[5]
Sitosterolemia
[89]
Smith-Lemli-Opitz syndrome (SLOS)
[89]
Purines / Pyrimidines Disorders
Adenosine deaminase deficiency
[5;91]
Adenylosuccinate lyase deficiency
[5;91]
β-Ureidopropionase deficiency
[5;36;92]
Dihydropyrimidinase deficiency
[5;91;93;94]
Dihydropyrimidine dehydrogenase deficiency
[5;91;93;94]
Molybdenum cofactor deficiency
[5;91]
Purine nucleoside phosphorylase deficiency
[5;91]
UMP synthase deficiency
[5]
Xanthine dehydrogenase deficiency
[5;91]
Vitamin related disorders
Biotinidase deficiency
[5;24]
Cobalamin Disorder Cbl-C
[95]
Vitamin B12 deficiency
[20]
Others
Cytochrome b deficiency
[80]
Dimethylglycine dehydrogenase deficiency
[5;42;96;97]
Fanconi syndrome
[48]
French type sialuria
[5;11]
45
Chapter 1.3
Table 2: Inborn errors of metabolism studied using body fluid NMR spectroscopy. (Continued)
Guanidinoacetate methyltransferase deficiency
[5;98]
Huntington's disease
[99]
Hypoacetylaspartia
[5;100]
N-Acetylaspartylglutamate accumulation in CSF
[5;25]
Porphyria
[48]
Trimethylaminuria (fish-odour syndrome)
[5;101-107]
Wilson disease
[108]
Amino acid disorders
Amino acids are metabolites containing an amino group (NH2) and a carboxylic
acid group (COOH). They are extensively metabolized and several inborn errors of
metabolism are located in amino acid metabolism. One of such pathways is the
biosynthesis of urea (urea cycle). Investigation of urea cycle disorders by 1H-NMR
spectroscopy was published for the urine of patients with citrullinaemia (OMIM:
215700), argininosuccinic aciduria (OMIM: 207900) and ornithine carbamoyl
transferase deficiency (OMIM: 311250) [57]. The diagnostic metabolites citrulline,
N-acetylcitrulline could be observed in the urine 1H-NMR spectrum of all patients
with citrullinaemia. These observations agree with those of Engelke et al. [11] who
reported an urinary citrulline and N-acetylcitrulline concentration of 825 and 518
µmol/mmol creatinine, respectively in a patient with argininosuccinate synthetase
deficiency (citrullinemia). The urine 1D spectrum of the patients with
argininosuccinic aciduria shows the characteristic metabolite argininosuccinate.
Orotic acid was detected in urine samples from three out of four patients with
ornithine carbamoyl transferase deficiency.
For the diagnosis of amino acid disorders cerebrospinal fluid is seldom used. The
metabolic profile of CSF may confirm a diagnosis. Table 2 gives an overview of the
inborn errors involving amino acid metabolism that have been studied with body
fluid NMR spectroscopy.
Carbohydrate related disorders
Carbohydrates may be found in body fluids in a soluble form as monosaccharides,
disaccharides, oligosaccharides and mucopolysaccharides and also as glycolipids.
The NMR spectrum of these molecules can be very complex. A paper by Moolenaar
[33] gives a table with resonances typical for a variety of monosaccharides and
disaccharides. Oligosaccharides accumulate in the urine in several lysosomal
diseases. Specific oligosaccharides have been isolated from thin layer
chromatography plates or otherwise purified and could subsequently be
characterized using NMR spectroscopy. In some cases this has contributed to
finding the primary defect in such disease. The most recent example is the
identification of a tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man
46
NMR spectroscopy of body fluids
structure that contributed to the identification of the primary defect in the subtype
IIb of the CDG syndrome [68] (Congenital Disorder of Glycosylation IIb) (OMIM:
606056).
Engelke et al have claimed the diagnosis of at least 5 different lysosomal diseases
in native urine with NMR spectroscopy [11]. The ganglioside concentration in the
urine of patients with GM1 and GM2 gangliosidosis escaped detection by NMR in
the native urine sample probably because of the low concentration. The group of
the mucopolysaccharidoses form a subgroup of the lysosomal diseases. The
accumulating mucopolysaccharides or glycosaminoglycans are long chains of
repeating saccharide units. Hochuli et al. have studied these compounds with twodimensional NMR spectroscopy but direct diagnosis in the native urine is not
feasible [109].
Organic acids
Organic acids are indicators of organic acidurias associated with various inborn
errors of metabolism. In 1985, Iles et al. [39] studied the organic acid metabolites
in urine from patients with propionic acidemia, methylmalonic aciduria, branched
chain ketoaciduria (maple syrup urine disease), isovaleric acidemia and glutaric
aciduria type I. The study provides a list with 1H-NMR chemical shifts (at pH 6.0) of
29 metabolites observed in the urine of the patients with the 5 selected organic
acidurias. Another list containing chemical shifts of many organic acid metabolites
was published by Lehnert and Hunkler [24]. They measured urine samples and
model compounds with NMR spectroscopy under standard pH conditions (pH
2.50). They investigated urine samples of 11 different inborn errors of metabolism
involving organic acids including three diseases from leucine metabolism. A recent
study gives the body fluid NMR characteristics for all known inborn errors of
leucine metabolism [47]. 1H-NMR spectroscopy of urine can easily discriminate
between these inborn errors of leucine metabolism and provide a correct the
diagnosis. Table 2 gives an overview of the available studies on organic acidurias.
Purines and pyrimidines
More than 12 enzyme defects in the metabolic pathways of purines and
pyrimidines are known to occur [110]. Wevers et al. [91] have studied body fluids
from 25 patients with 9 different inborn errors in these pathways. Characteristic
abnormalities could be demonstrated in the 1D 1H-NMR spectra of urine samples
of all patients. The only exception was case of adenine phosphoribosyl transferase
deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, could not
be seen under the NMR conditions used. In 2,8-dihydroxyadenine all protons
exchange rapidly with water and therefore this metabolite is NMR invisible. The
authors have provided a list of the most important 1H-NMR resonances from
relevant purine and pyrimidine metabolites measured at pH 2.5.
47
Chapter 1.3
Others
NMR spectroscopy can be used to find diseases characterized by the presence of
metabolites that cannot be detected with conventional screenings methods. The
available literature shows that NMR spectroscopy is the method of choice to
diagnose patients with trimethylaminuria or “fish odor syndrome” (OMIM: 602079).
This is a malodor syndrome caused by the accumulation of trimethylamine (TMA),
due to a deficiency of the liver enzyme flavin-containing monooxygenase 3
(FMO3). In urine of affected patients the TMA concentration is increased while the
trimethylamine N-oxide (TMAO) concentration is decreased. NMR spectroscopy
enables the simultaneous determination of these metabolites in urine (TMA:
doublet 2.98 ppm and TMAO: singlet at 3.54 ppm). Patients with milder mutations
can only be diagnosed with a loading test as the TMA concentration in a random
urine sample may be fully normal. Loading tests have been described with TMA
[111], with choline [112] and with fish meal [104]).
Guanidinoacetate methyltransferase (GAMT) deficiency (OMIM: 601240) deficiency
is a disorder in the biosynthesis of creatine. Guanidinoacetic acid (GAA) is the
characteristic accumulating metabolite in GAMT deficiency, the oldest of the three
creatine biosynthesis disorders known to date. 1H-NMR spectroscopy of urine and
CSF can be used for diagnosing this disorder [5;98]. The NMR spectrum of the
urine of a patient shows elevated GAA (doublet at 3.98 ppm). In CSF, creatine
(singlet resonances at 3.05 and 4.11ppm) and creatinine (singlet resonances at
3.13 and 4.29 ppm) are completely absent. Normally, they are always present in
CSF (reference value: creatine 25 – 70 µmol/l and creatinine 20 - 100 μmol/l [98]).
Accumulation of GAA in GAMT patients (CSF: ± 1 µmol/l; plasma: ± 10 µmol/l
[113]) is not visible in plasma and CSF 1H-NMR spectra due to overlap of other
resonances and the detection limit of the technique.
NOVEL DISEASES IDENTIFIED WITH BODY FLUID NMR
Three novel diseases were discovered by body fluid NMR spectroscopy, e.g.,
dimethylglycine dehydrogenase deficiency (OMIM: 605850), β-ureidopropionase
deficiency (OMIM: 606673) and a severe hypomyelination associated with
increased levels of N-acetylaspartylglutamate in CSF. In two other cases the
technique was strongly involved in the elucidation of the molecular defect, e.g.,
ribose 5-phosphate isomerase deficiency (OMIM: 608611) and congenital disorder
of glycosylation type IIb (OMIM: 606056).
The NMR findings in these novel diseases are shortly described below.
48
NMR spectroscopy of body fluids
Dimethylglycine dehydrogenase deficiency (OMIM: 605850)
In urine and plasma NMR spectra of an adult patient with a fish odor and muscle
weakness, high singlet resonances were observed at 2.93 and 3.80 ppm. 1H-NMR
spectroscopy of model compounds revealed that N,N-dimethylglycine (DMG)
caused these resonances. Addition of pure DMG to the urine sample and 13C-NMR
spectroscopy of the patient’s urine and an authentic reference solution of DMG
confirmed that the two singlets were caused by DMG. The concentration of DMG
amounted to 457 µmol/mmol creatinine in urine (reference range: 1 – 26) and 221
µmol/l in serum (reference range: 1 - 5). The high concentration of DMG was
caused by a deficiency of the enzyme dimethylglycine dehydrogenase (DMGDH)
converting DMG to sarcosine. A homozygous missense mutation was found in the
DMGDH gene of the patient [96].
β-Ureidopropionase deficiency (OMIM: 606673)
A new defect in pyrimidine metabolism found with in vitro 1H-NMR spectroscopy
was reported by Assmann et al. [92]. Characteristic resonances of 3ureidopropionic acid (triplet: 2.57 ppm; triplet: 3.36 ppm) and 3-ureidoisobutyric
acid (doublet: 1.13 ppm; multiplet: 2.69 ppm; AB protons: 3.26 nd 3.29 ppm)
were observed in high concentrations in the urine spectrum (861 and 718
μmol/mmol creatinine, respectively) [36]. One-dimensional and two-dimensional
NMR techniques were used to identify these metabolites. The accumulation of
these metabolites was caused by a deficiency of the enzyme β-ureidopropionase
(UP) converting 3-ureidoisobutyric acid and 3-ureidopropionic acid to,
respectively, β-aminoisobutyric acid and β-alanine. In this patient, Vreken et al.
[114] found compound heterozygosity for 2 splice acceptor site mutations in the
UPB1 gene, IVS1-2A-G and IVS8-1G-A. Van Kuilenburg et al. described a group of
patients with this defect [115].
Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate in CSF
Wolf et al. [25] described two unrelated girls with a severe hypomyelination of the
brain. Defects in the proteolipid protein (PLP) gene causing X-linked PelizaeusMerzbacher disease (PMD) could be ruled out. NMR spectroscopy of the CSF
revealed an increased concentration of the dipeptide N-acetylaspartylglutamic acid
(NAAG), a putative neurotransmitter that is thought to play a role in intercellular
signalling in the brain. NAAG concentration amounted to 95 and 197 µmol/l in the
2 patients (reference: < 4.4 µmol/l). The molecular and genetic basis of this novel
disease remains as yet unsolved. This is the first inborn error of metabolism where
a first key for understanding the molecular defect was found with NMR
spectroscopy of CSF.
49
Chapter 1.3
Ribose 5-phosphate isomerase deficiency (OMIM: 608611)
The combination of in vivo NMR spectroscopy of the brain with body fluid in vitro
NMR spectroscopy led to the recognition and characterization of the first ever
inborn error of polyol metabolism. In vivo NMR spectroscopy of the brain with a
severe white matter disease showed unknown resonances between 3.5 and 4.0
ppm. Body fluid NMR spectroscopy identified increased amounts of arabitol and
ribitol in all body fluids of the patient. Arabitol and ribitol in urine amounted to
1800 and 190 µmol/mmol creatinine (reference respectively: <98 and <11). In CSF
arabitol and ribitol concentrations were 5100 and 1200 µmol/l (reference range
respectively: <39 and <5 [33]). It could be shown that polyols caused the
abnormal resonances in the brain MR spectrum. This led to the discovery of the
defective enzyme ribose 5-phosphate isomerase, an enzyme in the pentose
phosphate pathway. Mutations in the corresponding gene could be demonstrated
in the DNA [116].
Congenital Disorder of Glycosylation type IIb (OMIM: 606056)
The group of van Coster found a patient who constantly excreted an unknown
tetrasaccharide in the urine. This compound could be shown with thin-layer
chromatography. After isolation NMR spectroscopy helped to identify this
compound as Glc(α1-2)Glc(α1-3)Glc(α1-3)Man. This led the way to the
characterization of a novel subtype of the so called CDG syndrome (Congenital
Disorder of Glycosylation type IIb). The defect is in the enzyme glucosidase I
(GCS1) involved in biosynthesis of N-glycans. The defect could be confirmed at the
enzyme level and the molecular genetic level [68]. Abnormal resonances from the
accumulating tetrasaccharide can be directly observed in a random sample of the
patient [5].
NOVEL PATHWAY: METHANETHIOL METABOLISM
Using one-dimensional and heteronuclear two-dimensional NMR techniques,
Engelke et al. [10] have shown that dimethyl sulfone (DMSO2) is a "regular"
constituent of CSF (Figure 7) and plasma. DMSO2 derives from dietary sources,
intestinal bacterial metabolism, and from human endogenous metabolism. In man,
methanethiol is converted into DMSO2. Increased plasma DMSO2 concentrations
are found in patients with methionine adenosyltransferase I/III (MAT I/III)
deficiency (OMIM: 250850). These findings led to the elucidation of a novel
metabolic pathway that was not known to occur in man (Figure 8).
50
of dimethyl sulfone (singlet 3.14 ppm) amounted to 18 µmol/L.
peak with the coordinates 3.14 (1H) / 44.3 (13C). The concentration
cerebrospinal fluid sample (pH=2.5). Dimethyl sulfone showed a
1D1H (upper) and 1H-13C HSQC (lower) spectrum of a representative
Figure 7
Both, dietary sources and putative endogenous metabolic pathways can potentially contribute the presence of
dimethyl sulfone in human body fluids.
The partial putative pathway leading to dimethyl sulfone in human body fluids.
Figure 8
Chapter 1.3
DRUGS AND FOOD COMPONENTS IN BODY FLUIDS
Beside age, other factors such as medication and diet may cause severe variations
in body fluid NMR spectra and hinder proper spectrum interpretation and
quantification. Some examples are described below.
Drugs
Valproic acid (Depakene®, Valproate, Valrelease®).
Several NMR studies [117-119] reported urine samples from patients who took the
anticonvulsant drug valproic acid. In man, valproic acid is extensively metabolized
into several products, and some are excreted into the urine. The urine 1H-NMR
spectra of these patients show many high resonances from 3-hydroxyvalproic acid
and valproic acid glucuronide. Azaroual et al. [118] reported a valproic acid
intoxification identified by NMR spectroscopy.
Vigabatrin (Sabril®)
The anti-epileptic drug vigabatrin is often used in patients suspected to have an
inborn error of metabolism. The 1H-NMR urine spectra of vigabatrin treated
patients show high vigabatrin resonances between 2.0 and 2.1 ppm caused by CH2
protons (multiplet). This vigabatrin multiplet interferes with the N-acetyl
resonances. Therefore correct interpretation and quantification of the N-acetyl
region of the 1H-NMR spectrum is impossible.
Ethosuximide (Zaronthin®)
Ethosuximide is a drug used to control seizures. A patient with guanidinoacetate
methyltransferase deficiency was treated with this drug and resonances of
ethosuximide (triplet: 0.86 ppm; singlet: 1.28 ppm; multiplet: 1.65 and AB: 2.68
ppm) could be observed in the CSF 1H-NMR spectrum of this patients [98]. Due to
the enzyme deficiency, creatine and creatinine resonances were absent in this
spectrum as expected.
Dimethyl sulfone (Methylsulfonylmethane, MSM)
NMR spectroscopy was performed on CSF from a 3-year-old boy with mental
retardation and loss of acquired skills. A high concentration of DMSO2 (singlet:
3.14 ppm) was observed in his 1D 1H-NMR CSF spectrum. At the time of the
lumbar puncture the patient had used a dietary supplement containing DMSO2
[10].
Other drugs that are observed in body fluid NMR spectra are acetaminophen
(Paracetamol®) [14;119-121], ampicillin [122], carbamazepine (Epitol®, Tegretol®)
[119], diethylcarbamazine (Hetrazan®) [123] and penicillin [14] and the drug
vehicle, propylene glycol [52]. In some cases also metabolites of the drugs can be
observed.
52
NMR spectroscopy of body fluids
Figure 9
500 MHz 1D 1H-NMR spectrum of urine from a healthy control after drinking Coca Cola light. Acesulfame K showed a
doublet at 2.11 ppm and a quartet at 5.67 ppm.
Food components
Acesulfame K
Acesulfame K is a sweetener about 200 times sweeter than sucrose. Acesulfame K
is not metabolized and it can be found in the urine 1H-NMR spectra of persons
who drink light products containing Acesulfame K, e.g., cola light (Figure 9). Other
food ingredients or their metabolites that are observed in body fluid NMR spectra
are ethanol in alcoholic drinks (triplet: 1.17 ppm; quartet: 3.64 ppm), TMA Noxide in fresh see fish (singlet: 3.54 ppm), trigonelline in coffee (singlet: 4.44
ppm; triplet: 8.10 ppm; singlet: 9.19 ppm) and Vitamin C (multiplet: 3.75 ppm;
multiplet: 4.07 ppm; doublet: 4.97 ppm).
53
54
TWO
N-ACETYLATED METABOLITES IN BODY FLUIDS
55
56
CHAPTER
2.1
N-Acetylated Metabolites in Urine:
Proton Nuclear Magnetic Resonance Spectroscopic
Study on Patients with Inborn Errors of Metabolism
Udo F.H. Engelke, Maria L.F. Liebrand-van Sambeek, Jan G.N. de Jong, Jules G.
Leroy, Éva Morava, Jan A.M. Smeitink and Ron A. Wevers
Clinical Chemistry 2004; 50(1): 58-66
57
58
N-acetylated metabolites in urine
Abstract
Background: There is no comprehensive analytical technique to analyze Nacetylated metabolites in urine. Many of these compounds are involved in inborn
errors of metabolism. In the present study, we examined the potential of proton
nuclear magnetic resonance (1H-NMR) spectroscopy as a tool to identify and
quantify N-acetylated metabolites in urine of patients with various inborn errors of
metabolism.
Methods: We performed 1H-NMR spectroscopy on a 500 MHz spectrometer. Using
a combination of one- and two-dimensional correlation spectroscopy (COSY) 1HNMR spectra, we were able to assign and quantify resonances of characteristic Nacetylated compounds products in urine of patients with 13 inborn errors of
metabolism.
Results:
The disease-specific N-acetylated metabolites were excreted at
concentrations >100 µmol/mmol of creatinine in the patients’ urine. In control
urine samples, the concentration of individual N-acetyl-containing compounds
was <40 µmol/mmol of creatinine. The combination of one- and two-dimensional
COSY NMR spectroscopy led to the correct diagnosis of nine different inborn errors
of metabolism. No abnormalities were observed in the spectra of urine from
patients with GM1- or GM2-gangliosidosis. We also determined the 1H-NMR
characteristics of N-acetylated metabolites that may be relevant to human
metabolism.
Conclusion:
spectroscopy may be used to identify and quantify Nacetylated metabolites of diagnostic importance for the field of inborn errors of
metabolism.
1H-NMR
59
Chapter 2.1
Introduction
Proton nuclear magnetic resonance (1H-NMR) spectroscopy of body fluids has been
used to diagnose inborn errors of metabolism [3;4;39]. The technique yields an
overview of proton-containing metabolites, is rapid and nondestructive, and
requires minimal sample pretreatment. In this report, we document the effective
use of 1H-NMR spectroscopy for the identification and quantification of N-acetylcontaining metabolites in body fluids of patients with various inborn errors of
metabolism. N-Acetylation occurs in many metabolic pathways, which makes the
detection of N-acetylated compounds important. Several diseases are known to be
associated with the accumulation of N-acetylated metabolites. Some of these have
been studied by 1H-NMR spectroscopy. The urinary excretion of N-acetylated
amino acids has been published for patients with inborn errors of amino acid
metabolism [124;125]. N-Acetylaspartic acid and N-acetylaspartylglutamic acid
are the laboratory marker metabolites in Canavan disease [126]. Other authors
have described one- and two-dimensional NMR experiments for structural
characterization and identification of N-acetylneuraminic acid-containing storage
products in lysosomal diseases [127-129]. These studies demonstrate that the
detection of N-acetylated metabolites assists in the diagnosis of various inborn
errors of metabolism. However, many N-acetylated compounds remain undetected
by the conventional analytical techniques used in metabolic screening laboratories.
The three equivalent N-acetyl protons resonate as a singlet in a small part of the
1H-NMR spectrum (1.9–2.2 ppm). Only a few other metabolites are known to cause
resonances in this area. The finding of singlet signals in a one-dimensional proton
NMR spectrum can thus be used as an indication of the presence of an Nacetylated metabolite.
We present here the NMR characteristics of many N-acetylated compounds
relevant for human metabolism. In addition, we provide NMR spectra for urines
representative for 13 inborn errors of metabolism that involve N-acetylated
compounds.
Materials and Methods
MODEL COMPOUNDS
Most model compounds were commercially available from Sigma or Aldrich. NAcetylated oligosaccharides from lysosomal storage diseases were isolated
preparatively from thin-layer chromatography (TLC) plates routinely used for
mono- and oligosaccharide analysis from urine of affected patients. Manα13Manβ1-4-N-acetylglucosamine (GlcNAc), Fuc-GlcNAc-Asn, aspartylglucosamine,
and two sialyloligosaccharides were isolated with a butanol–acetic acid–water (2:1:1
60
N-acetylated metabolites in urine
by volume) solvent system and visualized with orcinol–sulfuric acid (Fig. 1).
Because of an overlap with lactose, Manβ1- 4GlcNAc from the urine of a βmannosidosis patient was isolated with propanol–acetic acid–water (42.5:0.5:7.5
by volume) solvent system. The characteristic TLC bands were removed
preparatively from the silica-coated plate and measured as a model compound.
Figure 1
Thin-layer chromatogram of dextran and urine samples. The solvent system was butanol–acetic acid–water and bands were
visualized using orcinol-sulfuric acid staining.
Lanes: 1, dextran; 2, sample from a patient with α-mannosidosis; 3, sample from a patient with fucosidosis; 4, sample from a
patient with GM2-gangliosidosis; 5, sample from a patient with GM1-gangliosidosis; 6, dextran; 7, sample from a healthy
individual; 8, sample from a patient with aspartylglucosaminuria; 9, sample from a patient with sialidosis. The arrows indicate
the characteristic TLC bands.
URINE
Control urine samples were from 10 healthy individuals and from 50 diseased
patients. In the urine samples from diseased controls sent to our laboratory,
extensive metabolic screening had ruled out any relevant inborn errors of
metabolism.
Urine was centrifuged before analysis. We added 70 µL of a 20.2 mmol/l
trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (sodium salt; Aldrich) 2H2O
solution to 700 μL of urine as a chemical shift reference (δ = 0.00) and as a lock
signal. The pH of the urine was adjusted to 2.50 ± 0.05 with concentrated HCl.
Finally, we placed 650 µL of the sample in a 5-mm NMR tube (Wilmad Royal
Imperial).
61
Chapter 2.1
NMR MEASUREMENTS
High-resolution spectra for urine samples and the model compounds were
acquired at 500 MHz on a Bruker DRX and AMX spectrometer equipped with a
sample changer. The spin-lattice or longitudinal relaxation time (T1) values for Nacetylneuraminic acid, N-aspartylglucosamine, N-acetylaspartic acid, and Man(14)GlcNAc, as representative N-acetylated compounds, were determined in urine of
patients by the inversion-recovery technique. The T1 values amounted to 1.0, 0.9,
1.6, and 0.9 s, respectively.
ONE-DIMENSIONAL 1H-NMR SPECTROSCOPY
For urine, 128 transients were collected into 32 000 data points with a spectral
width of 6002 Hz. The H2O resonance was presaturated during the relaxation
delay (10 s). Shimming of the sample was performed automatically on the
deuterium signal. A sine-bell squared filter (SSB = 2) was used, and spectra were
Fourier-transformed after the free induction decay was zero-filled to 64 000 data
points. The phase and the baseline were corrected manually. The N-acetyl
resonances in the one-dimensional urine spectra were fitted semiautomatically to
a Lorentzian line shape model function. The integrals of these fits were used for
quantification by comparing them to the fit integral of creatinine (singlet at 3.13
ppm). For identification and quantification of the NMR spectra, 1D WinNMR and the
AMIX software were used (Bruker BioSpin).
A prerequisite for accurate quantitative determination of metabolites with proton
NMR spectroscopy lies in the length of the relaxation delay. We used a delay of 10
s, which is more than five times the T1 value of the N-acetyl protons in Nacetylaspartic acid, the compound with the longest T1 value among the Nacetylated compounds.
1H-1H
CORRELATION SPECTROSCOPY (COSY)
The spectral widths in the F1 and F2 axes were 6002 Hz, and 4000 data points
were collected in F2. For the urine samples, 256 increments and 16 scans per
increment were used. The relaxation delay was 2 s, and before the Fourier
transformation, a sine function was applied in both time domains. During the
relaxation delay, the water resonance was presaturated.
QUANTITATIVE DATA
To study the linearity and recovery, we added three N-acetylated metabolites, N-a
cetyltyrosine (cat. no. A-2513; Sigma), N-acetyltryptophan (cat. no. A-6376;
Sigma), and N-acetylaspartic acid (cat. no. A-5625; Sigma), separately to normal
human urine in concentrations of 0, 100, 500, and 3000 µmol/l. After addition of
these metabolites the creatinine concentration of the urine was 1.1 mmol/l. The
same experiment was repeated with a second urine with a creatinine concentration
of 2.0 mmol/l. For concentration calculations for the N-acetylated metabolites
62
N-acetylated metabolites in urine
using 1H-NMR spectroscopy, we used trimethylsilyl-2,2,3,3-tetradeuteropropionic
acid as the concentration reference, and the metabolite concentrations were
expressed in µmol/l.
Results
N-ACETYL RESONANCES IN URINE
A representative 1H-NMR spectrum of urine from a healthy individual is shown in
Figure 2. Methyl protons from N-acetyl groups characteristically give a singlet
resonance in a limited part of the 1H-NMR spectrum (1.9–2.2 ppm).
Figure 2
500 MHz 1H-NMR spectrum of urine
from a healthy volunteer.
The creatinine peak (singlet at 3.13
ppm) is scaled to 100. The inset show
the N-acetyl region in greater detail.
Table 1 provides an overview of the N-acetyl-containing metabolites detectable in
urine samples from healthy individuals and patients. Individual compounds can be
recognized by the chemical shift of the N-acetyl group CH3 protons and from
resonances deriving from other parts of the molecule. Neuraminic acid, Nacetylaspartic acid, and the N-acetyl groups in glycoproteins in urine from healthy
controls are known to contribute to the resonances in this area [6]. In addition, a
few metabolites without an N-acetyl group are known to resonate between 1.9 and
2.2 ppm. These include glutamine (β-CH2 multiplet at 2.15 ppm), proline (β-CH2
multiplet at 2.02 ppm), methionine (S-CH3 singlet at 2.13 ppm) [130], and acetic
acid (CH3 singlet at 2.08 ppm).
QUANTITATIVE DATA
The linearity was excellent for all three N-acetylated metabolites, N-acetyltyrosine,
N-acetyltryptophan, and N-acetylaspartic acid (slope, 0.94–1.09; intercept did not
63
Chapter 2.1
significantly deviate from zero; correlation coefficient >0.99). The recovery was in
90–110% for these compounds at all concentrations.
To study the reproducibility (within-run), we prepared and measured 10 samples
from the same urine on the same day. The within-run CV at 3000 µmol/l was <4%
for all three N-acetylated metabolites.
Table 1: 1H Resonances positions and multiplicity of N-acetyl containing metabolites.
Metabolites
Resonance position (ppm) and multiplicity
Acetyl
CH3 singlet (s)
Rest of the molecule
N-Acetylneuraminic acid (β anomer)
2.05
1.85 (dd); 2.26 (dd)
N-Acetylneuraminic acid (α anomer)
2.03
1.65 (dd); 2.72 (dd)
2.02, 2.03, 2.06
1.72 (dd); 2.67 (dd)
N-Acetylneuraminic acid (Bound AcNeu2→6)
a
GlcNAc-Asn
2.01
2.97 (mu); 3.83 (mu); 4.11 (tr); 5.08 (do)
(GalGlcNAc-)Asn
2.00
2.95 (mu); 3.54 (mu); 3.87 (mu); 4.10 (tr); 4.48 (do); 5.09 (do)
Manβ1-4GlcNAc
2.03
3.72 (mu); 3.89 (mu); 4.72 (do); 5.20 (do)
N-Acetylcitrulline*
2.03
1.52 (mu); 1.65-1.80 (mu); 3.12 (qu)
N-Acetylaspartic acid
2.03
2.95 (mu); 4.72 (mu)
N-Acetyltyrosine
1.94
3.07 (AB); 7.89 (AB)
Manα1-6Manβ1-4GlcNAc
2.03
3.88 (mu); 4.04 (do); 5.10 (mu); 5.20 (do)
Fuc-GlcNAc-Asn
2.01
1.21 (do); 2.94 (mu); 3.78 (do); 4.06 (tr); 4.12 (qu); 4.90 (do);
5.10 (do)
2.05, 205
β-GlcNAc(1-2)αMan(1-3)\
4.10 (mu); 4.19 (mu); 4.24 (mu); 4.53 (do); 5.11 (mu); 5.20
(do)
βMan(1-4)GlcNAc
β-GlcNAc(1-2)αMan(1-3)/
β-GlcNAc(1-2)αMan(1-3)\
2.04, 2.05, 2.06
βGlcNAc(1-4) - βMan(1-4)GlcNAc
4.10 (mu); 4.19 (mu); 4.24 (mu); 4.45 (do); 4.53 (do); 5.05
(mu); 5.11 (mu); 5.20 (do)
β-GlcNAc(1-2)αMan(1-3)/
GalNAcGalFuc (blood group A trisaccharide)
2.04
1.21 (do); 1.23 (do); 4.42 (mu); 5.13 (do); 5.15 (do); 5.18 (do);
5.29 (do); 5.38 (do); 5.45 (si)
Acetylcarnitine
2.14
3.19 (si)
N-Acetylglutamic acid
2.03
2.00 (mu); 2.19 (mu); 2.49 (tr); 4.38 (mu)
Bi-antennary octasaccharide
2.04, 2.05, 2.05
(in GM1-gangliosidosis urine)
N-Acetylaspartylglutamic acid
4.11 (do); 4.19 (do); 4.25 (do); 4.46 (do); 4.57 (do); 5.11 (mu);
5.20 (do)
2.04
2.01 (mu); 2.23 (mu); 2.48 (mu); 2.85 (AB); 4.44 (mu); 4.72
(mu)
N-Acetyltryptophan
1.92
3.31 (m); 4.70 (qu); 7.2-7.7 (varius)
a = si, singlet; do, doublet; tr, triplet; qu, quartet; mu, multiplet; dd, doublet doublet; AB, AB-system.
STRATEGY FOR QUANTITATIVE INTERPRETATION AND ASSIGNMENT
Because of the low concentrations of N-acetylated metabolites, assignment of
their singlet resonances in urine from healthy volunteers is generally not possible.
64
N-acetylated metabolites in urine
Metabolic diseases that involve N-acetylated compounds may be diagnosed if the
concentrations of the characteristic N-acetylated metabolites are high enough to
qualify them as abnormal and to allow their identification. Identification requires
the spectral information from the protons in the rest of the molecule (Table 1). As
a systematic approach, we quantified the highest singlet resonance between 1.9
and 2.2 ppm. Excluded from this quantification was the singlet for acetic acid
(mean chemical shift, 2.083 ppm; minimum, 2.081 ppm; maximum, 2.085 ppm; n
= 10). It was assumed that CH3 protons from N-acetylated metabolites cause the
resonances in this region.
Figure 3
Quantification of the N-acetylated compound with the highest concentration in the urine as function of age.
|, controls; z, urine of affected patients.
1, Salla disease; 2, French-type sialuria; 3, α-mannosidosis; 4, β-mannosidosis; 5, fucosidosis; 6, aspartylglucosaminuria; 7,
GM1-gangliosidosis; 8, GM2-gangliosidosis; 9, sialidosis; 10, Canavan disease; 11, citrullinemia; 12, tyrosinemia type I; 13,
tyrosinemia type II.
Figure 3 shows the concentrations of individual N-acetyl-containing compounds in
urine samples from 50 diseased controls and 10 healthy volunteers. The highest
concentration of an N-acetylated compound was generally <40 µmol/mmol of
creatinine. Higher concentrations were observed in children under 5 years.
Especially in the first month of life, values up to 200 µmol/mmol of creatinine
could be found. Figure 3 also shows data on urine samples from patients with 13
different inborn errors of metabolism involving N-acetylated compounds. Below
40 µmol/mmol of creatinine, the CH3 protons from the N-acetyl group are the only
resonances from most N-acetylated compounds that can be observed in the
spectra. Therefore, assignments based solely on the N-acetyl singlet were not
65
Chapter 2.1
possible. As shown in Figure 3, most patients with inborn errors of metabolism
excreted the N-acetylated metabolites typical for their diseases at concentrations
>100 µmol/mmol of creatinine. At such concentrations, the typical N-acetyl
compound could be assigned with greater certainty because the other protons of
the molecule contribute to the assignment. Two-dimensional COSY spectra may
help with further identification of relevant metabolites. As an example, Figure 4
shows how specific cross-peaks in a two-dimensional COSY spectrum of the
model compound Fuc-GlcNAc-Asn (Figure 4A) can help to identify this metabolite
in the urine of a patient with fucosidosis (Figure 4B). The cross-peaks derive from
the GlcNAc protons (3.83–5.10 and 3.78–4.90 ppm), the Asn protons (2.94–4.06
ppm), and the Fuc protons (1.21–4.12 ppm).
A
Figure 4
COSY spectra of model compound Fuc-GlcNAc-Asn
obtained from TLC chromatography (A) and urine
B
from a patient with fucosidosis (B).
The characteristic pattern of the model compound
Fuc-GlcNAc-Asn is shown in the circled cross-peaks.
66
N-acetylated metabolites in urine
INBORN ERRORS OF METABOLISM
Infantile sialic acid storage disorder (ISSD; OMIM 269920) and
Salla disease (OMIM 604369).
ISSD and Salla disease are both characterized by increased concentrations of free
sialic acid (N-acetylneuraminic acid) in the urine and by its storage in the
lysosomes of different tissues. The molecular basis of these diseases is a defect in
the lysosomal sialic acid transporter. Severe mutations in the gene lead to ISSD,
whereas milder mutations clinically present as Salla disease. Characteristically, a
20- to 200-fold increase in N-acetylneuraminic acid excretion in urine has been
described in infantile cases of the disease. Bound sialic acid typically is within the
range obtained for controls. One- and two-dimensional COSY 1H-NMR spectra
were obtained from urine of a 16-year-old patient with Salla disease. The onedimensional urine spectrum showed the presence of N-acetylneuraminic acid
[singlet at 2.05 ppm; 121 µmol/mmol of creatinine; reference values, 20.2 (11.6)
µmol/mmol of creatinine for ages >2 years [131]; Figure 5A]. The COSY spectrum
confirmed the presence of free N-acetylneuraminic acid by showing cross-peaks
from the pyranose ring protons of the carbon- 3 atom (1.85 and 2.26 ppm; data
not shown) [34].
French-type sialuria (OMIM 269921).
French-type sialuria is a metabolic disorder caused by a defect the enzyme uridine
diphosphate-N-acetylglucosamine 2-epimerase (EC 5.1.3.14), which is involved in
N-acetylneuraminic acid synthesis. The defect in the enzyme leads to failing
feedback inhibition by N-acetylneuraminic. Free N-acetylneuraminic acid is
massively increased in the body fluids of diseased patients. We investigated the
urine of a 6-year-old child with this disease. N-Acetylneuraminic acid excretion
(Figure 5B) was much higher than in the patient with Salla disease [4275
µmol/mmol of creatinine; reference values, 20.2 (11.6) µmol/mmol of creatinine
for ages >2 years [131]]. Because of the very high concentration of Nacetylneuraminic acid, even the pyranose ring protons (3.5–4.1 ppm) and the
nitrogen proton (doublet at 8.09 ppm) were clearly observed in the one- and twodimensional spectra (data not shown).
α-Mannosidosis (OMIM 248500).
Patients with a deficiency of the lysosomal enzyme α -mannosidase (EC 3.2.1.24)
have high concentrations of MannGlcNAc (n ≥ 2) oligosaccharides in the cells and
urine. Oligosaccharide TLC of the urine from an affected patient showed many
mannose-rich oligosaccharide bands. The major oligosaccharide band (Figure 1,
lane 2) typical for this defect was isolated preparatively from the silica-coated
plate and measured as a model compound by 1H-NMR. The compound was
identified by comparison with reference spectra as the trisaccharide Manα13Manß1-4GlcNAc [132]. One- and two-dimensional COSY 1H-NMR spectra were
obtained from urine of the patient. In the COSY spectrum, Manα1-3Manß1-
67
Chapter 2.1
4GlcNAc was recognizable as two cross-peaks (4.04–5.10 and 3.88–5.20 ppm),
and in the one-dimensional spectrum was recognizable as a singlet at 2.03 ppm
(Figure 5C) and doublet at 5.20 ppm. The urinary concentration of Manα13Manß1-4GlcNAc in our patient was 153 µmol/mmol of creatinine.
β-Mannosidosis (OMIM 248510).
The lysosomal enzyme β-mannosidase is involved in glycoprotein catabolism. A
deficiency of this enzyme leads to excessive urinary excretion of the disaccharide
Manß1-4GlcNAc. We measured urine samples from two brothers (ages 8 and 9
years) with ß-mannosidosis. Oligosaccharide TLC was performed on urine samples
from the boys. The Manß1-4GlcNAc band (data not shown) was isolated
preparatively from the silica-coated plate (100 µg) and measured as a model
compound by 1H-NMR. The spectrum showed the N-acetyl singlet (2.03 ppm), the
1α and 1β GlcNAc protons (doublets at 4.72 and 5.20 ppm, respectively), and
many resonances in the 3.4–4.1 region deriving from the GlcNAc and Man parts of
the disaccharide. The one-dimensional NMR spectra of their urine showed the
same singlets at 2.04 ppm (Figure 5D) and doublets at 5.21 ppm. The doublet at
4.72 ppm was not visible because of overlap of the water resonance. The COSY
spectra from the urine showed characteristic cross-peaks from the 1α and 1ß
GlcNAc protons (3.89–5.20 and 3.72–4.72 ppm, respectively; data not shown). The
Manß1-4GlcNAc concentrations in the urine of the brothers were 255 and 273
µmol/mmol of creatinine. Another, smaller singlet at 2.03 ppm was also observed
in the one-dimensional NMR spectra of the two brothers (56 and 122 µmol/mmol
of creatinine). Presumably this compound is sialyl-α2-6-mannosyl-ß1-4-Nacetylglucosamine ( 10% of the concentration of Manß1-4GlcNAc). Van Pelt et al.
have identified this trisaccharide in concentrated urine fractions of affected
patients [127].
Fucosidosis (OMIM 230000).
Because of a deficiency of lysosomal α-L-fucosidase (EC 3.2.1.51), patients with
fucosidosis accumulate fucose-containing glycolipids, oligosaccharides, or
glycoasparagines in the urine and tissue. Three characteristic oligosaccharides
were isolated from urine of an affected patient by TLC (Figure 1, lane 3). These
bands were removed from the TLC plate and dissolved in 2H2O. The fractions were
analyzed by 1H-NMR spectroscopy and compared with the literature [133]. The
one- and two-dimensional spectra of fraction 1 indicated the presence of FucGlcNAc-Asn (Table 1). Because of the low concentrations in the isolated fractions 2
and 3, interpretation was impossible. The characteristic COSY pattern of FucGlcNAc-Asn (cross-peaks at 1.21–4.12 and 2.94–4.06 ppm) was observed in the
spectrum for urine from an affected adult patient (Figure 4B). The urinary FucGlcNAc-Asn concentration was 109 µmol/mmol of creatinine (singlet at 2.01 ppm;
Figure 5E).
68
N-acetylated metabolites in urine
Figure 5
500 MHz 1H-NMR spectra of urine samples.
(A), Salla disease; (B), French-type sialuria; (C), α-mannosidosis; (D), β-mannosidosis; (E), fucosidosis; (F),
aspartylglucosaminuria; (G), GM1-gangliosidosis; (H), GM2-gangliosidosis; (I), sialidosis; (J), Canavan disease; (K),
citrullinemia; (L), tyrosinemia type I.
The creatinine peak (singlet at 3.13 ppm; not shown) in the urine spectra is scaled to 100.
69
Chapter 2.1
Aspartylglucosaminuria (OMIM 208400).
Aspartylglucosaminidase deficiency or aspartylglucosaminuria is an inborn error of
metabolism caused by the deficiency of the lysosomal enzyme N-aspartyl-ßglucosylaminidase (EC 3.5.1.26). This enzyme cleaves the N-acetylglucosamine–
asparagine linkage found in a variety of oligosaccharides and glycoproteins.
Patients with the disease excrete aspartylglucosamine (GlcNAc-Asn) as major
abnormal metabolite. In addition, several other glycoasparagines, such as Galß14GlcNAcß1-N-Asn, the mannose-rich derivative Man-Man-GlcNAc-GlcNAc-Asn,
and NeuAc-Gal-GlcNAc-Asn, can be excreted [134]. Urine samples from two
unrelated cases were available. TLC with orcinol–sulfuric acid showed the
abnormal Galß1-4GlcNAcß1-N-Asn band in these samples (Figure 1, lane 8). TLC
plates stained with ninhydrin showed the GlcNAc-Asn and the Galß1-4GlcNAcß1N-Asn band in the urine (data not shown). The NMR spectra of nearly pure
compounds were compared with reference spectra from the literature [135]. The
resonances of both compounds are shown in Table 1. The characteristic crosspeaks of both compounds were observed in COSY spectra of urine from both
patients. The GlcNAc-Asn concentrations (singlet at 2.02 ppm; Figure 5F) were
169 and 323 µmol/mmol of creatinine, respectively. The Galß1-4GlcNAcß1-N-Asn
(singlet at 2.01 ppm) concentrations were 27 (case 1) and 114 µmol/mmol of
creatinine (case 2).
GM1-Gangliosidosis (OMIM 230500).
A deficiency of β-D-galactosidase (EC 3.2.1.23) leads to GM1-gangliosidosis.
Oligosaccharides with a D-galactosyl group at the nonreducing end are increased
in the urine of affected patients. Three bands were isolated preparatively by TLC
from the urine of an affected patient (Figure 1, lane 5) and compared with spectra
from the literature [135]. The NMR spectrum of band 2 corresponded to the
biantennary octasaccharide [135], and the NMR spectrum of band 3 corresponded
to the triantennary decasaccharide [135] or the tetraantennary dodecasaccharide
[135]. For band 1, we could not find an exact match with reference spectra.
The one-dimensional NMR spectrum of urine from an affected patient (Figure 5G)
showed singlets between 2.00 and 2.04 ppm. The singlet with the highest
intensity (2.03 ppm) represented a N-acetylated storage compound with a
concentration of 34 µmol/mmol of creatinine.
GM2-Gangliosidosis (Sandhoff disease; OMIM 268800).
As a result of a deficiency in β-N-acetylhexosaminidase (EC 3.2.1.52),
oligosaccharides containing N-acetylglucosamine are excreted in the urine of
affected patients. Using TLC, we were able to preparatively isolate two bands from
the urine of an affected patient (Figure 1, lane 4). The NMR spectra of both
isolated bands showed resonances at 5.20, 5.11, and 4.53 and a singlet at 2.05
ppm. Band 2 differed from band 1 by an additional doublet at 4.46 ppm and two
more singlets (2.045 and 2.06 ppm). The chemical shift data point to
70
N-acetylated metabolites in urine
oligosaccharides with the structures β-GlcNAc-(1→2)-α-Man-(1-3)-[β-GlcNAc(1→2)- α-Man-(1-6)]-β-Man-(1→4)-GlcNAc for band 1 and β-GlcNAc-(1→2)-αMan-(1-3)-[β-GlcNAc-(1→4)][β-GlcNAc-(1→2)]-α-Man-(1-6)]-β-Man-(1→4)GlcNAc for band 2 [136].
The one-dimensional NMR spectrum for urine of an affected patient (Figure 5H)
showed singlets between 2.02 and 2.08 ppm. The singlet with the highest
intensity (2.06 ppm) represents a N-acetylated storage compound at a
concentration of 77 µmol/mmol of creatinine.
Sialidosis (OMIM 256550).
Sialidosis, or mucolipidosis I, is caused by a deficiency of the lysosomal enzyme
neuraminidase. The enzyme cleaves terminal α2→3 and α2→6 sialyl linkages of
various glycopeptides, oligosaccharides, and gangliosides. High concentrations of
sialyloligosaccharides (bound sialic acid) are found in urine of affected patients.
At least six singlet resonances with a concentration >50 µmol/mmol of creatinine
were found between 2.0 and 2.1 ppm by one-dimensional 1H-NMR spectroscopic
analysis of the urine of our patient (Figure 5I). The COSY spectrum of the urine
showed two cross-peaks: 1.72–2.67 ppm from the α2→6 sialyl linkages and 1.81–
2.67 ppm from the α2→3 sialyl linkages (data not shown). The singlet with the
highest intensity (2.03 ppm) was present at a concentration of 430 µmol/mmol of
creatinine.
Using TLC, we preparatively isolated two oligosaccharides from the urine of an
affected patient (Figure 1, lane 9). We observed two multiplets (1.75 and 2.66
ppm; cross-peak in the COSY spectrum) in the NMR spectra of both isolated
compounds. This allowed us to conclude that both isolated compounds have an
2→6 sialyl linkage. In the spectrum for band 1, three N-acetyl singlets resonated
at 2.02, 2.03, and 2.06 ppm, deriving from the two GlcNAc units and the
acetylneuraminic acid (NeuAc) unit. From its spectrum we could conclude that
band 1 probably is α-NeuAc-(2→6)-β-Gal-(1→4)-β-GlcNAc-(1→2)-α-Man(1→3)-β-Man-(1→4)-GlcNAc [137].
Band 2 differed in the chemical shift position of one acetyl singlet (2.05 instead of
2.03 ppm) and the presence of an additional signal at 4.11 ppm, which could
indicate compound V in the report by Dorland et al. [137].
Canavan disease (OMIM 271900).
Canavan disease is a leukodystrophy attributable to a deficiency of aspartoacylase
(EC 3.5.1.15) and belongs to the group of neurotransmitter diseases. We analyzed
urine from two unrelated patients. The one-dimensional 1H-NMR spectra for body
fluid showed the presence of high concentrations of N-acetylaspartic acid [Figure
5J; urine from case A, 1760 µmol/mmol of creatinine; urine from case B, 1474
µmol/mmol of creatinine; reference values for all ages, 6–36 µmol/mmol of
creatinine [138]].
71
Chapter 2.1
Citrullinemia (OMIM 215700).
Deficiency of the urea cycle enzyme argininosuccinate synthase (EC 6.3.4.5) leads
to increased concentrations of citrulline and N-acetylcitrulline in the urine
[124;139].
We analyzed two urine samples from a patient with citrullinemia. The urine 1HNMR spectrum showed citrulline multiplets (concentration, 825 µmol/mmol of
creatinine). We assigned the unusually high singlet at 2.03 ppm to Nacetylcitrulline by comparing the chemical shifts with reference spectra in the
literature [57]. N-Acetylcitrulline is not commercially available. The urinary Nacetylcitrulline concentrations were 441 and 518 µmol/mmol of creatinine (Figure
5K).
Tyrosinemia type I (OMIM 276700).
Tyrosinemia type I is caused by a deficiency of the enzyme fumarylacetoacetate
hydrolase (EC 3.7.1.2). High body fluid concentrations of tyrosine, 4hydroxyphenylacetic acid, 4-hydroxyphenyllactic acid, succinylacetone, and
methionine are characteristic of affected patients. We analyzed the urine of a 1year-old boy with this inborn error.
In one-dimensional 1H-NMR spectra of his urine, we observed high concentrations
of tyrosine and 4-hydroxyphenyllactic acid; the concentrations of betaine and
methionine were also high. In addition to these typical tyrosinemia type 1
metabolites, we found a high concentration of N-acetyltyrosine (singlet at 1.94
ppm; 195 µmol/mmol of creatinine; Figure 5L). We identified the compound by
comparing the spectrum with the model compound (singlet at 1.94 ppm; AB
system at 3.07 ppm and AB system at 7.89 ppm). Succinylacetone and
succinylacetoacetate, the biochemical hallmarks in tyrosinemia type I, could not be
demonstrated by NMR spectroscopy in this urine.
Tyrosinemia type II (OMIM 276600).
Deficiency of the cytosolic enzyme tyrosine aminotransferase (EC 2.6.1.5) forms
the primary defect of tyrosinemia type II. In the liver it leads to high concentrations
of tyrosine, 4-hydroxyphenyllactic acid, 4-hydroxyphenylacetic acid, 4hydroxyphenylpyruvic acid, and N-acetyltyrosine in body fluids. Urine from one
patient was analyzed. The concentration of N-acetyltyrosine in the urine of an
affected patient was 95 µmol/mmol of creatinine (singlet at 1.94 ppm). In another
patient (age, 0.2 years), we observed a much higher concentration of Nacetyltyrosine in the urine (>3000 µmol/mmol of creatinine). This patient received
parenteral nutrition containing N-acetyltyrosine. In premature infants (gestational
age at birth <36 weeks), urinary N-acetyltyrosine may be as high as 781
µmol/mmol of creatinine [140]. In addition, N-acetyltyrosine may be increased in
some liver diseases, in which it may be observed along with N-acetyltryptophan
(Table 1)
72
N-acetylated metabolites in urine
Discussion
N-Acetylated compounds are metabolites in many metabolic pathways and
therefore may relate to various inborn errors of metabolism. In this study, we gave
examples of 13 inherited diseases involving N-acetylated metabolites. At present,
a comprehensive analytical technique to detect and identify N-acetylated
compounds in body fluids is not available. However, N-acetylaspartic acid, the
biochemical hallmark in Canavan disease, is detected by organic acid analysis by
gas chromatography–mass spectrometry. Gas chromatography–mass spectrometry
can also be used for the accurate diagnosis of tyrosinemia by analysis for all
relevant metabolites, including N-acetyltyrosine. TLC is useful in the identification
of abnormal N-acetylated oligosaccharides characteristic of some of the lysosomal
storage disorders.
NMR spectroscopy of body fluids can provide an overview of N-acetylated
compounds. The protons of the N-acetyl group resonate in a specific narrow
region of the spectrum. For metabolic screening purposes, we used the highest
resonance in the N-acetyl region to identify samples with unusually high
concentrations of an N-acetylated metabolite (Figure 3). This approach allowed
different N-acetylated metabolites to be quantified in individual samples. For
example, in the urine of a young child, the singlet at 1.94 ppm gave the highest
concentration, whereas in an adult urine, the highest concentration was for the
singlet at 2.04 ppm. As shown in Figure 3, most patients with inborn errors of
metabolism excreted the N-acetylated metabolites typical for their diseases at
concentrations >100 µmol/mmol of creatinine. In 10 of the 13 inborn errors of
metabolism, this concentration was clearly higher than the upper limit of the
reference interval. In all of these cases the combination of one-dimensional
spectroscopy and COSY spectra allowed identification of the compound of interest.
This led to the correct diagnosis in 9 of the 13 cases. Exceptions included the
urines from patients with tyrosinemia type I and II and GM1- and GM2gangliosidosis.
In tyrosinemia type I, the excretion of succinylacetone and succinylacetoacetate in
urine is the diagnostic hallmark. To date, gas chromatography–mass spectrometry
remains the method of choice to determine succinylacetone and the other
characteristic metabolites and to diagnose tyrosinemia type I. For similar reasons
the same holds true for tyrosinemia type II.
Because of the relatively low urinary concentrations of the GM1- and GM2gangliosidosis metabolites (34 and 77 µmol/mmol of creatinine), assignment of
these metabolites in urine in affected patients was not possible, and the diagnosis
of GM1- and GM2-gangliosidosis was missed by 1H-NMR. NMR spectrometers with
higher fields (>500 MHz) and spectrometers using the increased sensitivity of
CryoProbe technology could probably detect these two inborn errors of
metabolism.
73
Chapter 2.1
In spite of pH standardization, assignments of structure based solely on the
chemical shift of the N-acetyl protons are insufficient because many acetyl singlets
may occur in urine. Therefore, characteristic COSY patterns of model compounds
are required to confirm the presence of individual N-acetylated metabolites in a
patient’s urine. For example, the acetyl protons of N-acetylaspartic acid and
Man(1-4)GlcNAc showed a singlet at the same chemical shift (2.03 ppm) in the
one-dimensional spectrum. Two-dimensional COSY NMR experiments showed for
each model compound different and characteristic cross-peaks produced by
protons elsewhere in the molecule (Table 1). Because of a change in structure,
bound N-acetylneuraminic acid can be distinguished from free N-acetylneuraminic
acid in two-dimensional COSY 1H-NMR spectra (Table 1). This makes it possible to
differentiate patients with isolated neuraminidase deficiency, who excrete bound
N-acetylneuraminic acid, from patients with Salla disease or French type sialuria,
who excrete excess free N-acetylneuraminic acid in urine [34]. However, in regions
with a high density of resonances, especially around the two-dimensional COSY
diagonal, cross-peaks sometimes show overlap and cannot be used for correct
assignments. Other two-dimensional NMR techniques, such as J-resolved
spectroscopy and total correlation spectroscopy, can be used to help with further
interpretation.
The use of medications can be a pitfall in the quantification of N-acetylated
metabolites. The antiepileptic drug Sabril® (vigabatrin) is often used in patients
suspected to have an inborn error of metabolism. The 1H-NMR urine spectra of
Sabril-treated patients show high Sabril resonances between 2.0 and 2.1 ppm
caused by CH2 protons (multiplet). This Sabril multiplet interferes with the N-acetyl
resonances. Therefore, correct interpretation and quantification of the N-acetyl
region of the 1H-NMR spectrum is impossible. Some drugs can be converted in the
body to their acetylated conjugates. Acetaminophen, for example, is metabolized
in the body and is excreted in the urine in various forms. The 1H-NMR spectrum of
urine may show acetaminophen (paracetamol; singlet at 2.14 ppm), 4glucuronosidoacetanilide (singlet at 2.15 ppm), N-acetyl-4-aminophenol sulfate
(singlet at 2.17 ppm), and N-acetyl-2-(N-acetyl-L-cysteinyl)-4-aminophenol
[120].
In conclusion, we have shown that careful inspection and quantitative
interpretation of the N-acetyl region of the 1H-NMR spectrum may identify an
abnormally high concentration of an N-acetylated metabolite. The technique is
uniquely suited for studying patients with inborn errors of metabolism, and our
data extend that to diseases involving N-acetylated metabolites. A combination of
one- and two-dimensional COSY 1H-NMR spectroscopy identified at least nine
different inborn errors of metabolism involving N-acetylated metabolites.
74
N-acetylated metabolites in urine
Acknowledgements
The 1H-NMR spectra were recorded at the Dutch hf-NMR facility at the Department
of Biophysical Chemistry, University of Nijmegen, The Netherlands (Prof. Sybren
Wijmenga, department head). We thank Jos Joordens for invaluable help and
technical assistance. We also thank Drs. Neidig and Fischer (Bruker BioSpin,
Karlsruhe, Germany) for invaluable help and improvements in the AMIX software.
75
76
CHAPTER
2.2
Severe hypomyelination associated with increased
levels of N-acetylaspartylglutamate in CSF
Nicole I. Wolf, Michèl A.A.P. Willemsen, Udo F.H. Engelke, Marjo S. van der
Knaap, Petra J.W. Pouwels, Inga Harting, Johannes Zschocke, Erik A.
Sistermans, Dietz Rating and Ron A. Wevers
NEUROLOGY 2004; 62: 1503-1508
77
78
N-acetylaspartylglutamate in CSF
Abstract
Background: Two unrelated girls had early onset of nystagmus and epilepsy,
absent psychomotor development, and almost complete
cerebral MRI. The clinical features and MR images of both
connatal form of Pelizaeus–Merzbacher disease (PMD),
recessive disorder caused by duplications or mutations of
gene (PLP).
absence of myelin on
patients resembled the
which is an X-linked
the proteolipid protein
Objective: To define a unique neurometabolic disorder with failure of myelination.
Methods and Results: 1H-NMR of CSF in both girls was performed repeatedly, and
both showed highly elevated concentrations of N-acetylaspartylglutamate (NAAG).
The coding sequence of the gene coding for glutamate carboxypeptidase II, which
converts NAAG to N-acetylaspartate (NAA) and glutamate, was entirely sequenced
but revealed no mutations. Even though both patients are girls, the authors
sequenced the PLP gene and found no abnormality.
Conclusions: NAAG is an abundant peptide neurotransmitter whose exact role is
unclear. NAAG is implicated in two cases of unresolved severe CNS disorder. Its
elevated concentration in CSF may be the biochemical hallmark for a novel
neurometabolic disorder. The cause of its accumulation is still unclear.
79
Chapter 2.2
Introduction
White matter disorders of infancy and childhood may cause severe neurologic
disability. The disorders can be divided into demyelinating and hypomyelinating
processes, depending on whether normal myelin is formed and subsequently
degraded or whether the process of normal myelin formation never or
incompletely occurs [141;142]. The differential diagnosis of white matter disorders
represents a major challenge, leaving many patients without a specific diagnosis.
There have been some efforts to further classify unsolved white matter disorders.
One approach takes typical changes of MRI into account to categorize MRI findings
systematically and thus, by recognizing typical patterns, to delineate new disease
entities [143]. New techniques such as diffusion-weighted imaging [144] and in
vivo 1H-NMR spectroscopy further differentiate white matter disorders. The latter
has been used in the elucidation of several new neurometabolic disorders
[70;145;146]. Another powerful tool in the investigation of white matter disorders
is in vitro 1H-NMR spectroscopy of CSF. It is able to detect a wide range of
metabolites and also has contributed to the delineation of new inherited inborn
errors of metabolism [33;36;42].
In this report, we describe two unrelated girls presenting with nystagmus,
intractable epilepsy, and severe psychomotor retardation. MRI showed almost
absent myelination of the brain. In vitro 1H-NMR spectroscopy of CSF revealed
strongly elevated N-acetylaspartylglutamate (NAAG). The phenotype resembles
Pelizaeus–Merzbacher disease (PMD) [147], which occurs in boys and is caused by
alterations of the proteolipid protein gene (PLP) on the X-chromosome [148].
However, PLP gene alterations were ruled out. We speculate that the marked
elevation of NAAG in CSF may indicate an unrecognized neurodegenerative
disorder, but the underlying genetic defect is still unclear.
Patients and Methods
CASE REPORTS
Patient 1 is an 8-year old girl, born after an uneventful pregnancy as the second
child of unrelated, healthy parents. The first symptom at age 6 weeks was a fine
rotatory nystagmus. At age 5 months, severe hypotonia was noted. Head growth
decelerated during the first 6 months of life. Epilepsy with secondarily generalized
tonic seizures was overt from age 24 months and resistant to therapy. The initial
hypotonia evolved into a severe spastic paraparesis with positive Babinski signs,
which was most prominent at age 2 years. Now, at age 8 years, the patient is again
hypotonic with some spastic features (scissoring of the legs). Tendon reflexes
were initially normal, at the age of 12 months clearly exaggerated, but
disappeared around the age of 5 years, suggesting the development of a
80
N-acetylaspartylglutamate in CSF
peripheral neuropathy. Feeding difficulties are prominent. An older sister has
slight mental retardation without abnormalities on cerebral MRI. Two younger
sisters are healthy. Parents declined further investigations in this child.
Patient 2 is a 3-year-old girl from healthy and unrelated parents. She has a healthy
older brother. At age 4 weeks, the parents noted head nodding and nystagmus. At
age 6 weeks, seizures started, which could not be controlled. Microcephaly
developed during the first 4 months. A gastric tube was inserted at the age of 2
months because of feeding difficulties, but the child failed to thrive. Neurologic
examination revealed severe hypotonia, no head control, and absent tendon
reflexes. No developmental milestones were achieved. The course of the disease
was further complicated by severe nonepileptic myoclonus and dystonia.
MRI
The first cerebral MRI of Patient 1 was performed at the age of 2 years, the second
at age 3.5 years (not shown), and the third at age 5 years. The cerebral MRI of
Patient 2 were obtained at ages 11 weeks and 2, 3, and 3.5 years. The imaging
protocol for the brain comprised sagittal and transverse T1-weighted (repetition
time [TR] 570 milliseconds, echo time [TE] 14 milliseconds) and T2-weighted (TR
3,000 milliseconds, TE 22, 60, and 120 milliseconds) spin echo imaging and
coronal or transverse FLAIR (fluid-attenuated inversion recovery) images (TR 9,000
milliseconds, TE 105 milliseconds, inversion time 2,200 milliseconds).
IN VITRO 1H-NMR SPECTROSCOPY OF CSF AND URINE
Sample preparation.
After deproteinization using a 10-kd filter (Sartorius, Goettingen, Germany), 20.2
mM trimethylsilyl-2,2,2,3- tetradeuteropropionic acid (TSP; sodium salt; Merck,
Whitehouse Station, NJ) was added to the samples for spectrum calibration and
metabolite quantification. Final TSP concentration was 0.8 mM in CSF and 1.8 mM
in urine. The pH was adjusted to 2.50 ± 0.05. NAAG as model compound was
commercially available from Sigma (A5930; St. Louis, MO).
One-dimensional 1H-NMR spectroscopy.
Single-pulse experiments were recorded using a 60° pulse at 298 K on a Bruker
AMX-500 spectrometer (Bruker Analytische Messtechnik, Zurich, Switzerland), a 5mm TXI 1H/15N/13C xyz gradient probe head, and 5-mm Wilmad Royal Imperial
NMR tubes (Aldrich, Milwaukee, WI). The temperature was set at 298 K. The sweep
width was 6002 Hz. For urine, 128 scans were recorded and for CSF 256 scans. Per
scan, 32K data points were sampled. The relaxation delay was 10 seconds, during
which the water resonance was presaturated. The spectra were analyzed using
one-dimensional WinNMR and AMIX software (Bruker Analytische Messtechnik). A
sine-bell-squared filter (SSB = 2) was used, the spectra were zero-filled to 64K
data points, and Fourier transformation was performed. Phase and baseline were
81
Chapter 2.2
corrected manually. The concentrations were calculated by comparing the peak
areas of the metabolites with those of creatinine in urine or TSP in CSF.
1H-1H
correlation spectroscopy.
The spectra were recorded at 500 MHz using 4K data points in F2 and a spectral
width of 6,002 Hz. For the CSF samples, 256 increments and 16 scans/increment
were used. The TR was 1.5 seconds, during which the water resonance was
presaturated. Prior to Fourier transformation, a sine function was applied in both
time domains.
IN VIVO 1H-NMR SPECTROSCOPY
Patient 2 underwent singlevoxel MR spectroscopy at age 3.5 years, as described
previously [70]. Volumes of interest (VOI) were selected within the “white matter”
of the centrum semiovale in both hemispheres (4 mL) and the midparietal cortex
(8 mL). In each VOI, a fully relaxed, short echo time (STEAM) spectrum
(TR/TE/mixing time = 6,000/20/10 milliseconds, 64 accumulations) was obtained.
Metabolite concentrations were calculated using LCModel [149;150] and expressed
as millimoles per liter of VOI. Data were compared with values obtained in agematched control subjects [151].
Figure 1
NAA and NAAG signaling and metabolism in the
CNS:
1
= NAA synthase
2
= NAAG synthase
4
= glutamate carboxypeptidase II
3
NAA
NAAG
= N-acetylaspartate
= N-acetylaspartylglutamate
AcCoA
= acetyl-coenzyme A
Ac
= acetate
Glu
Asp
82
= aspartoacylase
= glutamate
= aspartate
N-acetylaspartylglutamate in CSF
MUTATION ANALYSIS OF PLP AND FOLH1 GENES
Patient genomic DNA was extracted from leukocytes using standard procedures.
PLP gene analysis (amplification of individual exons and quantitative Southern
blotting) was done as previously described, with the exception that exon 1 was not
analyzed by restriction fragment length polymorphism analysis but by sequencing
analysis [152].
The FOLH1 gene (GenBank accession no. AF007544) encodes for the enzyme
glutamate carboxypeptidase II (GCP II; EC 3.4.17.21) involved in NAAG catabolism
(Figure 1). For analyzing this gene, the 19 exons and exon–intron boundaries were
amplified separately. Primer sequences used are available on request. Each primer
contained an additional 17mer corresponding to a universal recognition sequence
(for the forward primers P172: TATAGGGCGAATTGGGT; for the reverse primers
M13: GTAAAACGACGGCCAGT). After purification, the PCR products were cycle
sequenced with the appropriate 5'-labeled universal primers using a dye
terminator sequencing kit (Applied Biosystems, Foster City, CA). Samples were
analyzed on an ABI 3100 automatic sequencer (Applied Biosystems).
Results
LABORATORY AND ELECTROPHYSIOLOGIC STUDIES.
Routine blood hematology and chemistry panels were normal. Urinary excretion of
organic acids and amino acids, oligosaccharides, and free and bound neuraminic
acid was normal. Plasma amino acids, very long chain fatty acids, and phytanic
acid were normal. Isoelectric focusing of serum transferring gave a normal pattern.
Activities of lysosomal enzymes measured in leukocytes were normal. CSF glucose,
cells, albumin, and protein levels were within normal limits. Lactate in blood and
CSF was not increased. Still, a quadriceps muscle biopsy was performed in Patient
2 and gave normal results for muscle histology. Activities of the individual
complexes of the respiratory chain were normal. Chromosomal analysis showed a
normal 46, XX karyotype in both girls. Electrophysiologic studies were carried out
at age 5 years, after the disappearance of tendon reflexes, in Patient 1 and at age
5 months in Patient 2. Motor nerve conduction velocity was normal in both, but
sensory nerve conduction studies could not be interpreted properly in Patient 1.
Brainstem evoked auditory potentials were abnormal in both children, showing
normal wave I and absent waves II to V.
MRI.
With MRI of the brain performed on several occasions, hardly any myelin could be
detected, as indicated by the homogeneous high signal of white matter on T2weighted images and low signal on T1-weighted images. The only evidence of
myelin deposition was in the brainstem (Figure 2). The corpus callosum was very
83
Chapter 2.2
thin in both patients. The signals of cortical structures and deep gray nuclei were
normal. During the follow-up period, Patient 1 displayed atrophy of supratentorial
structures and to a lesser extent of the cerebellum. Patient 2 showed only mild
supratentorial atrophy and a normal cerebellum without change over time.
BODY FLUID 1H-NMR SPECTROSCOPY.
Proton 1H-NMR spectroscopy of CSF of both patients (Figure 3) revealed an
unknown singlet resonance at 2.04 ppm. Smaller resonances at 2.85 (AB system),
2.01, 2.23, 2.48, 4.44, and 4.72 ppm (multiplet resonances) deriving from the
same molecule helped to assign the responsible metabolite as NAAG in onedimensional and two-dimensional correlation spectroscopy (COSY) spectra.
Its identity was confirmed by measuring NAAG as model compound. Figure 3
shows the prominent 2.04 singlet resonance deriving from the N-acetyl moiety of
the molecule and minor resonances around 2.85 ppm (COSY spectrum not shown).
The concentration of NAAG repeatedly was strongly elevated in both patients
(Patient 1: 95 and 90 µmol/l; Patient 2: 197 and 146 µmol/l; reference 4.4 ± 1.7
µmol/l [1 SD] [153]). CSF N-acetylaspartic acid (NAA) in our patients was
consistently lower than 5 µmol/l (the in vitro NMR spectroscopy detection limit),
which is considered normal. An elevated concentration of NAAG could not be
demonstrated in the 1H-NMR spectrum of urine of our patients (not shown;
detection limit 50 µmol/mol of creatinine). Using 1H-NMR spectroscopy, we never
found NAAG levels higher than 12 µmol/l in a control group of 200 patients
clinically suspected to have a neurometabolic disorder. Additionally, we examined
CSF from patients both with defined (n = 16) and undefined (n = 5) disorders of
the white matter. The group of patients with a defined white matter disease
consisted of three children with X-linked adrenoleukodystrophy, two children with
Krabbe disease, one child with metachromatic leukodystrophy, three children with
Sjögren–Larsson syndrome (SLS), two children with vanishing white matter disease,
one child with Aicardi–Goutières syndrome, two children with a mitochondrial
disorder, and two children with Canavan disease. The highest NAAG level found
was 12 µmol/l in one of the patients with SLS. Among the patients with
unclassified white matter disease were three children with severe myelination delay
(all of them with severe epilepsy), one child with hypomyelination, cerebellar
atrophy, and progressive ataxia, and one child with elevated signal of the central
white matter, progressive spastic paraplegia, and mental retardation. The NAAG
concentrations in this group ranged from 2 to 11 µmol/l.
IN VIVO 1H-NMR SPECTROSCOPY.
In vivo 1H-NMR spectroscopy of white matter in Patient 2 showed an elevation of
absolute concentrations of the sum of NAA and NAAG (tNAA) and also of creatine
and phosphocreatine (tCr), resulting in a normal tNAA/tCr ratio. Choline was
decreased in white matter. In parietal gray matter, the absolute tNAA
84
N-acetylaspartylglutamate in CSF
concentration was elevated as well, but less than in white matter; the
concentration ratio with tCr was normal (tables 1 and 2). Myo-inositol was normal
in gray matter but increased in white matter.
Table 1: Absolute metabolite concentrations
Metabolites
Cortex
Patient
White Matter
Controls
Patient
Controls
tCr
7.1
6.4 ± 0.7
8.2 ± 0.1
5.0 ± 0.4
tNAA
9.4
8.2 ± 0.9
11.1 ± 0.5
7.2 ± 0.7
Cho
1.2
1.1 ± 0.2
1.3 ± 0.1
1.9 ± 0.2
Ins
4.3
4.7 ± 0.4
4.8 ± 0.1
3.7 ± 0.6
In vivo 1H-MR spectroscopy findings in Patient 2
Values are absolute concentrations in mmol/l.
* Control values are absolute concentrations expressed as means ± SD. Patient values in white
matter are also means ± SD obtained from two white matter spectra of right and left parietal
white matter. Cortex spectra were recorded in the parietal cortex and white matter spectra in
right and left parietal white matter.
tCR = creatine + phosphocreatine; tNAA = N-acetylaspartate + N-acetylaspartylglutamate;
Cho = choline-containing compounds; Ins = myo-inositol.
Table 2: Concentration ratios relative to tCr
Ratios
Cortex
White Matter
Patient
Controls
Patient
Controls
tNAA / tCr
1.33
1.28 ± 0.11
1.35 ± 0.04
1.45 ± 0.15
Cho / tCr
0.17
0.18 ± 0.02
0.16 ± 0.01
0.38 ± 0.03
Ins / tCr
0.61
0.73 ± 0.07
0.58 ± 0.00
0.74 ± 0.09
In vivo 1H-MR spectroscopy findings in Patient 2
Values are concentration ratios related to tCr.
* Control values are expressed as means ± SD. Patient values in white matter are also means ±
SD obtained from two white matter spectra of right and left parietal white matter. Cortex
spectra were recorded in the parietal cortex and white matter spectra in right and left parietal
white matter.
tCR = creatine + phosphocreatine; tNAA = N-acetylaspartate + N-acetylaspartylglutamate; Cho
= choline-containing compounds; Ins = myo-inositol.
PLP/FOLH1
Analysis of the PLP gene did not reveal either duplication of the whole gene or
other mutations in the coding region and the exon–intron boundaries. Sequencing
of the FOLH1 gene revealed a heterozygous polymorphism (c222C3T resulting in
H75Y) in exon 2 of Patient 1, but no pathogenic mutation. This polymorphism was
observed in 23 of 98 alleles from healthy volunteers (data not shown).
85
Chapter 2.2
Discussion
In this study, we describe two unrelated patients with almost absent CNS myelin,
intractable epilepsy, nystagmus, progressive microcephaly, and severe
psychomotor retardation. The phenotype could be compatible with PMD. Also, the
MRI appearance could be consistent with this diagnosis, as the typical appearance
of imaging studies in this disorder includes diffuse severe hypomyelination with a
homogeneous high signal in T2-weighted images [154-156]. However, PMD is an
X-linked recessive disorder, and PLP gene alterations were ruled out in both
patients.
We consider the markedly elevated concentrations of NAAG in CSF, found by in
vitro 1H-NMR spectroscopy, as the biochemical hallmark of a novel
neurodegenerative disorder in these patients. Several girls with symptoms similar
to the severe connatal form of PMD but without PLP alterations have been
described. This was interpreted as an autosomal recessive form of the disease with
a still unresolved genetic basis [157;158]. Therefore, the connatal PMD phenotype
without PLP alterations may represent a separate genetic entity. Sequencing of the
whole PLP gene (exons and intron–exon boundaries) was performed in both
patients and revealed neither point mutations nor duplication. Female carriers of
PLP gene alterations are usually asymptomatic. A few mildly affected female
patients with PLP gene alterations (including duplications and null mutations) have
been described [159-162]. In some families, they develop late-onset spastic
paraplegia [163;164]. The clinical and molecular genetic data in our patients thus
make PMD due to PLP gene alterations highly unlikely. In vivo 1H-NMR
spectroscopy of one of our patients revealed elevation of absolute levels of tNAA
in white and gray matter. In relation to creatine, tNAA levels were normal. Choline
was significantly reduced in white matter. Similar findings [165] as well as
conflicting results [155;166-169] have been reported in PMD patients. The
elevation of tNAA in white matter in our patients may be caused by a denser
packing of axons secondary to the lack of myelin sheets, thus indicating the
presence of intact axons in the absence of myelin. Alternatively, a high
concentration of NAAG could explain the tNAA elevation. The N-acetyl group of
NAAG resonates at 2.04 ppm and of NAA at 2.02 ppm. Because of its high
resolution, in vitro 1H-NMR spectroscopy is able to distinguish these resonances,
but this may often be difficult in in vivo 1H-NMR spectroscopy. In our white matter
spectra, however, a prominent NAAG signal could not be detected in spite of good
spectral resolution.
86
N-acetylaspartylglutamate in CSF
Figure 2
Cerebral MRI of Patient 1 (A) and Patient 2 (B), demonstrating almost complete absence of myelin in supra- and infratentorial
structures. Gray matter (cortex and deep nuclei) appears normal. The corpus callosum is string-like. Note the supratentorial and
cerebellar atrophy in Patient 1. Axial pictures are T2 weighted, sagittal pictures T1 weighted.
87
Chapter 2.2
As our data on other leukodystrophies show, the clear elevation of NAAG in our
two patients is not a nonspecific finding associated with white matter disorders in
general. A mild increase in the concentration of NAAG in CSF has been described
in combination with highly elevated concentrations of NAA in a case of Canavan
disease [170].
Figure 3
spectroscopy of CSF: (A) CSF of patient 1 with a strong N-acetyl resonance at 2.04 ppm and smaller resonances
around 2.85 ppm. (B) normal control.
1H-NMR
During a recent congress, NAAG levels were also reported to be elevated in body
fluids from boys with PMD [171]. However, the abstract gives details neither on the
clinical and genetic characteristics nor on the concentration of NAAG in body fluids
of the patients. There is no explanation as yet for this finding. Further data on the
NAAG concentration in body fluids of patients with PLP alterations are pivotal in
unraveling the pathophysiologic mechanisms underlying this phenomenon. NAAG
is one of the most abundant metabolites in vertebrate brain [172;173]. It is
synthesized by the enzyme NAAG synthase (N-acetylaspartate-L-glutamate ligase)
from NAA and glutamate and degraded by GCP II, formerly called NAALADase
(Figure 1) [174;175]. In the brain, the latter enzyme is exclusively expressed on
the membrane surface of astrocytes. NAA is thought to be involved in neuron-toglia signaling; NAAG is targeted to the astrocyte and thus might contribute to
neuron-to-astrocyte signaling, which might be important for processes as
myelination and maintenance of axons [173]. Based on the above, we
88
N-acetylaspartylglutamate in CSF
hypothesized that accumulation of NAAG in our patients could be caused by
deficiency of the NAAG-degrading enzyme, GCP II. Sequencing the coding region
of the corresponding FOLH1 gene, however, did not reveal pathogenic mutations.
Thus, GCP II deficiency is not likely for the NAAG accumulation in our patients,
although promoter mutations of the gene have not been ruled out. Very recently,
GCP II knock-out mice were found to exhibit no abnormalities of the CNS, and
NAAG degradation was normal in the brain tissue. The finding was explained by
the presence of another enzyme with NAAG peptidase capacity [176]. In humans as
well, there are enzymes with NAAG hydrolyzing activity besides GCP II, but it is not
known whether they are expressed in the CNS [177]. Impaired function of one of
those enzymes or a defect in the complex NAAG transport and receptor system
(Figure 1) could theoretically cause NAAG elevation in the CNS of our patients.
Another explanation could be that abnormal, severely reduced, or absent myelin
impairs the normal function of the NAAG degrading enzyme on the astrocyte
membranes and leads to secondary elevation of this metabolite. Elevated NAAG in
boys with PLP alterations might favor this hypothesis. NAAG metabolism and
molecular genetic studies of the genes involved should be a focus for further
pathophysiologic studies in CNS disorders with severe hypomyelination.
89
90
CHAPTER
2.3
NMR spectroscopy of aminoacylase 1 deficiency,
a novel inborn error of metabolism
Udo F.H. Engelke, Jörn Oliver Sass, Rudy N. Van Coster, Erik Gerlo, Heike Olbrich,
Stefan Krywawych, Jacqui Calvin, Claire Hart, Heymut Omran and Ron A. Wevers
NMR in Biomedicine 2007; in press
91
92
Aminoacylase 1 deficiency
Abstract
Aminoacylase 1 deficiency is a novel inborn error of metabolism. The clinical
significance of the deficiency is under discussion, as well as the possible
consequences of the defect for brain metabolism and function. This study includes
the five originally published cases as well as three novel ones. NMR spectroscopy
of urine, serum and cerebrospinal fluid has been used to study these patients. A
typical profile with 11 accumulating N-acetylated amino acids was observed in
urine from the patients. The concentration of most of the accumulating
metabolites is typically 100–500 µmol/mmol creatinine. Two additional minor N-
acetylated
metabolites
accumulating
remain
metabolites
are
unidentified.
<20
µmol/L
The
in
concentrations
serum
from
the
of
the
patients.
Interestingly we found no evidence of an increased concentration of N-acetylated
amino acids in the cerebrospinal fluid from one patient. Our data define
aminoacylase 1 deficiency at the metabolite level providing a specific urinary
profile of accumulating N-acetylated amino acids.
93
Chapter 2.3
INTRODUCTION
Proton NMR spectroscopy has been successfully applied to the field of inborn
errors of metabolism (IEM) [9]. The technique can be used for rapid identification
and quantification of metabolites in body fluids. Known, but also as yet unknown,
IEM have been diagnosed using NMR spectroscopy of body fluids [33;36;42].
Aminoacylase 1 deficiency is a novel inborn error of metabolism (OMIM: 609924).
In two recent papers, clinical and laboratory data from 5 children with excessive
urinary excretion of various N-acetylated amino acids have been described
[54;55]. The patients were found to have a deficiency of aminoacylase 1 (ACY1; EC
3.5.1.14) which catalyzes the deacetylation of N-acetylamino acids. In this paper,
we provide a more detailed description of the NMR spectroscopic findings of the
five original patients with ACY1 deficiency as well as three novel cases.
Four types of aminoacylase have been identified on the basis of substrate
specificity [178]. ACY1 is encoded by the gene aminoacylase 1 (ACY1) [54]. The
enzyme preferentially uses neutral aliphatic amino acids with a short-chain acyl
moiety as substrate [178]. These include the N-acetylated forms of serine,
threonine, alanine, methionine and glycine. The enzyme aminoacylase 2 (ACY2) or
aspartoacylase (EC 3.5.1.15) catalyzes the deacetylation of N-acetylaspartic acid
(NAA) into aspartic acid. Deficiency of this enzyme causes Canavan disease (OMIM
271900) and results in high concentrations of NAA in body fluids of affected
patients. Aminoacylase 3 (ACY3) acts preferentially on N-acylaromatic amino acids
and the enzyme acyl-lysine deacylase catalyzes the N-deacetylation of N5acetyllysine. A deficiency of ACY3 and acyl-lysine deacylase in humans has not
been reported.
In this paper, we present data at the metabolite level typical for ACY1 deficiency
and show that NMR spectroscopy can easily discriminate between ACY1 and ACY2
deficiency. Moreover, this study presents the NMR characteristics of a broad range
of N-acetylated amino acids relevant for the diagnosis of several other inborn
errors of metabolism [179].
MATERIALS AND METHODS
PATIENTS.
Eight unrelated patients with ACY1 deficiency were included in this study (Table 1).
Clinical details about patient 1 [55] and patients 2 – 5 (cases OS-104 II-1, OS-127
II-1, OS-124 II-1, OS-120 II-1 in [54], respectively) have been described
elsewhere. Patient 6 and 8 are children from independent families of English and
Pakistani ancestry. The case histories will be described separately (cases OS138II-1
94
Aminoacylase 1 deficiency
and OS-152II-1 [180]). Patient 7 is the third child of distantly related Pakistani
parents. His father, older brother and sister are well. His mother has learning
difficulties, apparently acquired at the age of 8 years, following an illness. At the
age of 13 weeks there were concerns that he was not fixing and following, thought
to be consistent with delayed visual maturation. At the age of 5 months he was
noted to be very floppy and was referred for investigation of global developmental
delay and failure to thrive. At this time he was found to have abnormal acetylated
amino acids in the urine with normal plasma amino acids and blood spot
acylcarnitine profile. Currently, at the age of 21 months, he has severe
developmental delay and failure to thrive (weight << 2nd centile). He remains
profoundly hypotonic with extremely poor head control. The delayed visual
maturation is improving although he now suffers from intermittent nystagmus.
The Canavan patient was a 1-year-old boy with neurological involvement, mental
retardation, demyelination and loss of acquired skills.
THE DIAGNOSIS.
In most patients, the diagnosis ACY1 deficiency (OMIM 609924) was confirmed at
the metabolite level (GC-MS and NMR spectroscopy of urine), at the enzyme level
(ACY1 deficiency in EBV transformed lymphocytes) and the molecular genetic level.
In patient 5 the defect was established at the metabolite and the molecular genetic
level only. In patient 7 the defect was established at the metabolite and the
enzyme level.
The diagnosis in the Canavan patient (ACY2 deficiency) was established on basis of
the clinical features and observations at the metabolite level (GC-MS and NMR
spectroscopy of body fluids). Further confirmation was obtained by enzymatic
analysis in fibroblasts.
AUTHENTIC STANDARDS.
Most N-acetylated amino acids, listed in Table 2 and 3, were commercially
available and were purchased at Sigma or Aldrich. Exceptions included Nacetylthreonine, N-acetylcitrulline, N-acetyltaurine and N-acetylisoleucine. These
compounds were synthesized by Chiralix (Chiralix B.V., Toernooiveld 100, 6525 EC
Nijmegen, The Netherlands), by acylation of the corresponding amino acid with
acetic acid anhydride in 1M aqueous sodium hydroxide [181].
SAMPLE PREPARATION.
All body fluids were centrifuged for cell separation and stored at low temperature
(-80°C for CSF; -20°C for urine and plasma) until NMR analysis. Plasma and CSF
samples were deproteinised using Sartorius Centrisart I filters (cut-off 10kDa;
Sartorius AG, Göttingen, Germany). Before use, the filter was washed twice by
centrifugation of water to remove glycerol. Urine was centrifuged before analysis.
An aliquot (20 µl for plasma and CSF; 70 µl for urine) of 20.2 mM trimethylsilyl2,2,3,3-tetradeuteropropionic acid (TSP, sodium salt; Aldrich) in D2O was added
95
120
90
70
150
310
160
160
360
250
290
120
150
160
nd
nd
2 (13.2)
2 (13.4)
3 (1.4)
3 (1.6)
4 (0.6)
5 (3.1)
6 (3.3)
7 (0.9)
7 (0.7)
7 (0.5)
8 (0.93)
8 (0.95)
8 (1.43)
Canavan
Ref[182]
nd
nd
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Leucine
N-Ac.
nd
nd
++
++
++
+
+
+
+
+
++
+
+
+
+
+
++
Asparagine
N-Ac.
nd
nd
270
290
290
540
490
590
250
280
740
230
280
170
190
190
510
Glutamic acid
N-Ac.
nd
nd
++
++
++
+
+
++
+
+
++
+
+
+
+
+
++
Glutamine
N-Ac.
nd
nd
120
50
50
nd
nd
nd
+
nd
nd
580
590
300
260
+
Nd
+
350
+
b
Nd
420
80
520
430
300
180
90
130
110
+
+
+
+
+
+
+
+
+
230
Glycine
Isoleucine
+
N-Ac.
N-Ac.
b
a
160
60
50
80
80
80
50
50
40
120
Methionine
N-Ac.
nd
nd
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Valine
N-Ac.
Artifact
Not detectable (< ± 5 µmol/mmol creatinine
b
nd
+ / ++ Semi-quantitative estimate from the 1D 1H NMR spectrum (+=10 – 250, and ++ = 250 - 500 µmol/mmol creatinine)
Quantification on S-CH3 singlet (Figure 1; peak 12)
90
2 (13.1)
a
200
Alanine
(age)
1 (0.3)
N-Ac.
Patient nr.
Canavan disease
nd
nd
220
210
220
280
260
350
160
210
390
150
<10
100
110
100
330
b
Serine
N-Ac.
nd
Nd
30
20
30
60
40
50
20
30
40
<40
1470
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
20
<10
b
<10
<10
<10
NAA
20
20
20
50
Threonine
N-Ac.
Table 1. Urine concentrations (μmol/mmol creatinine) of the N-acetylated amino acids from patients with ACY1 deficiency (1 – 8) and the patient with
Aminoacylase 1 deficiency
to 700 µL of the ultrafiltrate or urine, providing a chemical shift reference (δH =
0.00 ppm), a concentration reference and a deuterium lock signal. The pH of each
sample was adjusted to 2.50 ± 0.05 with concentrated HCl. Finally, 650 µL of the
sample was placed in a 5mm NMR tube (Wilmad Royal Imperial).
ONE-DIMENSIONAL 1H-NMR SPECTROSCOPY.
Body fluids samples were measured at 500 MHz on a Bruker DRX 500
spectrometer essentially as described before [183]. The acetyl methyl protons of
N-acetylated compounds have a T1 relaxation time of approximately 2 s [11].
Therefore, we used a recycle time of 10 s, which is 5 times the T1 value of Nacetylated metabolites.
The N-acetyl resonances in the 1-dimensional urine spectra were fitted
semiautomatically to a Lorentzian line shape model function. The integrals of
these fits were used for quantification by comparing them to the fit integral of
creatinine (singlet: 3.13 ppm) and concentrations were expressed as µmol/mmol
creatinine. If computer fitting was not possible on these singlets due to severe
overlap we estimated concentration ranges based on peak intensity of the N-acetyl
CH3 resonances and the creatinine CH3 resonance (Table 1: + / ++ semiquantitative estimate from the 1D 1H-NMR spectra).
For CSF, the N-acetylated amino acid resonances and the TSP singlet (nine
equivalent protons) were fitted semi-automatically with Lorentzian line shapes and
concentrations were calculated from the relative integrals of the fitted lineshapes
using the known concentration of TSP. Concentrations were expressed as
µmol/liter. For identification and quantification of the NMR spectra, Topspin and
AMIX software were used (Bruker BioSpin, Karlsruhe, Germany).
1H-1H
COSY SPECTROSCOPY. 2-dimensional COSY NMR urine spectra and all
model compound spectra were measured at 500 MHz on a Bruker DRX 500
spectrometer essentially as described before [47].
1H-13C
HSQC SPECTROSCOPY. Three HSQC urine spectra from different ACY1
patients (Patients 1, 2 and 6) were measured at 500 MHz on a Bruker AV 500
spectrometer using a triple-resonance inverse CryoProbeTM (Bruker), wherein the
preamplifier and the RF coils are cooled to low temperature. Furthermore, the
HSQC spectra were measured of the model coumpounds N-acetylalanine, Nacetylleucine, N-acetylasparagine, N-acetylglutamic acid, N-acetylglutamine, Nacetylmethionine, N-acetylisoleucine, N-acetylglycine, N-acetylvaline, N-acetylserine and N-acetylthreonine. Details about the measurement have been described
elsewhere [47].
GAS CHROMATOGRAPHY MASS-SPECTROSCOPY.
N-Acetyl amino acids were determined with GC-MS after liquid extraction from
urine and derivatization with BSTFA [55;182]) and/or diazomethane [54;184]).
97
Chapter 2.3
RESULTS
We analyzed 15 urine samples from eight patients with ACY1 deficiency (Table 1).
In Figure 1A, the urine 1H-NMR spectrum (1.3 – 3.3 ppm) from patient 6 is shown.
The major metabolites in this region and the chemical shifts at pH 2.5 are
creatinine (singlet: 3.13 ppm), creatine (singlet: 3.05 ppm), citric acid ([AB]2 spin
system: 2.99; 2.83 ppm) and alanine (doublet: 1.51 ppm). Furthermore, several
unusually high singlet resonances were observed in the region between 2.0 and
2.2 ppm (Figure 1A; insert). In this region, CH3 protons from N-acetyl groups
characteristically give a singlet resonance. In control urine samples, the highest
concentration of an N-acetylated metabolite is generally below 40 µmol/mmol
creatinine [11]. In the urine of patient 6 (Figure 1A), seven singlet resonances with
a concentration higher than 40 µmol/mmol creatinine were observed in this Nacetyl region. This pattern was found in the urine from all 8 patients with ACY1
deficiency. NMR measurements of model compounds confirmed that these singlet
resonances were caused by different N-acetylated amino acids. Resonances were
assigned from N-acetylalanine, N-acetylleucine, N-acetylasparagine, Nacetylglutamic acid, N-acetylglutamine, N-acetylmethionine, N-acetylisoleucine,
N-acetylglycine, N-acetylvaline, N-acetylserine and N-acetylthreonine (Table 2).
Table 1 gives the quantitative values for the N-acetylated amino acids.
IDENTIFICATION OF N-ACETYLATED AMINO ACIDS
In addition to the unusual singlets in the N-acetyl region, two triplets at 2.49 and
2.38 ppm and an unknown doublet at 1.41 ppm were observed in the onedimensional 1H NMR urine spectrum (Figure 1A; peak 14, 13 and 15, respectively).
These resonances were observed in the urine from all patients with ACY1
deficiency and are normally not present in urine spectra from healthy individuals.
1H-NMR of model compounds revealed that the doublet and singlet at 1.41 and
2.02 ppm, observed with an integral ratio 1:1 for the patient’s urine derive from
N-acetylalanine. This was confirmed in a spiking experiment. Additional
confirmation was provided by 2-dimensional NMR experiments (1H-1H COSY and
1H-13C HSQC) performed on an authentic reference solution of N-acetylalanine and
the patient’s urine. The characteristic COSY and HSQC patterns of N-acetylalanine
were observed in the 2-dimensional NMR spectra of the patient’s urine (Table 2 and
Figure 2C).
98
Aminoacylase 1 deficiency
Figure 1
One-dimensional 1H-NMR spectra (500 MHz) of urine measured at pH 2.5.
A. Urine of a patient with aminoacylase 1 deficiency with assignments as marked (Table II).
B. Urine of patient with aminoacylase 2 deficiency (Canavan disease). The concentration of NAA was determined to be
1470 μmol/mmol Creatinine.
99
Chapter 2.3
Figure 2
Two-dimensional NMR spectra of
the urine from a patient with
aminoacylase 1 deficiency.
A. and B.
Two regions from an 1H-13C HSQC
spectrum. The 1H/13C shift values
for the correlation peaks of the N-
acetylated amino acids are listed in
Table II
C. and D.
Two regions from an 1H-1H COSY
spectrum.
Thus, 1H and 13C chemical shifts are fully consistent with the assignment of Nacetylalanine. Similarly, the unknown triplets at 2.49 and 2.38 ppm were assigned
to N-acetylglutamic acid and N-acetylglutamine, respectively. Furthermore, the 2dimensional spectra confirmed the presence of the metabolites Nacetylmethionine, N-acetylglycine and N-acetylasparagine (Fig. 2A-D). For some
metabolites the singlet resonance from the CH3 protons of the N-acetyl group was
the only resonance from the metabolite in the 1-dimensional spectrum. In the
urine of some patients the β CH2 resonances from N-acetylasparagine could be
observed. The identification of N-acetylated forms of the branched chain amino
acids leucine, isoleucine and valine was based solely on the N-acetyl CH3 proton
resonances in the spiking experiments. GC-MS data confirm the presence of these
metabolites in clearly increased amounts in the urine of all patients [182].
In addition to the assigned singlet resonances, three smaller singlet resonances at
2.051, 2.075 and 2.108 ppm were observed in the 1H-NMR spectra of some
patients. The singlet at 2.051 probably derives from the N-acetyl protons of Nacetylneuraminic acid (reference values for urine, < 20 µmol/mmol creatinine),
which is always present in urine samples. Its concentration was within the
reference range for age in all patients. The singlets 2.075 and 2.108 could not be
assigned. These peaks were observed in all the urine samples of all 8 of our
patients. Therefore, these resonances probably derive from as yet unidentified
metabolites that must relate to the primary defect. Assuming three contributing
100
Aminoacylase 1 deficiency
protons the concentration of these metabolites is approximately 40 µmol/mmol
creatinine.
To obtain more information about the chemical shift reproducibility of the Nacetyl singlets we measured the chemical shift of these peaks in the 14 urine
samples from all patients at pH 2.50. All peaks show a minimal variation in the
chemical shift (< 0.005 ppm; Table 2), which allows reliable identification of the
metabolites.
QUANTIFICATION
Urine. In all patients, urine concentrations of the N-acetylated amino acids could
be determined from the 1-dimensional 1H-NMR spectra. Quantification of Nacetylglutamine and of N-acetyl forms of the branched chain amino acids, leucine,
isoleucine and valine, was difficult in some cases due to their rather low
concentration and due to overlap with other resonances. For example, in the 1HNMR urine spectrum of patient 7 the singlet of N-acetylleucine overlapped with
the singlet of N-acetylasparagine. In these cases the concentration could not be
calculated. Therefore, semi quantitative estimates are given for these metabolites
in table 1. Quantification of N-acetylmethionine was performed on the S-CH3
singlet (Figure 1; peak 12).
In almost all cases, N-acetylglutamic acid had the highest concentration in urine
varying between 170 and 740 µmol/mmol creatinine (reference value < 2
µmol/mmol creatinine [185]). Exceptions are the urine samples from patients 5
and 8, where N-acetylglycine or N-acetylasparagine were the most prominent
metabolites. In patient 8 this was found consistently in three independent
samples.
N-Acetylmethionine resonances were observed in the urine NMR spectra of all
patients. Exceptions were two urine samples from patient 7 (0.5 and 0.7 years)
that had been stored at –20ºC for six months. GC-MS analysis had shown clearly
increased
concentrations
of
N-acetylmethionine
in
these
urine
samples
immediately after collection. To test the hypothesis that N-acetylmethionine may
disappear from urine sample during storage, the NMR measurement of the urine
sample from patient 1 was repeated. Storage for 8 months at –20ºC resulted in a
decrease of N-acetylmethionine from 120 to 10 µmol/mmol creatinine. All other
N-acetylated amino acids showed the same concentrations in the two
measurements. The low excretion of N-acetylserine and N-acetylthreonine in
patient 3 (< 10 µmol/mmol creatinine) suggests that also these metabolites may
have disappeared due to storage artifacts. Therefore we measured a second urine
from patient 3. In this sample, the concentration of N-acetylserine and N-
acetylthreonine was in the same range as in the other patients with ACY1
deficiency (Table 1).
101
Chapter 2.3
Table 2. NMR assignmentsa (urine, pH 2.5) for N-acetylated amino acids
characteristic of aminoacylase 1 deficiency
Metabolite
Assignment
δ 1H
N-Acetylalanine
N-Acetyl CH3
β CH3
α CH
1.41 d
N-Acetylleucine
N-Acetylasparagine
N-Acetylglutamic acid
N-Acetylglutamine
N-Acetylmethionine
N-Acetyl CH3
δ CH3
δ’ CH3
γ CH
β CH2
α CH
N-Acetyl CH3
β+β’ CH2
α CH
N-Acetyl CH3
γ CH2
β+β’ CH2
α CH
N-Acetyl CH3
γ CH2
β+β’ CH2
α CH
N-Acetyl CH3
γ +γ’’ CH2
β+β’ CH2
α CH
S-CH3
N-Acetylisoleucine
N-Acetyl CH3
δ CH3
β’ CH3
γ CH2
β CH
α CH
N-Acetylglycine
N-Acetyl CH3
α CH2
N-Acetylvalined
N-Acetyl CH3
γ+γ’ CH3
β CH
α CH
N-Acetylserine
N-Acetylthreonine
N-Acetyl CH3
β+β’ CH2
α CH
N-Acetyl CH3
γ CH3
β CH
α CH
δ
13C
Peak no.c
2.018 s (2.015-2.020)b
24.6
1
4.31 m
19.2
51.9
15
2.029 s (2.027-2.031)
24.6
2
0.93 d
25.1
0.88 d
1.65 m
23.5
27.4
1.65 m
42.4
2.031 s (2.029-2.033)
24.7
4.33 m
54.9
2.83 AB
39.7
2.034 s (2.032-2.036)
24.6
2.00 m, 2.20 m
28.9
4.68 m
2.49 t
4.39 m
?
33.1
24.6
1.99 m, 2.20 m
29.5
34.3
4.36 m
55.4
2.040 s (2.038-2.042)
24.7
2.00 m, 2.15 m
33.0
2.58 m, 2.64 m
32.4
4.49 m
55.0
2.106 s (2.105-2.106)
5
13
6
17.1
12
2.043 s (2.042-2.046)
24.6
7
0.92 d
17.9
0.88 t
13.6
1.45 m, 1.24 m
27.6
4.23 m
60.9
1.91 m
39.2
2.050 s (2.048-2.051)
24.6
8
2.064 s (2.062-2.066)
24.6
9
2.17 m
32.8
3.95 d
0.95 t
4.21 dd
44.4
20.1
61.8
2.068 s (2.067-2.070)
24.7
4.48 m
58.2
2.099 s (2.097-2.100)
24.7
4.37 m
70.3
3.92 AB
1.21 d
4.42 m
64.3
21.9
61.4
a: Chemical shifts in ppm relative to TSP; multiplicities are: s, singlet; d, doublet; q, quartet, qt,
quintet, m, multiplet; AB, AB system (second-order coupling)
b: Variation in the chemical shift
c: Numbers refer to the peaks observed in the urine 1-dimensional 1H-NMR spectrum (Fig. 1A)
102
4
14
55.3
2.039 s (2.036-2.040)
2.38 t
3
10
11
Aminoacylase 1 deficiency
Serum. From six patients (patient 2-7), we analyzed serum samples using 1dimensional 1H-NMR spectroscopy. Proline also has resonances in the N-acetyl
region (multiplet deriving from the ß-CH2 protons). Due to its high concentration
in serum (40-332 µmol/L [110]) and the high degree of splitting identification and
quantification of other metabolites in this region is hampered. We have quantified
the highest peak between 2.0-2.1 ppm in the serum samples, assuming N-acetyl
protons cause this peak. Excluded from this quantification was the singlet for
acetic acid at 2.08 ppm. The maximal concentration was found to be 20 µmol/L.
This implies that the concentration of individual N-acetylated amino acids in
serum of the patients must have been lower than 20 µmol/L.
Cerebrospinal fluid. We also analyzed a CSF sample from patient 7. The N-acetyl
region in the 1H-NMR spectrum in control CSF samples shows several singlet
resonances. In an earlier study [3], these singlet resonances were reported as
common but unidentified resonances in 40 CSF samples (unknown peaks x12
[singlet 2.07], x11 [singlet 2.04 ppm] and x10 [singlet at 2.02 ppm]). These
singlets may derive from N-acetylserine (2.07 ppm), N-acetylaspartylglutamic acid
(2.04 ppm), N-acetylglutamic acid (2.03 ppm) and N-acetylalanine (2.02). Spiking
experiments with pure N-acetylated compounds were inline with these
assignments. We established reference values from 10 normal CSF samples. These
amounted to <1 – 15 µmol/l. The concentration of N-acetylaspartylglutamic acid
in normal CSF is comparable with the observation of Wolf et al. [25]. In CSF of
patient 7 with aminoacylase 1 deficiency we found normal concentrations for all
eleven N-acetylated amino acids that accumulate in this disease. N-Acetylserine,
N-acetylaspartylglutamic acid, N-acetylglutamic acid and N-acetylalanine
amounted to 10, 4, 6 and 4 µmol/l, respectively.
AMINOACYLASE 2 DEFICIENCY (CANAVAN DISEASE) (OMIM: 271900)
The urine from an affected patient was measured by 1-dimensional 1H-NMR
spectroscopy and compared with the urine spectrum from a patient with ACY1
deficiency (Fig. 1A and B). The urine spectrum of the CD patient (Fig. 1B) showed
the presence of high concentrations of NAA (1470 µmol/mmol creatinine;
Reference: 6–36 µmol/mmol creatinine). The singlet at 2.03 ppm (N-acetyl
protons) and the characteristic β CH2 proton resonances at 2.95 ppm confirm the
presence of NAA in the urine of the patient. Figure 1 shows that NMR spectroscopy
can easily discriminate between ACY1 and ACY2 deficiency.
103
Chapter 2.3
OTHER N-ACETYLATED AMINO ACIDS
In order to improve assignment in the N-acetyl region of body fluid NMR spectra
other N-acetylated amino acids were measured. Table 3 provides on overview of
fourteen N-acetylated amino acids. Some of these metabolites assist in the
diagnosis of various inborn errors of metabolism as indicated in Table 3.
Table 3. 1H chemical shiftsa (pH 2.5) for N-acetylated amino acids observed in urine of patients with
various inborn errors of metabolism and other relevant to human metabolism.
Metabolite
Assignment
δ 1H
N-Acetylarginine
N-Acetyl CH3
δ CH2
γ CH2
β+β’ CH2
α CH
2.04 s
N-Acetyl CH3
β+β’ CH2
α CH
2.03 s
N-Acetyl CH3
Aspartyl:
β+β’ CH2
α CH
Glutamic acid:
γ+γ” CH2
β+β’ CH2
α CH
2.04 s
- Canavan disease
2.85 AB
- Severe hypomyelination associated with increased levels
N-Acetyl CH3
δ CH2
γ CH2
β+β’ CH2
α CH
2.03 s
N-Acetyl CH3
β+β’ CH2
α CH
2.07 s
N-Acetyl CH3
β+β’ CH2
α CH
1.99 s
C2H, ring
8.59 s
N-Acetylaspartic acid
N-Acetylaspartylglutamic
acid
N-Acetylcitrulline
N-Acetylcysteine
N-Acetylhistidine
C4H, ring
N-Acetylisoleucine
N-Acetylleucine
N2-Acetyllysine
104
See table 2
See table 2
N-Acetyl CH3
ε CH2
δ CH2
γ CH2
β+β’ CH2
α CH
Inborn errors of metabolism
3.21 q
1.66 m
1.91 m, 1.77 m
4.32 m
- Canavan disease (Figure 1B)
2.95 m
4.72 m
4.72 dd
- Pelizeus Merzbacher Disease
of N-acetylaspartyl-glutamate in CSF[25]
2.01 m, 2.23 m
2.48 m
4.44 dd
3.12 t
- Citrullinemia
1.55 m
1.86 m, 1.73 m
4.30 m
2.97 m
4.57 m
3.15m
- Histidinaemia
4.67m
7.29 s
- Maple Syrup Urine Disease
2.03 s
3.00 m
1.67 m
1.44 m
1.88 m, 1.75 m
4.30 m
- Maple Syrup Urine Disease
- Hyperlysinaemia
- Lysinuria
Aminoacylase 1 deficiency
Table 3. 1H chemical shiftsa (pH 2.5) for N-acetylated amino acids observed in urine of patients
with various inborn errors of metabolism and other relevant to human metabolism. (Continued)
N5-Acetyllysine
N-Acetylornithine
N-Acetylphenylalanine
N-Acetylproline
N-Acetyltaurine
N-Acetyltryptophan
N-Acetyltyrosine
N-Acetylvaline
N-Acetyl CH3
ε CH2
δ CH2
γ CH2
β+β’ CH2
α CH
1.97 s
N-Acetyl CH3
δ CH2
γ +γ’ CH2
β CH2
α CH
2.04 s
N-Acetyl CH3
β+β’ CH2
α CH
1.93 s
H, ring
7.33 various
3.17 q
1.55 m
1.41 m, 1.89 m
1.41 m, 1.89 m
3.84 t
3.05
1.01
1.94
4.35 m
- Phenylketonuria (PKU)
3.12 m
4.61 m
N-Acetyl CH3
δ CH2
γ CH2
β+β’ CH2
α CH
2.11 s
N-Acetyl CH3
1.99 s
S-CH2
3.55 m
3.42 m
2.00 m
2.00 m
4.40 dd
N-CH2
3.08 t
N-Acetyl CH3
β+β’ CH2
α CH
1.92 s
H, ring
7.2 – 7.7 Various
- Liver diseases
3.31 m
4.70 qu
N-Acetyl CH3
β+β’ CH2
α CH
1.94 s
- Tyrosinemia type I
4.57 m
- Liver diseases
H, ring
7.89 AB
See table 2
3.07 AB
- Tyrosinemia type II
- Maple Syrup Urine Disease
a: Chemical shifts in ppm relative to TSP; multiplicities are: s, singlet; d, doublet; q, quartet, qt, quintet, m, multiplet
DISCUSSION
This study describes the in vitro NMR spectroscopic investigations at the
metabolite level on eight unrelated patients with ACY1 deficiency. The 1dimensional 1H-NMR urine spectra from these patients show a characteristic
metabolic profile, mainly due to the N-acetyl singlets deriving from N-acetylated
amino acids. Using a combination of one dimensional 1H and two dimensional 1H1H COSY and 1H-13C HSQC NMR experiments, resonances of 11 N-acetylated
amino acids could be assigned and quantified or semi-quantified. The profile of
105
Chapter 2.3
the accumulating N-acetylated amino acids is consistent with the enzyme
specificity of ACY1. The enzyme preferentially uses neutral aliphatic amino acids
with a short-chain acyl moiety as substrate [178]. These include the N-acetylated
amino acids of serine, threonine, alanine, methionine and glycine. N-Acetylated
branched chain amino acids can also be a substrate for ACY1 [178]. This explains
the presence of N-acetylated forms of leucine, valine and isoleucine in the urine of
our patients. The same holds for the N-acetylated forms of the basic amino acids
glutamine and asparagine and the acidic amino acid glutamic acid. NAcetylaspartic acid (=NAA) is not a substrate for ACY1 [178]. NAA is one of the
most abundant amino acid in the human brain. There, it is catabolized by a special
aminoacylase (ACY2) to acetate and aspartate. NAA therefore does not accumulate
in body fluids of the patients with ACY1 deficiency. N-Acetylated aromatic amino
acids are not good substrates for ACY1 [178]. Aminoacylase 3 and acyl-lysine
deacylase (EC 3.5.1.17) are two other enzymes involved in the breakdown of Nacetylated amino acids [178]. Aminoacylase 3 acts preferentially on N-acylaromatic
amino
acids,
e.g.
N-acetyltryptophan, N-acetyltyrosine, Nacetylphenylalanine and N-acetylhistidine and the enzyme acyl-lysine deacylase
catalyzes the N-deacetylation of N5-acetyllysine. Accordingly, none of these Nacetylated amino acids were observed in increased concentration in the urine of
the ACY1 deficient patients. A deficiency of aminoacylase 3 has never been
described. It is to be expected that body fluid NMR spectroscopy would easily pick
up the resonances of N-acetylated aromatic amino acids and therefore may be a
good technique to diagnose patients with aminoacylase 3 deficiency. Also Nacetylated aromatic amino acids will be picked up in GC-MS analysis of organic
acids [182].
Differences in excretion levels were observed between various N-acetylated amino
acids in the urine of our patients. Table 1 shows that the N-acetylated form of
glutamic acid had the highest concentration in all patients except patients 5 and 8
where N-acetylglycine or N-acetylasparagine were the prominent diagnostic
metabolites. Such variations may relate to the specific mutations of a patient but
higher number of patients would be required to prove this hypotheses. In general,
the excretion levels of the N-acetylated amino acids do not correlate with serum
concentrations of the corresponding free amino acid. Most of the accumulating Nacetylated amino acids are excreted in a concentration range between 100-500
µmol/mmol creatinine. Exceptions are the N-acetylated forms of the branched
chain amino acids and threonine and methionine. The excretion of Nacetylthreonine for instance ranges between 20 and 60 µmol/mmol creatinine. It
may be that these N-acetylated amino acids are accepted as a substrate by one of
the other aminoacylases. In addition, the concentration of the N-acetylated amino
acids in our patients may partially reflect the extent of N-acetylation of specific
amino acids at the N-terminus of human proteins. In mammals N-terminal
acetylation of amino acids occurs on approximately 80-90% of proteins [186].
106
Aminoacylase 1 deficiency
Severe neurological involvement has been observed in individual patients with
ACY1 deficiency [54;55]. However, CSF of only 1 patient was available for analysis
and therefore the observations on this sample are preliminary. We found normal
concentrations of N-acetylated amino acids in CSF of patient 7. Further
measurements of CSF of affected cases are required to confirm our observation.
Our observation may reflect that ACY1 deficiency does not lead to accumulation of
N-acetylated amino acids in the brain. It will be of interest to see whether
accumulation of N-acetylated amino acids can be demonstrated in the brain of
these patients with in vivo MRS. ACY1 gene expression has been found in brain
tissue and ACY1 protein and enzyme activity have been demonstrated in brain
[54;178]. Normal levels of N-acetylated amino acids in CSF in patients with ACY1
deficiency question the role of the enzyme in brain tissue. In ACY2 deficiency
(Canavan disease) NAA does accumulate in CSF (380 µmol/l; reference value: < 2
µmol/l [3;187]). The relation between the molecular defect and the neurological
signs and symptoms is obvious in Canavan disease. In ACY1 deficiency, this is less
obvious even more so when we take into account the asymptomatic case described
by Sass et al [54], patient 3 in this study. However, this patient still is at very
young age and may develop clinical signs and symptoms later in life.
Initially, the high amounts of N-acetylated amino acids in the urine of patients 1 –
8 were detected by routine GC-MS analyses. Organic acid analysis with GC-MS is
appropriate for identifying individuals with ACY1 or ACY2 deficiency. However, not
all relevant N-acetylated amino acids can be detected with GC-MS in the urine of
ACY1 patients. Depending on the derivatization procedure used N-acetylglutamine
[54;55], N-acetylasparagine [54;55], N-acetylserine [54] or N-acetylthreonine [54]
were missed by GC-MS. NMR spectroscopy does not require extensive sample
treatment and using a combination of one- and two-dimensional NMR spectra, we
were able to detect 11 N-acetylated amino acids in the urine of ACY1 deficient
patient. Moreover, most N-acetylated amino acids could be quantified or semi
quantified.
In conclusion, we have shown that NMR spectroscopy can be used to identify and
quantify characteristic N-acetylated amino acids in urine from patients with ACY1
deficiency. The technique can discriminate reliably between deficiencies of
aminoacylase 1 and aminoacylase 2.
107
108
THREE
THE COMBINATION OF
IN VIVO AND IN VITRO NMR SPECTROSCOPY
109
110
CHAPTER
3.1
Dimethyl sulfone in human cerebrospinal fluid and
blood plasma confirmed by one-dimensional 1H
and two-dimensional 1H-13C NMR
Udo F. H. Engelke, Albert Tangerman, Michèl A.A.P. Willemsen, Detlef Moskau,
Sandra Loss, S. Harvey Mudd and Ron A. Wevers
NMR in Biomedicine 2005; 18(5): 331-336
111
112
DMSO2 in CSF and blood plasma
Abstract
1H-NMR
spectroscopy at 500 MHz was used to confirm that a previously
unidentified singlet resonance at 3.14 ppm in the spectra of cerebrospinal fluid
and plasma samples corresponds to dimethyl sulfone (DMSO2). A triple resonance
inverse cryogenic NMR probe, with pre-amplifier and the RF-coils cooled to low
temperature, was used to obtain an 1H-13C HSQC spectrum of CSF containing 8 µM
(753 ng/ml) DMSO2. The 1H-13C correlation signal for DMSO2 was assigned by
comparison with the spectrum from an authentic reference sample. In plasma and
CSF from healthy controls, the concentration of DMSO2 ranged between 0 and 25
µmol/l. The concentration of DMSO2 in plasma from three of four patients with
severe methionine adenosyltransferase I/III (MAT I/III) deficiency was about twice
the maximum observed for controls. Thus, DMSO2 occurs as a regular metabolite
at low micromolar concentrations in cerebrospinal fluid and plasma. It derives
from dietary sources, from intestinal bacterial metabolism and from human
endogenous methanethiol metabolism.
113
Chapter 3.1
INTRODUCTION
1H-NMR
spectroscopy of body fluids is a powerful tool in the field of inborn errors
of metabolism. The technique provides an overview of proton-containing
metabolites, is rapid, non-destructive and requires minimal sample treatment. For
most compounds detected, quantitative analysis is possible. A prerequisite for the
analysis of body fluid with NMR spectroscopy lies in the correct assignments of
resonances. Several papers have reported assignments of resonances in 1H-NMR
spectra of body fluids [3;4;17].
However, some resonances are still unassigned. In earlier papers we identified 16
unassigned resonances in cerebrospinal fluid (CSF) [3] and 14 in plasma [4]. In this
study, we concentrate on the origin of the unidentified singlet resonance at 3.14
ppm that was described as unknown in both studies [3;4]. Here, we assign this
singlet resonance to the six equivalent protons in dimethyl sulfone (DMSO2), using
one-dimensional and heteronuclear two-dimensional NMR techniques. We present
reference values for DMSO2 in human plasma and CSF. Furthermore, we document
an increased concentration of DMSO2 in plasma from three out of four unrelated
patients with methionine adenosyltransferase I/III (MAT I/III) deficiency. Our data
demonstrate the common presence of DMSO2 in human blood and CSF and show
that DMSO2 in body fluids derives partly from dietary sources, partly from bacterial
metabolism and partly from endogenous human methanethiol metabolism.
EXPERIMENTAL
CONTROL SUBJECTS
CSF samples were obtained from 30 patients (age range 0–78 years) suspected to
suffer from neurometabolic disease. The clinical samples were sent to our
laboratory for metabolic screening. Appropriate analysis ruled out immunologic
and chronic infectious diseases as well as disorders caused by toxic agents. For
these subjects normal results were obtained for metabolic screening tests of urine
(amino acids, organic acids and lysosomal diseases), and the CSF samples were
classified as ‘controls’. Plasma samples were obtained from 15 healthy adult
volunteers (age range 24–66 years; seven male and eight female), who gave their
informed consent.
114
DMSO2 in CSF and blood plasma
PATIENTS WITH MAT I/III DEFICIENCY
Plasma samples were available from four severely and two mildly affected MAT
I/III-deficient patients (OMIM 250850). All six patients have mutations in the gene
MAT1A on chromosome 10q224 [188] that encodes the catalytic subunit of the
two native MAT isoenzymes (EC 2.5.1.6), MAT I and MAT III [189].
SAMPLE PREPARATION
Plasma and CSF samples were deproteinized using a 10 kDa filter (Sartorius).
Before use, the filter was washed twice by centrifugation of water to remove
glycerol. A 20 µl aliquot of 20.2mM trimethylsilyl-2,2,3,3- tetradeuteropropionic
acid (TSP, sodium salt; Aldrich) in 2H2O was added to 700 µl of the ultrafiltrate,
providing a chemical shift reference (δ=0.00), a concentration reference and a
deuterium lock signal. The pH of the ultrafiltrate was adjusted to 2.50±0.05 with
concentrated HCl. Finally, 650 µl of the sample was placed in a 5mm NMR tube
(Wilmad Royal Imperial).
ONE-DIMENSIONAL 1H-NMR SPECTROSCOPY
Plasma and CSF were measured at 500MHz on a Bruker DRX 500 spectrometer
equipped with a triple-resonance inverse (TXI) 1H{15N, 13C} probehead and
equipped with x, y, z gradient coils. 1H spectra were acquired as 128 transients in
32K data points with a spectral width of 6002 Hz. The sample temperature was
298K and the H2O resonance was presaturated by single-frequency irradiation
during a relaxation delay of 10 s, and a 90º excitation was used. Shimming of the
sample was performed automatically on the deuterium signal. To improve the
spectral resolution, the samples were spun (7 Hz) during the measurements. The
resonance linewidths for TSP and metabolites were <1 Hz. A π/2-shifted sine-bell
window function was applied to the FID. Fourier transformation was performed
after zero-filling to 64K data points. The phase and the baseline were corrected
manually. The DMSO2 singlet resonance (six equivalent protons from the two
methyl groups) and the TSP singlet (nine equivalent protons) were fitted semiautomatically with Lorentzian line shapes. The concentration of DMSO2 was
calculatedfrom the relative integrals of the fitted lineshapes using the known
concentration of TSP.
1H-13C
HETERONUCLEAR SINGLE-QUANTUM CORRELATION (HSQC) SPECTROSCOPY
An HSQC spectrum of one CSF sample was measured at 500MHz on a Bruker AV
500 spectrometer using an inverse triple-resonance cryoprobe (Bruker), whereby
the preamplifier and the RF coils were cooled to low temperature. The sequence
was optimized for a 1J CH-coupling constant of 130 Hz. FIDs (1536 transients) were
acquired with 2K time-domain data points (t2), an acquisition time of 0.128s, a
115
Chapter 3.1
recycle time of 2.128s and 96 t1 increments. The spectral widths were 16 ppm (1H)
and 70 ppm (13C), and the total measurement time was 5 days and 10 h.
RESULTS
LINEARITY, RECOVERY AND DETECTION LIMIT
To study linearity and recovery we added DMSO2 in known concentrations (0, 32,
62, 94 and 195 µmol/l) to a pool of control CSF samples. The recovery was 90–
100% at all concentration levels. The linearity and correlation coefficient were
excellent (95% confidence interval of the slope, 0.882–0.996; correlation
coefficient, r>0.99). The detection limit at S/N=3 for DMSO2 was determined to be
0.5 µmol/l (1H-NMR at 500 MHz, 128 transients; using the conventional 5mm TXI
probehead operating at ambient temperature).
ONE-DIMENSIONAL 1H-NMR SPECTROSCOPY
An expanded region (2.50–3.20 ppm) of the 1H-NMR spectrum of a control CSF
sample is shown in Figure 1 for pH 7.5 (lower panel) and pH 2.5 (upper panel). The
major metabolites in this region and the chemical shifts at pH 2.5 are creatinine
(singlet, 3.13 ppm), creatine (singlet, 3.05 ppm) and citric acid ([AB]2 spin system,
2.99, 2.83 ppm). The singlet resonance at 3.14 ppm was observed in an earlier
CSF study as unidentified resonance x14 and was assigned to DMSO2 as described
below [3].
Figure 1
1H-NMR
spectra (500 MHz) of a
representative
CSF
sample
measured at pH 2.5 (upper panel)
and pH 7.5 (lower panel). The
concentration of dimethyl sulfone
was determined to be 18 µmol/l
116
DMSO2 in CSF and blood plasma
IDENTIFICATION OF DMSO2
NMR spectroscopy was performed on CSF from a 3-year-old boy with mental
retardation and loss of acquired skills. Routine metabolic screening of the
patient’s body fluids showed no abnormalities. However, an unusually high
concentration of the metabolite corresponding to the unknown singlet x14 was
observed in the one-dimensional 1H-NMR CSF spectrum (data not shown) [3]. This
observation could not be confirmed with a repeat CSF sample. It was then
discovered that at the time of the first lumbar puncture this patient had used a
dietary supplement containing DMSO2. 1H-NMR of the authentic compound DMSO2
in H2O at pH 2.5 showed a singlet at 3.14 ppm. The addition of pure DMSO2 to a
control CSF sample (spiking) resulted in an increase of the singlet at 3.14 ppm.
Additionally, one-dimensional 1H-NMR spectra of a CSF sample and reference
solution of DMSO2 in H2O were recorded at pH 2.5 and 7.5. Both samples exhibited
a singlet resonance which shifted from 3.138 ppm at pH 2.5 to 3.157 ppm at pH
7.5 (Figure 1). These data provide strong evidence for the assignment of DMSO2 as
source of the singlet x14. Furthermore, at pH 7.5, characteristic shifts were
observed for the creatinine peak (3.13 at pH 2.5 to 3.05 ppm at pH 7.5) and citric
acid [AB]2 system (2.99, 2.83 ppm at pH 2.5–2.70; 2.54 ppm at pH 7.5).
CONFIRMATION OF DMSO2 BY 1H/13C HSQC TWO-DIMENSIONAL NMR
SPECTROSCOPY
Additional confirmation of the DMSO2 in body fluid was provided by 1H-13C HSQC
two-dimensional NMR experiments performed on an authentic reference solution
of DMSO2 and a control CSF sample containing 8 µmol/l DMSO2. The twodimensional spectrum of the reference sample exhibited a correlation peak with
the coordinates 3.14 (1H)/44.3 (13C) (data not shown), and a resonance was
observed at the same position in the two-dimensional spectrum of control CSF
sample in addition to correlation signals for the other metabolites shown in Figure
1. Thus, 1H and 13C chemical shifts and the pH dependence of the 1H shift are fully
consistent with the assignment of the previously unknown metabolite as DMSO2.
OCCURRENCE AND CONCENTRATION OF DMSO2 IN CSF AND PLASMA
We observed the DMSO2 singlet resonance in 28 of 30 clinical CSF samples
examined, consistent with the results reported earlier (35 out of 40 samples) [3].
Furthermore, DMSO2 was detected in 11 of 15 plasma samples from a different
group of healthy volunteers. No age and sex dependency for DMSO2 was found in
blood and CSF. Figure 2 summarizes DMSO2 concentration determined for
individual uncorrelated plasma and CSF samples. The highest concentration
amounted to 25 µmol/l in CSF (mean±SD, 11.3±6.2) and 24 µmol/l in plasma
117
Chapter 3.1
(mean±SD, 8.8±7.3). A much higher concentration of DMSO2 in plasma and CSF
was observed for the one patient receiving a dietary supplement containing DMSO2
(plasma, 624 µmol/l; CSF, 574 µmol/l). We also measured the concentration of
DMSO2 in plasma CSF sample pairs obtained within 1 h from four control patients
not included in the groups presented in Figure 2. In all four pairs, the
concentration of DMSO2 was slightly higher in plasma (plasma/CSF values, each in
µmol/l: 7:2; 15:8; 13:11; 20:15). Correlation analysis (Fischer’s r to z) gave a
correlation coefficient r=0.928 (p=0.10, n=4).
Figure 2
Quantification of DMSO2 in plasma and CSF from adults.
({) Plasma from healthy adult volunteers (n=15); (…)
CSF samples classified as ‘controls’ (n=30); („) plasma
from severely affected patients with MAT-deficiency
(methionine>300 µmol/l; n=4); (z) plasma from mildly
affected patients with MAT-deficiency (methionine<
300 mmol/l; n=2).
Values of zero correspond to DMSO2 levels below the
detection threshold of 0.5 µmol/l
DMSO2 IN PLASMA FROM MAT I/III-DEFICIENT PATIENTS
MAT catalyzes the biosynthesis of S-adenosylmethionine (AdoMet) from
methionine. Patients with the disease have high concentrations of methionine and
methionine sulfoxide in urine and plasma. The normal trans-sulfuration pathway
is defective in these patients and methionine degradation via the transamination
pathway is increased [190] (Figure 3). Methionine transamination metabolites
accumulate abnormally only when plasma methionine concentrations exceed 300–
350 µM [191]. Dimethyl sulfide (DMS) is such an intermediate in this pathway. We
measured the DMSO2 concentration in plasma from different unrelated patients
with MAT I/III deficiency. In four severe patients, the methionine level in plasma
was higher than 300 µmol/l. Here, the concentration of DMSO2 was significantly
higher than for the 15 controls (p=0.04, one-sided Wilcoxon test; Figure 2). For
one patient with severe deficiency and two patients with mild deficiency
(methionine <300 µmol/l), the plasma concentration of DMSO2 was within the
control range. Some of the samples from MAT I/III-deficient patients were stored
118
DMSO2 in CSF and blood plasma
at -70ºC for several years so that elevated DMSO2 concentrations could be the
result of in vitro oxidation of DMS during storage. To test this hypothesis, DMS
was added to plasma at concentrations of 30 and 60 µmol/l in a closed 15 ml
septum vial. Storage for one week at room temperature did not result in the
formation of detectable DMSO2 and the added DMS was recovered quantitatively
(data not shown). Thus, in vitro oxidation of DMS is unlikely to be a source of
DMSO2. The one-dimensional 1H spectra from the plasma obtained from the six
patients all showed the presence of the methionine singlet at 2.13 ppm and the
methionine sulfoxide singlet at 2.74 ppm (normally not present). Dimethyl
sulfoxide (DMSO) (singlet at 2.72 ppm) was not detectable in patient plasma.
Figure 3
Transamination pathway for methionine and metabolic steps leading to dimethyl sulfone. Methionine adenosyltransferase I/III
deficiency (OMIM 250850) results in a strong reduction (bar) in methionine flux through the trans-sulfuration pathway.
119
Chapter 3.1
DISCUSSION
The results of this study confirm the presence of DMSO2 as a common metabolite
in human CSF and plasma. The higher detection sensitivity achievable with
CryoProbe technology allowed the 1H-13C signal of 8 µmol/l DMSO2 to be assigned
in an HSQC spectrum of a control CSF sample. As early as 1959 Williams [192]
suggested that a possible fate of DMS would be oxidation to DMSO2. To our
knowledge Williams and Burstein were the first and only authors to describe the
presence of DMSO2 in urine. They reported concentrations of 4–11 mg/day (~20–
120 µmol/l) [193] in urine of healthy volunteers. Jacob et al. [194] mentioned
DMSO2 in blood of an adult man (2 µM). Until now, DMSO2 had not been described
as a regular metabolite in CSF. Commodari et al. [20] described a singlet
resonance at 3.17 ppm at pH 9.6 in two out of eight CSF samples (peak Y) and
Wevers et al. [3;4] reported this singlet at 3.14 ppm (pH 2.50) in plasma and CSF
spectra (peak u10 in plasma and peak x14 in CSF). However, in none of these in
vitro NMR studies could this singlet be definitively assigned to DMSO2. Our data
for the patient receiving DMSO2 in a dietary supplement suggest that intestinal
DMSO2 can enter the blood and subsequently reach the CSF. Moreover,
threepapers have reported DMSO2 in the human brain, as detected by in vivo 1HMRS [195-197]. In all cases the presence of this metabolite resulted from
ingestion of a dietary supplement containing DMSO2. Milk [198] and port wine
[199] are also possible sources of DMSO2, and we confirmed its presence in milk
(20 µmol/l). Until now, it was unknown if other food sources or beverages
contained DMSO2. Another exogenous source of DMSO2 is the sulfur oxidation of
dietary DMS via DMSO to DMSO2 [200]. DMS has been found in cooked vegetables
[193], beer (0.2–2.3 µmol/ l) [201], rice (21–319 µmol/kg) [202] and port wine (up
to 0.8 µmol/ l) [199;203]. However, the relatively low concentrations of DMSO2 and
DMS found in food and beverages cannot explain the concentrations of DMSO2
found in CSF and plasma. Therefore, it appears likely that DMSO2 can also be a
product of endogenous metabolism. In the gut methanethiol is formed either from
methionine or from methylation of hydrogen sulfide, and subsequent methylation
of methanethiol results in the formation of DMS [204]. It has been shown that
methanethiol can be converted to DMS. Furne et al. [205] have postulated that
conversion of methanethiol to thiosulfate and sulfate (via formation of H2S) is
much more predominant in rat intestine than conversion of methanethiol to DMS.
Some of the methanethiol directly enters the blood [206]. Once in the blood,
methanethiol is nearly quantitatively oxidized by erythrocytes to formic acid and
sulfate (ca. 99%; Figure 4). Only 1% of the total methanethiol is metabolized to
DMS by the enzyme thiol methyltransferase [206]. DMS may then be further
oxidized to DMSO and DMSO2. Methanethiol, DMS, DMSO and DMSO2 all seem able
to enter the blood.
120
DMSO2 in CSF and blood plasma
Subcutaneous injection of DMS leads to the excretion of DMSO and DMSO2 in the
urine of rabbits [200]. Rhesus monkeys treated with DMSO had high
concentrations of DMSO2 in their blood [207]. The authors concluded that part of
the DMSO was also metabolized to DMS, as illustrated by a particular sweetness of
the breath of the monkeys. These data are strongly indicative for both intestinal
bacterial metabolism of methanthiol and also from endogenous human
methanethiol metabolism as possible sources for body fluid DMSO2. Human
endogenous methanethiol conversion into DMSO2 is further supported by our
finding of an increased plasma DMSO2 concentration in patients with MAT I/III
deficiency. In these patients the normal trans-sulfuration pathway of methionine is
defective and the transamination of methionine occurs more extensively (Figure 3),
especially when plasma methionine concentrations exceed 300 µM. These patients
may have high levels of DMS, a product of the transamination pathway, in their
breath [191]. In three out of four plasma samples from severely affected patients
with MAT I/III deficiency, the DMSO2 plasma concentration was about twice the
maximum value for controls.
Figure 4
Both dietary sources and putative endogenous metabolic pathways can potentially contribute to the presence of dimethyl
sulfone in human body fluids
121
Chapter 3.1
This elevation is most likely the result of oxidation of elevated levels of DMS via
DMSO to DMSO2 (Figure 4) and indicates that at least some fraction of body fluid
DMSO2 may derive from methionine metabolism. In one severely affected patient
with MAT I/III deficiency, the DMSO2 concentration fell within the normal range. An
explanation for this phenomenon might be that in this patient the methylation of
methanethiol to dimethyl sulfide is partly blocked, resulting in normal DMSO2
values. The patient receiving a dietary DMSO2 supplement exhibited high
concentration of DMSO2 in both CSF and plasma (ca. 600 µmol/l). The plasma: CSF
DMSO2 ratio of ca. 1.09 suggests rapid equilibration of this metabolite between
blood and CSF compartments. Our results are consistent with other MRS studies
that show that dietary DMSO2 accumulates in the brain [195-197]. Plasma and CSF
studies using dogs showed high brain concentrations of DMSO after in vivo
administration [208], indicating that the transfer of DMSO from blood into CSF is
also possible. In conclusion, we have shown that DMSO2 is a common metabolite
in human CSF and plasma, occurring at concentrations of 0–25 µmol/l.
Furthermore, we have shown that the DMSO2 concentration may be higher in the
plasma of severely affected MAT I/III-deficient patients, and that the DMSO2
detected in body fluids probably derives from both dietary sources and
endogenous metabolism.
122
CHAPTER
3.2
Methylsulfonylmethane (MSM) ingestion causes a
significant resonance in proton magnetic resonance
spectra of brain and cerebrospinal fluid
Michèl A.A.P. Willemsen, Udo F.H. Engelke, Marinette van der Graaf
and Ron A. Wevers
Neuropediatrics 2006; 37(5): 312-314
123
124
MSM in brain
Abstract
The use of methylsulfonylmethane (MSM), available as an ‘over-the-counter’
dietary supplement, led to the occurrence of an abnormal resonance at 3.15 ppm
in the in vivo brain proton MR spectrum as well as the in vitro cerebrospinal fluid
NMR study of a 4-year-old girl. The concentration of this compound amounted to
1.2 mmol/l in brain tissue and 1.7 mmol/l in cerebrospinal fluid. Our findings
illustrate that ingestion of exogenous compounds, e.g. in medication, food or
‘innocent’ supplements, may lead to abnormal resonances in spectroscopy studies
that might be difficult to assign.
125
Chapter 3.2
Patient
A 4-year-old girl and her 6-year-old brother both suffered from a progressive
demyelinating sensomotor polyneuropathy that started at the age of 1½ years and
6 months respectively. The neurological disorder in the boy additionally included
mild mental retardation and mild bilateral pyramidal tract involvement. Extensive
work-up had never led to a classifying diagnosis in these siblings. The girl
underwent routine magnetic resonance (MR) imaging and proton MR spectroscopy
investigations in a 3T Magnetom Trio MR System (Siemens, Erlangen, Germany)
using a standard CP head coil. Single voxel MR spectroscopy was performed using
PRESS volume selection (TE/TR=20/5000 ms) with and without water suppression.
After this procedure a lumbar puncture was performed. Nuclear magnetic
resonance (NMR) spectroscopy was performed on CSF using methods described
previously [10]. Initially the parents had reported that the girl did not use any
medication at that time, but after the investigations (see below) they told us that
the girl had methylsulfonylmethane as a dietary supplement.
Figure 1
3T single voxel proton MR spectrum (PRESS, TE/TR=20/5000 ms) of occipital gray matter (7.2 ml) clearly showing a resonance of
methylsulfonylmethane (MSM) at 3.15 ppm between the methyl signals of choline (3.2 ppm) and creatine (3.0 ppm). The voxel
location is shown in the MR image.
126
MSM in brain
Results
MR imaging showed normal cerebral anatomy and myelination. Proton MR
spectroscopy of cerebral white matter, basal ganglia as well as occipital gray
matter (Figure 1) showed an abnormal resonance at 3.15 ppm in an otherwise
normal spectrum. The abnormal resonance could be attributed to the presence of
methylsulfonylmethane (MSM; synonyms: dimethyl sulfone, sulfonylbismethane).
An approximation of the amount of MSM in occipital gray and periventricular white
matter was determined by comparison of the signal area of the resonance of
dimethyl sulfone at 3.15 ppm and the methyl signals of creatine (3.03 ppm) and
choline (3.22 ppm). Assuming comparable attenuation of the three resonances due
to relaxation and assuming choline and creatine contents of 0.88 mM and 6.9 mM
respectively, in occipital gray matter [209], the MSM concentration at this location
was estimated 1.2 to 1.3 mM. A similar calculation for the voxel in periventricular
white matter resulted in an MSM concentration of 1.1 to 1.3 mM. These
concentrations show that the MSM is equally distributed among gray and white
matter. Cerebrospinal fluid (CSF) analysis revealed normal values for cells, protein,
glucose, lactate and pyruvate. Nuclear magnetic resonance (NMR) spectroscopy
was performed on CSF and showed the singlet resonance of MSM at 3.14 ppm (at
pH 2.5; Figure 2). The concentration of MSM in CSF was 1.7 mmol/l.
Figure 2
In vitro NMR spectrum of cerebrospinal fluid showing a resonance of dimethylsulfone (methylsulfonylmethane, MSM) at 3.14 ppm
(pH 2.5).
127
Chapter 3.2
Discussion
In vivo and in vitro proton MR spectroscopy (of the brain and CSF, respectively),
generally referred to as 1H-MRS and body fluid NMR spectroscopy, are relatively
novel techniques that are used to investigate patients who suffer from known and
unknown neurological disorders. The study of peak heights of known resonances
and the appearance of ‘novel’ resonances has proven to be very useful in the
elucidation of pathophysiological processes in brain disorders. Sometimes,
however, the interpretation of the spectral abnormalities can be difficult, especially
when abnormal resonances cannot simply be assigned to known metabolites. A
recent study in our own group concentrated on a hitherto unknown resonance at
3.14 ppm observed in CSF NMR spectra of some patients [10]. The resonance was
assigned to methylsulfonylmethane or dimethylsulfone which appears to occur in
low concentration (0 – 25 µmol/l) as a physiological metabolite in human CSF [10].
Shortly after this study we encountered the in vivo spectrum of the patient
described here. The interpretation of her cerebral proton MR spectrum was
therefore simple, but might otherwise have led us to search for an explanation for
the abnormal resonance in this family with two children with an unknown – most
probably genetic – disorder.
MSM may derive from dietary sources, from intestinal bacterial metabolism and
from human endogenous methanethiol metabolism [10;210]. The molecule can
easily cross the blood-brain barrier. After ingestion of ‘generally advised dosages’
it can be demonstrated in millimolar concentrations in brain tissue and in CSF
which is more than thousand-fold the normal physiological concentration
[10;195-197;210]. Comparable results of cerebral proton MR spectroscopy have
previously been described in a small number of adults [196;197] and in only one
(5-year-old) child [195], all using MSM. We are surprised about the high
concentrations that occur in the central nervous system after ingestion of MSM in
‘recommended’ dosages. Apparently, its accumulation is tolerated without adverse
effects [10;195-197;210].
The occurrence of an abnormal resonance at 3.15 ppm on cerebral proton MR
spectroscopy (or CSF NMR) should remind the investigator to check the use of
MSM as dietary supplement in the patient. More in general, in vitro and in vivo MR
spectroscopy might incidentally reveal puzzling results due to exogenous
metabolites like medication or food additives. Other compounds that may be
encounted as remarkable or “confusing” resonances in in vivo and in vitro MR
spectra are e.g., propanediol (propylene glycol) and ethanol [3;211;212].
128
CHAPTER
3.3
NMR spectroscopic studies on the late onset form
of 3-methylglutaconic aciduria, type I and other
defects in leucine metabolism
Udo F.H. Engelke, Berry Kremer, Leo A.J. Kluijtmans, Marinette van der Graaf, Éva
Morava, Ference J. Loupatty, Ronald J.A. Wanders, Detlef Moskau, Sandra Loss,
Erik van den Bergh and Ron A. Wevers
NMR in Biomedicine 2006; 19(2): 271-278
129
130
3-Methylglutaconic aciduria type I
Abstract
A diagnosis of 3-methylglutaconic aciduria type I (OMIM: 250950) based on
elevated urinary excretion of 3-methylglutaconic acid (3MGA), 3-methylglutaric
acid (3MG) and 3-hydroxyisovaleric acid (3HIVA) was made in a 61-year-old
female patient presenting with leukoencephalopathy slowly progressing over more
than 30 years. The diagnosis was confirmed at the enzymatic and molecular level.
In vivo brain MR spectroscopic imaging (MRSI) was performed at 3.0 T, and onedimensional and two-dimensional in vitro NMR spectroscopy of body fluids of the
patient was performed at 11.7 T. Additionally, we measured 1D 1H-NMR spectra of
urine of seven patients with a total of four different inborn errors of leucine
metabolism. Increased concentrations of 3HIVA, 3MGA (cis and trans) and 3MG
were observed in the NMR spectra of the patient’s urine. In the cerebrospinal fluid,
the 3HIVA concentration was 10 times higher than in the plasma of the patient and
only the cis isomer of 3MGA was observed. In vivo brain MRSI showed an abnormal
resonance at 1.28 ppm that may be caused by 3HIVA. Comparison of 1H-NMR
spectra of urine samples from all eight patients studied, representing five different
inborn errors of leucine metabolism, showed that each disease has typical NMR
characteristics. Our leukoencephalopathy patient suffers from a late-onset form of
3-methylglutaconic aciduria type I. In the literature, only very few adult patients
with this conditions have been described, and 3HIVA accumulation in white matter
in the brain has not been presented before in these patients. Our data
demonstrate that 1H-NMR spectroscopy of urine can easily discriminate between
the known inborn errors of leucine metabolism and provide the correct diagnosis.
131
Chapter 3.3
Introduction
3-Methylglutaconic aciduria type I (OMIM: 250950) is an inborn error of leucine
metabolism caused by the deficiency of the enzyme 3-methylglutaconyl-CoA
hydratase (3MGH; EC 4.2.1.18). This enzyme catalyzes the conversion of 3methylglutaconyl-CoA
to
3-hydroxy-3-methyl-glutaryl-CoA
in
leucine
metabolism (step E, Figure 1).
Figure 1
Metabolic pathway of leucine.
3-Methylglutaconyl CoA hydratase deficiency (solid square) results in
accumulation of 3MGA, 3MG and 3HIVA.
A, branched-chainamino-acid transaminase (EC 2.6.1.42);
B, branched-chain 2-keto acid dehydrogenase (EC 1.2.4.4);
C, isovaleryl-CoA dehydrogenase (EC 1.3.99.10);
D, 3-methylcrotonyl CoA carboxylase (EC 6.4.1.4);
E, 3-methylglutaconyl CoA hydratase (EC 4.2.1.18);
F, 3-hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4);
G, enoyl-CoA hydratase (EC 4.2.1.17).
The metabolic phenotype of this disease is characterized by increased excretion of
3-methylglutaconic acid (3MGA), 3-methylgutaric acid (3MG) and 3hydroxyisovaleric acid (3HIVA) in body fluids. Four other inborn errors of leucine
metabolism are known and in vitro 1H-NMR spectra of body fluids have been
reported for several of these [3;24;27;32;39], but not for 3MGH deficiency. In vivo
MR spectroscopy has also been used for studying inborn errors of metabolism. In
1H-MR spectra of the brain, various metabolites such as creatine, glutamine,
glutamate,
choline,
myo-inositol,
N-acetylaspartylglutamate
and
N-
132
3-Methylglutaconic aciduria type I
acetylaspartate can be observed in healthy persons. Several metabolic
abnormalities have been documented with in vivo MR spectra of the brain, e.g. in
patients with creatine biosynthesis defects [145], succinate dehydrogenase
deficiency [213], Salla disease[214], Canavan disease [215], Sjögren Larsson
syndrome [216], N-acetylaspartic acid deficiency [146] and ribose 5-phosphate
isomerase deficiency [116]. In this paper, we describe in vivo and in vitro NMR
spectroscopic investigations of a 61-year-old woman with a 3MGH deficiency and
a leukoencephalopathy which has been progressing very slowly over more than 30
years. Furthermore, we demonstrate for the first time the presence of increased
3HIVA in the brain in this disease. Our data show that 3HIVA in human body fluids
may derive from brain metabolism.
Materials and Methods
IN VIVO MR SPECTROSCOPY
MR measurements were performed on a 3.0 T MR spectrometer (Magnetom Trio;
Siemens Medical Solutions, Erlangen, Germany), using the standard head coil. T2weighted images were acquired in three perpendicular planes using a TGSE
sequence with an echo time of 110 ms a repetition time of 2500 ms, turbofactor
11 and an EPI factor of 3. Two-dimensional proton MRSI was carried out using
PRESS volume pre-selection of a transversal slice through the ventricles with WET
water suppression. Sequence parameters were TR= 2500 ms, TE= 30 ms, slice
thickness= 15 mm, field-ofview= 220mm and spectral width= 1 kHz. The Kspace was sampled in a weighted, elliptical way (20 × 20 points) followed by zerofilling to 32 × 32 points and multiplication using a 50% Hamming filter before
Fourier transformation. This results in an apparent voxel size of 0.7 cm3 and an
effective voxal size of 2.7 cm3. Time domain data (1024 points) in the
spectroscopic domain were processed using the standard Siemens software, which
included removal of the residual water signal, multiplication by a Hanning filter
(center= 0 ms, width= 400 ms), zero-filling to 2K data points, Fourier
transformation, baseline correction, phase correction and curve fitting using
Gaussian lineshapes.
IN VITRO NMR SPECTROSCOPY
Authentic standards.
All authentic standards of the metabolites accumulating in the investigated inborn
errors of metabolism, except isovalerylglycine, 3-methylcrotonylglycine, 3MGA
and 3MA, were commercially available and were purchased at Sigma.
133
Chapter 3.3
Sample preparation.
Plasma and cerebrospinal fluid (CSF) samples were deproteinised using a 10 kDa
filter (Sartorius). Before use, the filter was washed twice by centrifugation of water
to remove glycerol. Urine was centrifuged before analysis. An aliquot (20 µl for
plasma and CSF; 70 µl for urine) of 20.2mM trimethylsilyl-2,2,3,3tetradeuteropropionic acid (TSP, sodium salt; Aldrich) in D2O was added to 700 µl
of the ultrafiltrate or urine, providing a chemical shift reference (δH= 0.00 ppm), a
concentration reference and a deuterium lock signal. The pH of each sample was
adjusted to 2.50 ± 0.05 with concentrated HCl. Finally, 650 µl of the sample were
placed in a 5mm NMR tube (Wilmad Royal Imperial).
One-dimensional 1H-NMR spectroscopy.
Body fluid samples were measured at 500MHz on a Bruker DRX 500 spectrometer
with a triple-resonance inverse (TXI) 1H {15N, 13C} probehead equipped with X,Y,Z
gradient coils. 1H spectra were acquired as 128 transients in 32K data points with
a spectral width of 6002 Hz. The sample temperature was 298K; the H2O
resonance was presaturated by single-frequency irradiation during a relaxation
delay of 10 s; a pulse width of 7 µs was used (corresponded to a 90º excitation
pulse). Shimming of the sample was performed automatically on the deuterium
signal. To improve the spectral resolution, the samples were spun (7 Hz) during
the measurements. The resonance linewidths for TSP and metabolites were <1 Hz.
A π/2-shifted sine-bell window function was applied to the FID. Fourier
transformation was performed after zero-filling to 64K data points. The phase and
the baseline were corrected manually. The 3HIVA, cis 3MGA, trans 3MGA
resonances and the TSP singlet (nine equivalent protons) were fitted semiautomatically with Lorentzian line shapes and concentrations were calculated from
the relative integrals of the fitted lineshapes using the known concentration of
TSP.
1H–13C
HSQC spectroscopy.
An heteronuclear singlequantum correlation (HSQC) spectrum of the patient’s
urine was measured at 500MHz on a Bruker AV 500 spectrometer using a tripleresonance inverse CryoProbe TM (Bruker), wherein the preamplifier and the RF
coils are cooled to low temperature. The sequence was optimized for 1JCH=130 Hz.
FIDs (1536 transients) were acquired with 2K time-domain data points (t2), an
acquisition time of 0.128 s, TR=2.128 s and 96 t1 increments. The spectral widths
were 16 ppm (1H) and 70 ppm (13C), and the total measurement time was 5 days
and 10 h.
1H–1H
correlation (COSY) spectroscopy.
A magnitude-mode COSY spectrum of the patient’s urine was acquired on a
500MHz Bruker Avance spectrometer equipped with a TCI cryo probe. The spectral
134
3-Methylglutaconic aciduria type I
widths in the F1 and F2 domains were 5500 Hz. A total of 2K data points were
collected in t2, 256 t1 increments with 32 transients per increment were used, and
the relaxation delay was set to 2 s. Before the Fourier transformation, a sine-bell
function was applied in both time domains. During the relaxation delay, the water
resonance was presaturated.
CASE REPORT
The clinical history of the patient and the findings at MR imaging of the brain will
be reported in more detail separately. The patient is a 61-year-old woman with
leukoencephalopathy progressing slowly over more than 30 years, manifesting as
optic neuropathy, mild ataxia and mild spasticity of the legs. For a long time the
patient was misdiagnosed as suffering from multiple sclerosis. The patient did not
have oligoclonal immunoglobulin bands in the CSF. T2-weighted MR images
showed extensive white matter lesions of the patient’s brain.
THE DIAGNOSIS
The diagnosis of 3-methylglutaconic aciduria type I (OMIM: 250950) was
confirmed at the metabolite level (gas chromatography for organic acid analysis of
urine; NMR spectroscopy of body fluids), at the enzyme level (3MGH deficiency in
fibroblasts) and the molecular genetic level. The 3MGH activity in homogenates of
the patient’s fibroblasts was undetectable (controls; 2.1±0.7 nmol/min/mg
protein). Analyses of the AUH gene of the patient revealed a heterozygous
559G→A (G187S) and a heterozygous 650G→A (G217D) missense mutation in
exons 5 and 6, respectively. Other forms of 3-methylglutaconic aciduria, namely
types II–IV, were excluded on clinical and biochemical grounds. Lactic acid in body
fluids of the patient was unremarkable.
Results
IN VIVO NMR SPECTROSCOPY
MR imaging of the brain revealed a leukoencephalopathy. Therefore, in vivo MR
spectroscopic imaging was performed of the patient’s brain. Four MRSI voxels
positioned in the posterior lateral subcortical periventricular white matter are
shown in Figure 2. Resonances from total creatine (creatine and
phosphocreatinine; N-CH3 at 3.04, N-CH2 at 3.95 ppm), GABA, glutamine and
glutamate (2.19–2.4 ppm), total cholines [N-(CH3)3 group of choline,
phosphocholine and glycerophosphocholine at 3.2 ppm], myo-inositol (H1,3 at
3.53 ppm, H4,6 at 3.61 ppm) and N-acetylaspartate (NAc at 2.02 ppm, Hβ1 at
2.50 ppm, Hβ2 at 2.68 ppm) were observed. Compared with voxels 1 and 2, voxel
3 showed a reduced NAA:tCho signal ratio, as often seen in patients with
leukoencephalopathy [217;218]. In all spectra a singlet resonance at 1.28 ppm
with an unusually small line-width of 7Hz was observed. Acyl chain protons in
135
Chapter 3.3
mobile lipids and methyl protons of lactic acid are known to contribute to
resonances in this area. It is unlikely that lactic acid causes the peak at 1.28 ppm
because the methyl protons of lactic acid resonate as a doublet at 1.33 ppm. Lipid
signals are known to resonate at 1.3 ppm (methylene protons) and 0.85 ppm
(methyl protons). However, generally, the lipid methylene signals have a broader
line width than the 7 Hz observed in the patient’s brain.
Figure 2
A 3.0T T2 weighted brain image (upper right) and four MRSI
voxels (apparent voxel size=0.7 cm3) positioned in the
posterior lateral subcortical periventricular white matter
(upper left) showing a resonance of 3-hydroxyisovaleric acid.
Compared with voxels 1 and 2, voxel 3 (lower) showed a
reduced NAA:tCho signal ratio. Furthermore, the spectrum
contains resonances of the normal brain metabolites total
creatines (tCr), myo-inositol (myo-Ins), γ-aminobutyric acid
(GABA), glutamine and glutamate (Glx)
In addition, the resonance at 0.85 ppm visible in Figure 2 may also originate from
methyl groups of proteins and amino acids. Furthermore, no lipid contamination
originating from the skull region is expected to contribute to the spectra from this
location. Still, we cannot rule out the possibility that some lipid signal contributes
to the intensity at 1.28 ppm. It was then discovered by in vitro NMR spectroscopy
that the patient had an abnormally high concentration of 3HIVA in her CSF (172
µmol/l, reference value, <10µmol/l). At pH 2.5, 3HIVA gives a singlet resonance at
1.33 ppm (six protons of the two methyl groups) and a singlet resonance at 2.55
ppm (two protons of the methylene group). Since brain pH values range from 6.96
to 7.05 [219], a one-dimensional (1D) 1H-NMR spectrum of a reference solution of
3HIVA in H2O was recorded at pH 7.0. The singlet resonance from the six
136
3-Methylglutaconic aciduria type I
equivalent protons of the two methyl groups shifted from 1.33 ppm at pH 2.5 to
1.27 ppm at pH 7.0. These data provide evidence supporting the assignment of
3HIVA as the source of the resonance at 1.28 ppm in the in vivo MR brain
spectrum. Owing to overlap of glutamate/glutamine (Glx) the two equivalent
protons of the methylene group of 3HIVA (singlet: 2.37 ppm at pH 7) could not be
observed in the in vivo MR brain spectrum. The singlet resonance at 1.28 ppm and
the total creatine (tCr) singlet resonance (3.04 ppm; three equivalent protons) in
the in vivo spectrum were fitted semiautomatically with Gaussian line shapes.
Assuming that only 3HIVA and no lipid contribute to the peak at 1.28 ppm then
the concentration of 3HIVA can be estimated from the relative integrals of the
fitted lineshapes. Based on the reference value of 5.5 mmol/l for total creatine in
occipital white matter [150], 3HIVA was calculated to be 0.9 mmol/l, about five
times the concentration in CSF.
IN VITRO NMR SPECTROSCOPY
Expanded regions (1H-axis: 1.00–3.25 ppm/13C-axis: 15.0–55.0 ppm) of the 1D 1H
and the two-dimensional (2D) 1H–13C HSQC 2D NMR spectra of the patient’s urine
at pH 2.5 are shown in Figure 3. The major metabolites in this region and the
1H/13C shift values for the correlation peaks are creatinine (singlet: 3.13/38.8),
creatine (singlet 3.05/40.0), citric acid ([AB]2 spin system 2.99; 2.83/46.5),
dimethyl amine (triplet 2.71/37.8), acetic acid (singlet 2.08/23.5), alanine (doublet
1.51/18.7) and lactic acid (doublet 1.41/22.4). Furthermore, unusually high
concentrations of 3HIVA, cis 3MGA, trans 3MGA and 3MG were observed in the
urine spectra. These characteristic 3-methylglutaconic aciduria metabolites were
assigned and quantified as described below, and their structures and NMR data are
summarized in Table 1.
Figure 3
500 MHz 1D 1H spectrum (upper panel)
and 2D
1H-13C
HSQC spectrum (lower
panel) of the patient’s urine (pH=2.5).
The insert shows the region between
2.2 and 1.9 ppm in greater detail.
137
Chapter 3.3
3HIVA
1H-NMR
of model compounds in H2O at pH 2.5 revealed that the two unusually
intense singlets at 1.33 and 2.55 ppm, observed with integral ratio 3:1 for the
patient’s urine (Figure 3), derived from 3HIVA (Table 1). In two different urine
samples of the patient the concentration of 3HIVA amounted to 63 and 61µmol/
mmol creatinine [control urines from normal adults (n=12), mean 8, range 3–15].
Also CSF and plasma of the patient showed increased concentrations of 3HIVA
(CSF, 172 µmol/l, reference value <10 µmol/l; plasma 20 µmol/l, reference value
<10 µmol/l).
Table 1: NMR assignments (urine, pH 2.5) for metabolites characteristic of 3methylglutaconic aciduria, type I
Pos.
δH a
Mult.
δC
2
4, 4’
2.55
s
50.2
3-Methylglutaric acidC
3
6
2.35
m
3-Methylglutaconic
2
4
6
3.65
d
41.9
41.4
5.96
m
121.9
115.4
2
4
6
3.28
d
48.4
47.4
5.85
m
122.2
115.4
Metabolite
3-Hydroxyisovaleric
acid
acid
“Cis”
3-Methylglutaconic
acid
“Trans”
Structure
1.33
1.01
1.99
2.14
s
δ Cb
31.1
d
d
d
28.6
21.6
22.2
14.2
a Chemical
shifts in ppm rel. TSP; multiplicities are: s, singlet; d, doublet; m, multiplet.
shift predictions made by ChemOffice 2004.
c Spectrum not yet completely interpreted.
b Chemical
Cis- and trans-3MGA
3MGA is not commercially available as a model compound. Therefore, 1D 1H, 2D
COSY and 2D HSQC experiments on a urine sample of the patient were used to
make the assignments for the spin systems of the two isomers of this metabolite
(Table 1). It is expected that the carboxyl group attached to the C4 of the double
138
3-Methylglutaconic aciduria type I
bond will exert a downfield shift on methyl or methylene (C6 or C2) in a cis
position compared with the trans position relative to the carboxyl. Therefore, the
doublet resonances at 1.99 (4J4,6=1 Hz) and 2.14 (4J4,6= 1 Hz) ppm were assigned
to the methyl protons of the cis and trans forms of 3MGA, respectively. With this
assignment of the isomers the methylene protons on C2 are downfield-shifted for
the cis isomer as expected, and the order of shifts for H4, C2 and C6 agree with
predictions made by ChemOffice 2004 software. For example, the predicted and
observed 13C shift for C2 are 41.4 and 41.9 ppm (cis-3MGA) and 47.4 and 48.4
ppm (trans-3MGA). These assignments for the 3MGA isomers agree with those of
Iles et al. [32] Lehnert and Hunkler [24] incorrectly assigned the H4 multiplet at
5.96 ppm to trans 3MGA and the multiplet at 5.85 to cis 3MGA. In two different
urine samples of the patient the concentration of 3MGA was 94 (cis/trans values:
62/32) and 141 (cis/trans values: 90/51) µmol/mmol creatinine [normal adult
range: 1.0–6.5 µmol/mmol creatinine [220]]. In CSF, only the cis form of 3MGA
was observed (125 µmol/l, reference <10µmol/l). In plasma no 3MGA was
observed. All samples have been adjusted to pH 2.5 with concentrated HCl so that
cis/trans interconversion could be an artifact due to the pH changes. To test this a
urine sample was recorded at pH 9 and pH 2.5. Both spectra showed the same
cis/trans ratio of 3MGA. Thus, pH changes are unlikely to be a source of 3MGA
cis/trans interconversion.
3MG
3MG is also not commercially available as a model compound. Again, 1D and 2D
COSY NMR experiments on a urine sample of the patient allowed the assignments
of this metabolite (Table 1). The doublet resonance at 1.01 ppm, observed in the
methyl region of the 1D 1H-NMR urine spectrum, was assigned to the methyl
protons of 3MG. In the COSY spectrum, this doublet showed a cross peak at 2.35
ppm (Figure 4). This multiplet resonance derives from the single CH proton of
3MG. The chemical shift of the resonances of 3MG agree exactly with those of
Lehnert and Hunkler [24]. However, these authors incorrectly assigned the methyl
protons of 3MG as a multiplet. Because of the low concentration of 3MG in urine,
13C assignments of 3MG in the 1H-13C HSQC spectrum were not possible. In two
different urine samples of the patient the concentrations of 3MG were 15 and 12
µmol/mmol creatinine [reference adult 0.5±0.2 µmol/mmol creatinine [220]]. In
CSF and plasma no 3MG was observed.
139
Chapter 3.3
Figure 4
500MHz 1H 2D COSY spectrum of the patient’s urine (pH=2.5). The cross peak of 3methylglutaric acid is shown in the insert for the selected region (gray box).
OTHER INBORN ERRORS OF LEUCINE METABOLISM
We measured urine samples from seven patients suffering from four other
diseases involving leucine metabolism. Resonance assignments were based on
previouslyreported spectra [24] and on 1D and 2D spectra of 2-hydroxyisovaleric
acid, 2-oxoisovaleric acid, 3-methyl-2-oxo-valeric acid, 3-hydroxyisovaleric acid
and 3-hydroxy-3-methylglutaric acid dissolved in H2O (pH 2.5).
The 1D 1H-NMR spectrum of urine from a patient with maple syrup urine disease
(Figure 1, enzyme B) showed the oxo acids 3-methyl-2-oxo-valeric acid (1713
µmol/mmol creatinine), 2-oxoisovaleric acid (558 µmol/mmol creatinine) and 2oxoisocaproic acid (2951 µmol/mmol creatinine) as well as the presence of 2hydroxyisovaleric acid (2622 µmol/mmol creatinine). High concentrations of
isovalerylglycine (4895 µmol/mmol creatinine) and 3HIVA (1505 µmol/mmol
creatinine) could be observed in the 1H-NMR spectrum of urine from a patient with
isovaleric acidemia (Figure 1, enzyme C). In two unrelated patients with 3methylcrotonyl CoA carboxylase deficiency (Figure 1, enzyme D), unusually high
concentrations of 3HIVA (6400 and 6300 µmol/mmol creatinine) and 3methylcrotonylglycine (345 and 800 µmol/mmol creatinine) were observed in the
urine 1D 1H-NMR spectra. Furthermore, we measured urine samples from three
patients with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (Figure 1, enzyme
F). In all 1H-NMR spectra we observed high concentrations of 3-hydroxy-3-
140
3-Methylglutaconic aciduria type I
methylglutaric acid (3365, 2037 and 4259 µmol/mmol creatinine), 3HIVA (877,
918 and 1136 µmol/mmol creatinine), 3MG (382, 219 and 441µmol/mmol
creatinine) and 3MGA (2921, 2249 and 3638 µmol/mmol creatinine). In all three
urine samples a cis/trans 3MGA ratio of 2:1 was observed. Table 2 provides an
overview of all five known inborn errors of leucine metabolism and their
diagnostically important metabolites with the 1H chemical shift data observed in
urine samples from affected patients (pH 2.5).
Table 2: 1H chemical shifts (pH 2.5) for metabolites accumulating in urine for inborn errors
of leucine metabolism (Figure 1; B–F)
Metabolite
δH and Mult.
a
Affected enzyme
b
in Figure 1
2-Hydroxyisovaleric acid
0.88d; 0.98d; 2.06m; 4.09d
B
2-Oxoisocaproic acid
0.94d; 2.09m; 2.61d
B
3-Methyl-2-oxovaleric acid
0.95t; 1.09d; 1.45m; 2.92m
B
2-Oxoisovaleric acid
1.12d; 3.02m
B
Isovalerylglycine
0.94d; 2.18m; 3.94d
C
3-Hydroxyisovaleric acid
1.33s; 2.55s
3-Methylcrotonylglycine
1.86d; 2.03d; 3.97d; 5.78m
3-Methylglutaconic acid (cis)
1.99d; 3.65d; 5.96m
E, F
3-Methylglutaconic acid (trans)
2.14d; 3.28d; 5.85m
E, F
3-Methylglutaric acid
1.01d; 2.35m
E, F
3-Hydroxy-3-methylglutaric acid
1.41s; 2.71d
F
C, D, E, F
D
a Chemical shifts in ppm rel. TSP; multiplicities are: s, singlet; d, doublet; m, multiplet s, singlet; d,
doublet; m, multiplet.
b Disorders: B, maple syrup urine disease; C, isovaleric acidemia; D, 3-methylcrotonyl CoA carboxylase
deficiency; E, 3-methylglutaconyl CoA hydratase deficiency; F, 3-hydroxy-3-methylglutaryl-CoA lyase
deficiency.
141
Chapter 3.3
Discussion
This study describes in vivo and in vitro NMR investigations of a 61-year-old
patient with 3MGH deficiency and a slowly progressing leukoencephalopathy. In
the literature 12 children between 0 and 14 years of age have been reported with
3MGH deficiency. Some papers have reported leukoencephalopathy in these
patients [221]. In none of these studies was MR spectroscopy performed. In vitro
NMR and in vivo MR spectra showed that our patient had high levels of 3HIVA in
her CSF and probably in her brain. Several NMR experiments confirmed the
presence of 3HIVA, 3MGA and 3MG in body fluids of our patient. NMR
spectroscopy of body fluids can provide an overview of proton-containing
metabolites and the technique can be used for quantification. Lehnert and Hunkler
[24] were the first to perform NMR spectroscopy on urine samples and model
compounds under standardarized pH conditions (pH 2.50). They used 1H-NMR at
250 MHz to investigate urine samples of 13 different inborn errors of metabolism,
including three diseases from leucine metabolism: isovaleric acidemia, 3methylcrotonyl-CoA carboxylase deficiency and 3-hydroxy-3-methylglutaryl-CoA
lyase deficiency. Iles et al. [32] reported 1D and 2D COSY 1H-NMR spectra of urine
from twins with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Until now,
body fluids of patients with type I 3MGH deficiency have not been studied with
NMR spectroscopy. The combination of 1D 1H, 2D 1H-1H COSY and 1H-13C HSQC
spectra allowed identification of the resonances of 3MGA, 3MG and 3HIVA in urine.
Moreover, 1D spectra were used to quantify these metabolites. This led to the
correct diagnose of 3-methylglutaconic aciduria. 3-Hydroxy-3-methylglutaric acid
was not detectable in urine, thus excluding 3-hydroxy-3-methylglutaryl- CoA
lyase deficiency.
We had the opportunity to study urine samples of seven patients with four other
inborn errors of leucine metabolism. As shown in Table 2, for all diseases NMR
spectroscopy allowed identification of the diagnostically important metabolites
and the correct diagnosis. In most studies, only data obtained from urine samples
have been reported. We had the opportunity to analyze plasma as well as CSF from
our patient. In plasma, slightly increased 3HIVA was the single unusual finding
observed in theNMR spectrum. This may be explained by the rapid and efficient
renal clearance of 3HIVA, 3MGA and 3MG. In an earlier paper we reported a 3HIVA
concentration of 540 µmol/l in CSF from a patient with 3-methylcrotonyl-CoA
carboxylase deficiency [3] (Figure 1, enzyme D). This concentration was about
three times higher than in the CSF of our current patient (172 µmol/l). The
CSF:plasma ratio of 3HIVA for our patient was about 8.6. Duran et al. reported a
CSF:plasma 3HIVA ratio of ca. 1.7 in one patient with 3-methylcrotonyl-CoA
carboxylase deficiency [222] (Figure 1; enzyme D). These data may indicate that
CSF 3HIVA in our patient derives from brain leucine metabolism (Figure 1).
Moreover, in vivo MR spectra suggested that our patient had high levels of 3HIVA
in her brain. Yudkoff et al. [223] have demonstrated the transamination of leucine
142
3-Methylglutaconic aciduria type I
into 2-oxoisocaproic acid in rat brain and Bixel et al. [224;225] showed the
presence of branched-chain aminotransferase (Figure 1; enzyme A), branchedchain a-keto acid dehydrogenase (Figure 1; enzyme B) and b-methylcrotonyl-CoA
carboxylase (Figure 1; enzyme D) in astroglial cells from rats [225]. This suggests
that leucine, which is involved in the synthesis of the neurotransmitter glutamate
[223], can be degraded into acetoacetate in the human brain (Figure 1). However,
to our knowledge, the presence of 3MGH activity in brain cells has not been
described in the literature.
Biotinidase deficiency (OMIM: 2532620) is a form of multiple carboxylase
deficiency in which the formation of the cofactor biotin from biocytin via
biotinidase is inhibited. Patients with this disease may have high concentrations of
3HIVA, 3-methylcrotonylglycine, methylcitric acid and lactic acid in their body
fluids. Biotinidase deficiency usually presents with neurological problems. Three
symptomatic patients had CSF/plasma 3HIVA ratios ranging between 4.3 and 8.4
[222]. In a fourth patient the CSF/plasma ratio was 27.8. This patient had severe
chronic neurological problems. The authors speculated that 3HIVA may have a
specifically harmful effect on the brain. Our study shows an accumulation of 3HIVA
in the brain of our patient with 3-methylglutaconic aciduria while we found no
evidence for accumulation of 3MGA in the in vivo spectrum. However, we cannot
exclude an overlap of 3MGA resonances with other resonances in the in vivo
spectrum. The concentration in CSF of 3HIVA was higher than the concentration of
3MGA in our patient. The leukoencephalopathy in our patient is in line with the
hypothesis that 3HIVA may be the key factor in the pathogenesis of central
nervous system pathology in biotinidase deficiency and 3-methylglutaconic
aciduria.
1D 1H NMR spectroscopy can be used to quantify both the cis and trans form of
3MGA in body fluids. 3-Methylcrotonyl CoA carboxylase (Figure 1; enzyme D) is a
stereospecific enzyme and produces only trans 3-methylglutaconyl-CoA. Lynen et
al. [226] reported isomerization of cis/trans 3-methylglutaconyl-CoA under
alkaline conditions and Kelley et al. [220] showed that the choice of conditions for
derivatization by GC-MS might influence the cis/trans ratio of the free acid. A
cis/trans 3MGA ratio of 2:1 was observed in all urine samples from the three
patients with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Iles et al.
reported a similar ratio in urine NMR spectra of twins with 3-hydroxy-3methylglutaryl-CoA lyase (Figure 1; enzyme F) deficiency [32], which we could
confirm in this study on three patients with this defect. In our patient with 3MGCoA hydratase deficiency, a cis/trans 3MGA ratio of ca. 2:1 was observed in both
urine samples, while in CSF only cis-3MGA was observed. Different cis/trans ratios
for 3MGA could be the result of spontaneous cis/trans interconversion of the free
acid according to the environment in different organs or body fluids. However, this
patient’s urine NMR spectra recorded at pH 9 and pH 2.5 showed the same
cis/trans 3MGA ratio. This suggests that the pH is unlikely to be a source of
143
Chapter 3.3
cis/trans interconversion. Another explanation for the occurrence of only cis 3MGA
in CSF may be the presence of a brain-specific isoform of 3-methylcrotonyl CoA
carboxylase which may have stereospecificity. As yet, there is no other evidence
for a separate brain isoform. Further investigations are necessary to explain this
finding.
In conclusion, we have shown that NMR spectroscopy can be used to identify and
quantify the characteristic 3-methylglutaconic aciduria metabolites 3MGA, 3MG
and 3HIVA in body fluids. Furthermore, our data suggest that 3HIVA accumulates
in brain of a patient with 3-methylglutaconic aciduria, type I. Our data show that
3HIVA and 3MGA may derive from brain metabolism.
Acknowledgements
The authors thank Dr Jörn Oliver Sass (Stoffwechsellabor, Zentrum für
Kinderheilkunde- und Jugendmedizin, Universitätsklinikum Freiburg, Germany) for
providing urine samples from patients with 3-hydroxy-3-methylglutaryl-CoA
lyase deficiency.
144
CHAPTER
3.4
Leukoencephalopathy in adult-onset
3-methylglutaconic aciduria type I
Berry Kremer, Udo F.H. Engelke, Janneke Schuuring, Marinette van der Graaf,
Ference J. Loupatty, Ronald J.A. Wanders, Jouke Mellema and Ron A. Wevers
Submitted for publication
145
146
Leukoencephalopathy in adult-onset 3-methylglutaconic aciduria type I
Abstract
We describe a 61-year-old woman with 3-methylglutaconic aciduria type I (3methylglutaconyl-CoA hydratase deficiency) and a very slowly progressive
leukoencephalopathy over more than 30 years, manifesting as optic neuropathy,
mild ataxia and leg spasticity.
Organic acid analysis and in vitro NMR
spectroscopy of body fluids revealed elevated 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid suggesting 3-methylglutaconyl-CoA
hydratase deficiency as confirmed enzymatically. In vivo MR spectroscopy revealed
an increase of brain intraparenchymal 3-hydroxyisovaleric acid. Mutation analysis
demonstrated heterozygosity for two novel missense mutations (559G→A;
650G→A) in the AUH gene (3-methylglutaconyl-CoA hydratase). This patient is the
oldest patient ever described with 3-methylglutaconic aciduria type I.
147
Chapter 3.4
Introduction
3-Methylglutaconic aciduria comprises a group of metabolic disorders biochemically characterized by elevated urine levels of 3-methylglutaconic acid (3MGA) and
3-methylglutaric acid (3MG). To date, four distinct forms have been recognized.
3-Methylglutaconic aciduria type I (OMIM 250950) is caused by a deficiency of 3methylglutaconyl-CoA hydratase (3MGH, E.C. 4.2.1.18) [227]. This enzyme
catalyzes the fifth step in leucine catabolism, which is the conversion of 3methylglutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA. The hydratase
deficiency is associated with symptoms varying from mildly delayed speech development to neurologic, cardiac and hepatic abnormalities that until now have
been described in young children, only. Patients with other forms of 3methylglutaconic aciduria (Barth syndrome, OMIM 302060; Costeff syndrome,
OMIM 258501; unspecified 3-methylglutaconic aciduria, OMIM 250951) have
normal hydratase activity [227]. 3MGH is encoded by the AUH gene [228;229].
Mutation analysis of AUH in seven reported patients revealed protein function
abolishing mutations [221;227-229].
We describe a patient with a protracted adult onset 3-methylglutaconic aciduria
type I, heterozygous for two novel missense mutations in AUH. As far as we are
aware, this is the first adult patient ever diagnosed with this condition.
Case Report
Since age 35, after giving birth to her second child, this woman had experienced a
progressive visual decline, initially with recurrent episodes of rapid deterioration
and subsequent improvement. Initial neurological and ophthalmologic
examinations, at age 37, revealed a visual acuity of 0.6 and 0.8 of the right and
left eye, respectively; optic disc pallor and atrophy bilaterally, slight gait and limb
ataxia and bilateral extensor responses of the toes. The electroretinogram
indicated rod dysfunction. CSF studies revealed no abnormalities. Over the years,
her visual acuity gradually deteriorated with fluctuations. Repeated Goldmann
perimetry showed progressive concentric visual field constriction and an expanding blind spot. Ataxia and pyramidal abnormalities increased slightly, while
orobucal dyskinesia was noted at the age of 40 years. A neurological re-evaluation
at age 61, revealed: preserved light / dark perception; severe optic disc pallor;
slight pendular nystagmus; minimal dysarthria; mild limb ataxia with severe gait
ataxia and frequent falls despite preserved ambulation; increased tendon reflexes
with absent ankle tendon reflexes and extensor responses of the toes. Nerve
conduction studies at that age were still normal but routine 1.0 Tesla MR-imaging
revealed marked supratentorial symmetric confluent subcortical white matter
abnormalities (Figure 1A demonstrates a subsequent 3.0 Tesla image). Routine
148
Leukoencephalopathy in adult-onset 3-methylglutaconic aciduria type I
biochemical and hematological analyses were completely normal. The CSF
contained a normal cell count, a total protein content of 392 mg/l, lactate of 1731
µmol/l, without intrathecal immunoglobulin synthesis and no abnormal IgG bands.
Figure 1
In vivo MR spectroscopic imaging.
T2-weighted MR images show extensive white matter lesions of the patient’s brain (Panel A). The in vivo spectrum of an
MRSI voxel (3T, PRESS, TE/TR= 30/2500 ms) positioned in the posterior lateral subcortical periventricular white matter is
shown in Panel B. (The voxel position is marked in white rectangles (Panel A)). The spectrum shows an unusual singlet
resonance deriving from 3-hydroxyisovaleric acid. Furthermore, the spectrum shows a reduced NAA/tCho signal ratio as
often seen in patients with leukoencephalopathy.
Analysis of organic acids in the urine of the patient repeatedly showed strongly
elevated 3MGA and 3-hydroxyisovaleric acid (3HIVA) and increased 3MG. Using
proton NMR spectroscopy with a 500 MHz Bruker spectrometer both the cis and
the trans form of 3MGA could be consistently demonstrated in urine (in two
samples the cis-form: 62 and 90 µmol/mmol creatinine and the trans-form: 32
149
Chapter 3.4
and 51 µmol/mmol creatinine; reference for total 3MGA 1.0-6.5 for adults [220]).
Details on the identification of cis and trans 3MGA with body fluid NMR
spectroscopy have been published elsewhere [47]. Urinary 3MG amounted to 15
and 12 µmol/mmol creatinine in two samples (reference 0.5±0.2 µmol/mmol
creatinine [220]). In addition 3HIVA was increased (63 and 61 µmol/mmol
creatinine; reference < 15 µmol/mmol creatinine).
The in vitro NMR spectrum of the CSF revealed the presence of only the cis-form
of 3MGA (125 µmol/l; not detectable in normal CSF). This suggests that the transform is not synthesized in the brain or is immediately converted to the cis-form.
3HIVA in CSF amounted to 172 µmol/l (reference <10). 3MGA could not be
detected in plasma of the patient and the plasma concentration of 3HIVA (20
µmol/l; reference <10) was substantially lower than in CSF, suggesting that both
compounds are synthesized in the brain of the patient. Figure 2 compares the
proton NMR spectra of urine and CSF of the patient.
In vivo brain MR spectroscopy on a 3.0 Tesla MR imaging system revealed a
resonance at 1.28 ppm in our patient with an unusual small line width of 7 Hz.
This resonance was present in abnormal white matter and is absent in normal
brains (Figure 1B). At physiological pH 3HIVA resonates at 1.28 ppm [47]. As lipid
resonances usually have a broader line width the unusual resonance at 1.28 ppm
may be caused by 3HIVA. Assuming that no lipids contribute to the signal at 1.28
ppm the estimated concentration of 3HIVA in the brain of our patient is 0.9
mmol/l which is about five times higher than in the CSF.
To determine whether the elevated urinary metabolite levels were caused by a
deficiency of 3MGH we measured the activity of this enzyme in cultured human
skin fibroblasts and in lymphocytes as described elsewhere using 20-50 mg/l of
lymphocyte protein. 3MGH activity [230] in fibroblasts and lymphocytes of the
patient was undetectable (reference values fibroblasts 2.1 ± 0.7 nmol/min/mg
protein and lymphocytes 1.8 ± 0.2 nmol/min/mg protein).
In addition, we performed mutation analysis for AUH on genomic DNA derived
from fibroblasts and could identify a heterozygous 559 G>A (G187S) and a
heterozygous 650 G>A (G217D) missense mutation in exons 5 and 6, respectively.
150
Leukoencephalopathy in adult-onset 3-methylglutaconic aciduria type I
Figure 2
Body fluid 1H-NMR spectra of the patient’s CSF and urine at 500 MHz (A and B, respectively).
The inserts show the region between 2.2 and 1.95 ppm in greater detail.
151
Chapter 3.4
Discussion
This case demonstrates four features of 3-methylglutaconic aciduria type I. The
patient is unique in terms of age of presentation and perhaps of onset of her
disease;
3-methylglutaconic
aciduria
type
I
may
present
as
a
leukoencephalopathy; in vivo MR spectroscopy may demonstrate increased brain
tissue levels of 3-hydroxisovaleric acid; and we describe two novel mutations. 3Methylglutaconic aciduria type I (3MGH deficiency) is a very rare recessive disorder
of leucine metabolism that usually presents in infancy or early childhood with
either severe metabolic derangements, or, alternatively, as a mild psychomotor
retardation. Until now 12 patients have been reported, with 1 case diagnosed at
neonatal screening and asymptomatic at age 2½ years [221;228;231-239]. From
the literature the natural course of the disease is unclear. Our patient suggests
that if affected individuals survive acute metabolic crises in infancy, the disease
may develop as a very slowly progressive neurodegenerative disorder with perhaps
a normal or only slightly reduced life expectancy. Its ultimate phenotype may be a
progressive demyelinating leukoencephalopathy that in our patient had long been
misdiagnosed as multiple sclerosis. Although MRI reports describe predominant
basal ganglia gray matter lesions [231], white matter lesions have been mentioned
as well [221;231;232;238] The leukoencephalopathy in our patient may be caused
by 3MGA. Alternatively 3HIVA may be the causative factor. Duran has speculated
about a toxic effect of accumulating 3HIVA on the brain in patients with
biotinidase deficiency and patients with 3-methylcrotonyl CoA carboxylase
deficiency [222]. The obvious accumulation of 3HIVA in the brain of our patient is
a further clue towards the potential toxic effects of this metabolite on the brain.
An intriguing finding in our patient is the severe optic neuropathy, probably due to
demyelination as well. Optic neuropathy has been described as a feature of 3methylglutaconic aciduria type III (Costeff syndrome). The urinary 3MGA
concentration is lower in Costeff patients than in type I cases. Costeff syndrome is
caused by a mutation in the OPA3 gene [240]. 3HIVA does not accumulate in
Costeff syndrome. The function of the protein encoded by the OPA3 gene is
unknown. Therefore the optic neuropathy in Costeff syndrome may either be due
to the accumulating 3MGA and/or the 3MG or to an as yet unknown metabolic
consequence of the defect in the OPA3 gene.
We demonstrate that MR spectroscopy may reveal an increase in 3HIVA in 3MGH
deficiency. Resonances from 3MGA and 3MG were not observed in the in vivo
spectrum but these may have been obscured by other, predominant, metabolites.
AUH encodes 3MGH that catalyzes the conversion 3-methylglutaconyl-CoA to 3hydroxy-3-methylglutaryl-CoA [227]. Until now, 6 mutations have been
described: one exon 9 skipping splice acceptor mutation; a mutation in the second
nucleotide of the intron 9 splice donor sequence; a missense mutation, a point
deletion and an insertion that all three introduce premature stop codons and an
152
Leukoencephalopathy in adult-onset 3-methylglutaconic aciduria type I
exon 7 missense mutation [221;228;229]. Mutation analysis of our patient’s
genomic DNA revealed two novel missense mutations, substituting two glycine
residues at position 187 and 217 by the amino acids serine and aspartate,
respectively. BLAST analysis with the mRNAs of human AUH [Genbank:
NM_001698], human short-chain enoyl-CoA hydratase [Genbank: NM_004092]
and the hydratase domain of the human mitochondrial trifunctional protein
[Genbank: NM_000182] revealed at least 400 related proteins of the hydratase
family of different origins. All of these proteins contained conserved glycine
residues at positions 187 and 217, suggesting that the glycines at positions 187
and 217 are critical for enzyme function. Indeed, hydration of an enoyl-CoA substrate by 3MGH proceeds through an intermediate which is stabilized via hydrogen
bond interactions with two peptide NH-groups [241]. These amino groups are
positioned in two distinct, but conserved regions that are an intrinsic part of the
active site pocket. Glycine 187 of 3MGH is located in one these regions.
Mutagenesis studies of this region in human short-chain enoyl-CoA hydratase revealed that substitution of glycine with proline resulted in a 106-fold decrease in
hydratase activity, whereas the overall protein production of this mutant remained
unaffected [242]. One can easily imagine that substition of glycine with a polar
serine residue, as described in our patient, will result in a similar effect and, thus,
a strongly decreased activity of 3-methylglutaconyl-CoA hydratase.
The three-dimensional structure of 3-methylglutaconyl-CoA hydratase is a dimer
of trimers [243]. Glycine 217 is located in a short flexible region (7 nucleotides)
which is surrounded by two α-helices; one helix contains a catalytic glutamate
residues, whereas the other is involved in subunit interactions to form the trimers.
Both the catalytic activity and trimer formation depend on hydrogen bond
interactions. Substitution of glycine 217 with the charged amino acid residue
aspartate could alter the conformation of the flexible region thereby disrupting
the distance required for optimal hydrogen bond interaction. As a result, trimer
formation and catalytic activity could be restrained. Indeed, in lysates of
fibroblasts and lymphocytes from our patient we have found no residual 3MGH
activity. This strongly suggests that these mutations abolish the activity of the
enzyme. Expression studies have to be done in the future to prove this
unambiguously.
153
154
FOUR
LIPID METABOLISM
155
156
CHAPTER
4.1
Diagnosing inborn errors of lipid metabolism using
1H Nuclear Magnetic Resonance spectroscopy
Udo F.H. Engelke, Marlies Oostendorp, Michèl A.A.P. Willemsen and Ron A. Wevers
Clinical Chemistry 2006; 52: 1395 – 1405
157
158
Inborn errors of lipid metabolism
Abstract
Background: Many severe diseases are caused by defects in lipid metabolism. As a
result, patients often accumulate unusual lipids in their blood and tissues and
proper identification of these lipids is essential for correct diagnosis. In this study,
we investigated the potential use of proton nuclear magnetic resonance (1H-NMR)
spectroscopy to simultaneously identify and quantify (un)usual lipids present in
the blood of patients suffering from different inborn errors of lipid metabolism.
Methods: Blood plasma or serum lipids were extracted in 2:1 (v/v) chloroform-
methanol. After addition of the non-volatile chemical shift and concentration
reference compound octamethylcyclotetrasiloxane, 1H-NMR measurements were
performed on a 500 MHz spectrometer. Assignments were based on literature,
computer simulations and reference spectra of relevant authentic standards.
Results: Spectra of normal plasma samples allowed the identification of nine lipid
species. For cholesterol and triglyceride levels, a good correlation was found
between conventional methods and 1H-NMR. Furthermore, four inborn errors of
lipid metabolism were investigated (three in sterol metabolism and one in fatty
acid metabolism). NMR analysis led to a correct diagnosis for all four diseases,
while the concentration of the diagnostic metabolite could be determined for
three.
Conclusions: We showed that 1H-NMR spectroscopy of blood plasma or serum
lipid extracts can be used to accurately identify lipids. Identification was also
possible for unusual lipids in the blood of patients suffering from an inborn error
of lipid metabolism. Additionally, simultaneous lipid quantification was possible
for the first time. The technique is therefore applicable in clinical diagnosis and
follow up.
159
Chapter 4.1
Introduction
Proton nuclear magnetic resonance (1H-NMR) spectroscopy is a versatile technique
that can be used in a wide range of disciplines. The best known medical
applications are in vivo magnetic resonance imaging and spectroscopy, but in vitro
1H-NMR spectroscopy of body fluids has also been used to diagnose inborn errors
of metabolism (e.g. [3;4;11]). In contrast to conventional techniques, NMR can
detect the majority of all proton containing metabolites in a single experiment of
approximately 15 minutes. Furthermore, it is a non-destructive method and
requires little to no sample preparation.
Currently, blood plasma or serum samples are either measured directly [7;23;244]
or after deproteinization by ultrafiltration [4]. The advantage of using the latter
method is that broad, overlapping protein resonances are removed, thereby
yielding a highly resolved spectrum in which only the water soluble, low molecular
mass metabolites are observed. Unfortunately, this also limits NMR as a diagnostic
tool for inborn errors of metabolism, to diseases involving accumulation or
absence of these relatively small metabolites.
There are also many severe diseases that are caused by inherited defects in the
metabolism or biosynthesis of different fatty acids and sterols. As a result,
unusual lipids are often present in the blood and tissues of affected patients.
Accurate identification and quantification of these metabolites is essential for
correct diagnosis of the disease.
1H-NMR has several major advantages over GC-MS and LC-MS for analysis of
lipids and sterols. First, authentic standards are usually not required once the
chemical shifts of the biologically relevant species are known (see [245] for the
chemical shifts of many C27-sterols and their acetyl derivatives). Secondly, lipid
identification is almost unequivocal if a few distinct resonances are resolved.
Finally, sample preparation for 1H-NMR measurements can be fairly simple,
whereas the conventional biochemical analysis of unusual lipids in body fluids can
be complicated and time-consuming, sometimes involving derivatization steps
and a combination of several types of chromatography.
Using one-dimensional (1D) 1H-NMR spectroscopy of intact blood plasma, several
lipid signals can be detected [23]. The technique can be used to determine the
relative amounts of high, low and very low density lipoprotein cholesterol using
complex mathematical line fitting techniques [244]. However, the diagnostic
markers for inborn errors in lipid metabolism remain undetected due to their low
concentrations, overlap with other metabolites and protein derived interferences.
Here, we describe a simple procedure based on the Folch-extraction [246] to
isolate all lipids from blood plasma or serum samples using a 2:1 (v/v)
chloroform-methanol extraction medium. Casu et al. [247] reported the NMR
analysis of lipids extracted from erythrocytes and plasma of humans. Furthermore,
1H-NMR spectroscopy has been applied successfully in the diagnosis of the SmithLemli-Opitz syndrome [248;249]. This study demonstrates for the first time that
160
Inborn errors of lipid metabolism
several serum lipids can be quantified simultaneously using 1H-NMR spectroscopy.
The clinical usefulness of the technique is shown by successfully applying it to
four inherited disorders in lipid metabolism (Smith-Lemli-Opitz syndrome,
cerebrotendinous xanthomatosis, sitosterolemia and Refsum disease).
Materials and Methods
AUTHENTIC STANDARDS
All authentic standards of the metabolites accumulating in the investigated inborn
errors of metabolism, except 8DHC and 8-lathosterol, were commercially available
and were purchased at Sigma. Authentic standards of cholesterol, 7-lathosterol,
oleic acid, linoleic acid, palmitic acid and stearic acid were purchased at Sigma as
well.
SAMPLE PREPARATION
Blood plasma or serum samples were obtained from healthy controls and patients
suffering from an inborn error of lipid metabolism at the University Medical Centre
Nijmegen, The Netherlands. All samples were provided anonymously after routine
diagnostic screening was performed and were kept frozen until NMR analysis. All
lipid material was extracted from 1 mL of blood plasma or serum using a Folch
extraction [246] optimized for blood plasma [250]. Per extraction, 30 mL of a 2:1
(v/v) chloroform-methanol extraction medium was used. The extraction was
performed in a 50 ml capped Teflon® centrifuge tube (Nalgene, Rochester, NY).
The water-methanol layer and denaturated protein precipitate were discarded,
while the chloroform layer was evaporated to dryness using an AS290 automatic
Speedvac concentrator (Savant Instruments, Farmingdale, NY). Subsequently, the
extract was redissolved in approximately 650 µl CDCl3 for NMR analysis. The use
of fresh chloroform and methanol during sample preparation is important. Aged
chloroform can contain phosgenes, which can react with the analytes and hence
lead to incorrect results.
Unfortunately,
the
conventional
chemical
shift
reference
compound
tetramethylsilane (TMS) was not suitable as concentration reference due to its high
volatility. Therefore, octamethylcyclotetrasiloxane (OMS; Fluka), which has a
boiling point of 448 K and a chemical shift of 0.094 ppm compared to TMS, was
used instead. Pipetting an amount of OMS dissolved in CDCl3 to the sample could
not be performed, because the use of plastic pipet tips with chloroform solutions
lead to sample contamination. Furthermore, chloroform leakage from the pipet tip
can cause significant volume, and hence concentration, differences. Therefore,
OMS concentrations were determined by carefully weighing the amount added to
161
Chapter 4.1
the sample. The final sample was placed into a standard 5 mm NMR-tube (Wilmad
Royal Imperial).
NMR SPECTROSCOPY
All high-resolution 1H-NMR spectra were carried out at 298 K on a Bruker DRX 500
MHz spectrometer with a triple-resonance inverse (TXI) 1H {15N, 13C} probehead
and equipped with x, y, z gradient coils. Shimming of the samples was performed
automatically on the deuterium signal. The resonance linewidth for OMS was < 1
Hz in all spectra. For the one-dimensional spectra, 64 transients were recorded
into 32000 data points using a spectral width of 6010 Hz and a 6 second recycle
delay. A pulse width of 5 µs was used (corresponded to a 90° excitation pulse). An
inversion recovery experiment revealed a T1 of 2.6 s for OMS.
Data were processed and analyzed using MestReC version 4.4.1
(www.mestrec.com). The Free Induction Decay was apodized with a sine square
filter and subsequently Fourier transformed after zero filling to 64 k points. The
phase was corrected manually and metabolite signals were integrated (for peaks
showing complex J-splitting) or fitted semi-automatically with a Lorentzian line
shape (singlets only). The resulting areas were compared with the area of OMS to
determine metabolite concentrations.
Two-dimensional 1H-1H correlation (COSY) spectra were recorded using a spectral
width of 6010 Hz in both dimensions, 256 and 2000 data points in F1 and F2,
respectively, 16 scans per increment and a recycle delay of 6 seconds. Prior to
Fourier transformation, both time domains were apodized using a sine bell
function and zero filled once. Resonance assignments were based on literature
[245;251-253], one and two-dimensional spectra of the authentic standards in
chloroform and computer simulations using ACD/HNMR Predictor version 2.03
(ACD/labs, Toronto, Canada). Furthermore, available authentic standards of the
accumulating metabolites were added to patient samples to confirm their
assignment. Chemical shift values in this study are referenced to the chemical shift
of OMS at 0.094 ppm.
GAS CHROMATOGRAPHY
For gas chromatography of sterols samples were extracted in pentane and
subsequently derivatized using N,O-bis(trimethylsilyl)trifluoroacetamide (Fluka)
and pyridine. GC analysis was performed using a CP-Sil-19 CB column
(Chrompack, Bergen op Zoom, The Netherlands) on a Hewlett-Packard 6890 GC
equipped with a flame ionization detector [254].
In the conventional analysis of plasma phytanic acid, all lipids were first extracted
in 1:1 (v/v) chloroform-methanol. After evaporation of the organic solvent, the
fatty acids were esterified using methanolic HCl. The resulting fatty acid methyl
esters were subsequently extracted in hexane and analyzed with gas
chromatography using the same equipment as described above for sterol analysis.
162
Inborn errors of lipid metabolism
CORRELATION STUDY
For the correlation study, 15 patient plasma samples were selected from routine
samples of the clinical chemistry department to give a wide range of cholesterol
and triglyceride values. They were selected anonymously and the results were not
available to the person (M.O.) doing the 1H-NMR analysis. Cholesterol and
triglycerides had been measured enzymatically in 15 and 12 samples, respectively,
using standard reagent kits on an AEROSET System (Abbott laboratories, Illinois)
according to instructions of the manufacturer [255;256].
For 1H-NMR analysis, samples were prepared as described above. Statistical
analysis was performed using Passing and Bablok regression analysis [257]. The
within run coefficient of variation of the NMR method was determined by
preparing 9 separate samples from the same blood serum and subsequently
measuring their cholesterol and triglyceride content. Sample preparation and NMR
measurements were carried out in one session.
INBORN ERRORS OF LIPID METABOLISM
The patient population consisted of:
• 3 cases of the Smith-Lemli-Opitz syndrome (SLOS)
• 3 cases of cerebrotendinous xanthomatosis (CTX)
• 1 case of sitosterolemia
• 1 case of the Refsum disease.
The patient material from the SLOS, CTX and sitosterolemia patients was obtained
prior to treatment and therefore represents diagnostic samples. Six samples were
obtained from the follow-up during therapy of a patient with Refsum disease. For
every inborn error, previous detection of the accumulating metabolite as well as
mutation analysis in the relevant gene had confirmed the diagnosis.
The diseases are shortly described below.
SLOS. The Smith-Lemli-Opitz syndrome is caused by a deficiency of the enzyme
7-dehydrocholesterol reductase (EC 1.3.1.21), which catalyzes the conversion of
7-dehydrocholesterol (7DHC, Figure 1) to cholesterol; the final step in cholesterol
biosynthesis [258]. As a result, 7DHC accumulation and low cholesterol levels are
observed in SLOS patients. Furthermore, 8-dehydrocholesterol (8DHC, Figure 1)
can be detected in the blood of affected patients, resulting from isomerization of
7DHC to 8DHC.
CTX. CTX is caused by a defect in the CYP27A gene, which encodes the
mitochondrial enzyme sterol 27-hydroxylase (EC 1.14.13.15) [259]. This leads to a
block in bile acid synthesis, resulting in the accumulation of unusual bile alcohols
in urine and cholestanol in blood.
163
Chapter 4.1
Sitosterolemia. Sitosterolemia, is characterized by elevated levels of the plant
sterols β-sitosterol, campesterol and stigmasterol (Figure 1) in blood and tissues.
Mutations in the ABCG5 and ABCG8 genes, which both encode for half-transporter
proteins, have been identified in sitosterolemia patients [260]. In healthy subjects,
approximately 5% of the 200-300 mg daily consumed plant sterols is absorbed
and almost all plant sterols are rapidly excreted in the bile. Sitosterolemia patients
absorb between 15 and 60% of the ingested plant sterols and excrete only very
little [259-261]. The mean β-sitosterol level in patients is approximately hundred
fold increased [259].
Refsum disease. In Refsum disease patients, phytanic acid α-oxidation can not
take place due to a defect in the enzyme phytanoyl-CoA hydroxylase (EC
1.14.11.18) [262]. Phytanic acid is totally exogenous in origin. It derives from the
bacterial metabolism of chlorophyll in ruminants. Phytanic acid (3,7,11,15tetramethylhexadecanoic acid, Figure 1) accumulates in blood and tissues of
affected patients [263]. Phytanic acid levels may reach values up to 1300 µmol/l
(normal <10 µmol/l) in plasma, where it is incorporated in triglycerides [264].
Refsum patients are generally treated with a low phytanic acid diet [264].
Furthermore, plasmapheresis treatment can be used to rapidly lower plasma
phytanic acid levels.
Figure 1
Structures of the metabolites accumulating in the
investigated inborn errors of lipid metabolism,
including relevant atom numbering. For the plant
sterols (g-i), only the side chain is shown. Their ring
structure is identical to cholesterol.
a cholesterol; b cholestanol; c 7DHC; d 8DHC; e 7lathosterol;
f
8-lathosterol;
g
β-sitosterol;
campesterol; i stigmasterol; j phytanic acid.
164
h
Inborn errors of lipid metabolism
Results
NORMAL LIPID RESONANCES
The one-dimensional 1H-NMR spectrum of the blood plasma lipid extract of a
healthy volunteer is shown in Figure 1. Resonance assignments were based on
previously reported spectra [245;251-253], one and two-dimensional spectra of
pure cholesterol, oleic acid, linoleic acid, palmitic acid and stearic acid dissolved in
deuterated chloroform and supporting computer simulations. In total, 25 lipid
related resonances could be assigned, leading to the identification of nine
molecular species, i.e. free cholesterol, esterified cholesterol, 7-lathosterol,
triglycerides, spingomyelin, choline, glycerophospholipid, phosphatidylcholine and
fatty acids (esterified plus non-esterified; Table 1).
Although the spectrum in Figure 2 shows many overlapping signals from fatty acyl
chains, several distinct groups can be identified, such as methyl, methylene, allylic
(=CHCH2–), olefinic (–CH=CH–) and diallylic (=CHCH2CH=) protons (Table 1). Since
these groups resonate at approximately the same frequency for every fatty acid, it
is impossible to differentiate between the different fatty acids present in the
sample. However, the use of methylene/diallylic and methylene/olefinic intensity
ratio as an estimate for the average fatty acid chain length and number of double
bonds, respectively, has been reported [251].
On the contrary, the C18H3 protons of cholesterol and its precursor 7-lathosterol
are nicely resolved (See Figure 1 for molecular structures and atom numbering)
and since the cholesterol C19 H3 group shows separate singlets for free and
esterified cholesterol, it is possible to determine their relative amounts
(approximately 60-70% of the total cholesterol is normally esterified). The C3
proton shows individual signals for esterified and nonesterified cholesterol as
well, but unfortunately, the latter partially overlaps with the methanol
contamination in Figure 2. Additionally, the C21 H3 and C26 H3/C27 H3 proton
resonances can be distinguished, although they moderately overlap with fatty acid
methyl groups.
Finally, cross peaks between the allylic, olefinic and diallylic protons can be readily
observed in a two-dimensional 1H-1H COSY spectrum of a healthy control, as well
as cross peaks between the different protons of the glycerol backbone (data not
shown). The crowded spectral region between 0.5 and 2.5 ppm could not be
completely assigned due to overlap problems and complex J-splitting of the high
number of cross peaks. However, changes in the observed cross peak pattern can
still contribute to the diagnosis of an inherited metabolic disease (see below).
165
Chapter 4.1
Table 1: 1H resonance assignments with chemical shifts, multiplicity, and J-coupling constants for
signals identified in the lipid extract of blood plasma taken from healthy controls (n = 3)a.
Peak
number
1
1H
shift (ppm)
0.53
2
0.68
3
0.86 / 0.87
5
7
4
6
Multiplicity b ; J (Hz)
Assignment
Total 7-lathosterol C18H3
s
Total cholesterol C18H3
s
Total cholesterol C26H3 / C27H3
2x d; 6.6
0.91
Total cholesterol C21H3
d; 6.5
1.02
Esterified cholesterol C19H3
0.88
1.01
Fatty acyl chain CH3(CH2)n
t; 6.9
Free cholesterol C19H3
s
s
c
8
1.05 – 1.19
Multiple cholesterol protons
m
9
1.24 – 1.37
Fatty acyl chain (CH2)n
m
10
1.42 – 1.55
Multiple cholesterol protons
m
11
1.55 – 1.65
Fatty acyl chain –CH2CH2CO
m
12
1.79 – 1.88
Multiple cholesterol protons
m
13
1.98 – 2.09
Fatty acyl chain –CH2CH=
m
15
16
2.77 – 2.87
3.32 / 3.35
Fatty acyl chain =CHCH2CH=
17
3.48 – 3.57
Free cholesterol C3H
19
3.96
14
18
20
21
22
2.24 – 2.35
3.81
4.15 / 4.29
4.32 – 4.43
4.57 – 4.65
23
5.17 – 5.24
20
5.26
21
5.29 – 5.43
Fatty acyl chain –CH2CO
m
Sphingomyelin and choline
m
N(CH3)3 d
2x s (largely overlapping)
m
Phosphatidylcholine N-CH2 d
Glycerophospholipid backbone C3H2
Glycerol backbone C1H2 / C3H2 c
Phosphatidylcholine
PO-CH2 d
Esterified cholesterol C3H
c
Glycerophospholipid backbone C2H d
Glycerol backbone C2H
c
Fatty acyl chain –HC=CH–
s (broad)
d
s (broad)
m (AB spin system)
m (broad)
m
m (broad)
p; 5.7
m
a; Peak numbers correspond with Fig. 1. Chemical shifts are referenced to OMS (δ = 0.094 ppm). All assignments were based on
spectra of authentic standards, on the literature, or on computer simulations, unless specified differently.
b; s, singlet; d, doublet; t, triplet; p, pentet; m, multiplet.
c; Assignment based on the literature and spectral simulation.
d; Assignment based solely on the literature.
166
tube-cleaning procedures, respectively. The peak labeled
13C
corresponds to the
13C
satellite of peak 2. The chemical shift reference compound OMS resonates at 0.094 ppm.
The inset shows an expanded scale for the 0.52–0.58 ppm region. Peak assignment is provided in Table 1. Methanol (MeOH) and ethanol (EtOH) contamination resulted from extraction and
1-Dimensional 1H-NMR spectrum of the blood plasma lipid extract of a healthy female volunteer.
Figure 2
Chapter 4.1
CORRELATION STUDY
Cholesterol and triglyceride concentrations were measured in 15 and 12 blood
plasma samples, respectively, using conventional enzymatic analysis and 1H-NMR
spectroscopy. In the latter method, peaks 2 and 20 of Figure 2 were used for
cholesterol and triglyceride quantification, respectively. The obtained results were
compared using Passing and Bablok regression analysis [257], which showed a
good correlation for both (Figure 3).
Figure 3
Comparison
concentrations
conventional
of
a
in
cholesterol
blood
enzymatic
and
plasma
analysis
b
triglyceride
determined
and
by
1H-NMR
spectroscopy. Solid lines correspond to the regression
lines calculated with Passing and Bablok regression, while
dashed lines indicate the 95% confidence intervals.
For cholesterol, the regression line had a slope of 0.90 (95% confidence interval
0.83 – 1.07) and an intercept of 0.15 (-0.66 – 0.48) mmol/l, while for the
triglycerides a slope of 1.02 (0.88 – 1.13) and an intercept of -0.04 (-0.31 – 0.10)
mmol/l were obtained. No significant deviation from linearity (P>0.10) was found
for both metabolites. However, it is clear that there is a slightly increasing
deviation with increasing cholesterol concentration (Figure 3). This is likely due to
a less efficient extraction at concentrations exceeding ~6 mmol/l and may be
overcome by using a larger volume of 2:1 (v/v) chloroform-methanol. Another
explanation might be the effect of a partial saturation of OMS. As the recycle time
is not 5 times the T1-relaxtion time, OMS will be partially saturated. However, the
methyl singlets of cholesterol and sterols have a similar T1-relaxtion as OMS
(around 3 s). Therefore we estimate that the error made by partial saturation
cannot be very significant.
168
Inborn errors of lipid metabolism
The within run coefficient of variation was determined using 9 samples, which
were prepared from the same blood serum containing 7.6 mmol/l cholesterol and
1.4 mmol/l triglyceride (values determined by NMR). For cholesterol and
triglyceride concentrations, a within run coefficient of variation of 7.9% and 6.8%
was found, respectively.
The detection limit may vary for different metabolites, since it is dependent on the
number of equivalent protons contributing to the NMR signal, the peak splitting
pattern, the number of scans and the field strength of the spectrometer. The
detection limit for the C18 H3 singlet of cholesterol at 0.68 ppm is estimated to be
approximately 10 µmol/l, assuming that the peak can be distinguished when the
signal to noise ratio is ≥3.
INBORN ERRORS OF METABOLISM
To assess the diagnostic ability of 1H-NMR spectroscopy of lipid extracts, several
samples from patients with a known inborn error of lipid metabolism were
investigated. Three diseases involved errors in sterol metabolism, while one
disease is caused by a defect in the breakdown of an unusual branched chain fatty
acid. Table 2 gives characteristic resonance frequencies of different metabolites.
Table 2: Characteristic resonance assignments, including multiplicity and J-coupling constants (Hz)
used for identification of metabolites accumulating in several inborn errors of lipid metabolism1.
Metabolite
Assignment
1H
shift
(ppm)
Multiplicity2
(J-coupling)
Inborn error
7DHC
C-18 H3
0.618
s
SLOS
Cholestanol
C-18 H3
0.645
s
CTX
7-Lathosterol
C-18 H3
0.534
s
CTX, lathosterolosis4
8DHC3
C-18 H3
C-19 H3
8-Lathosterol3
ß -Sitosterol
s
0.817
C-19 H3
0.810
C-18 H3
0.680
C-18 H3
s
s
0.606
s
C-26 H3/C-27 H3/C-29 H3
0.800–0.850
m
C-24 H
1.685
Phytanic acid (esterified to
glycerol)
C-16 H3/C-17 H3
C-18 H3/C-19 H3
C-20 H3
1
0.651
m
0.844
d (6.6)
0.867
0.9273,
s
SLOS
CTX
Sitosterolemia
Refsum disease
d (6.6)
5
d (5.6)
Chemical shifts are referenced to OMS (δ = 0.094 ppm). Values were determined in patients and confirmed by use of authentic
standards, unless stated otherwise.
2
3
4
5
s, singlet; d, doublet; m, multiplet.
Tentative assignment; no authentic standard available.
No patient material available.
Resonates at higher ppm value in pure compound (see text).
169
Chapter 4.1
Smith-Lemli-Opitz syndrome (SLOS; OMIM 270400)
Figure 4b shows the 0.55 – 0.75 ppm region of the 1D 1H-NMR spectrum of the
blood plasma lipid extract of a 5 months old boy suffering from SLOS. It clearly
differs from the spectrum of a healthy volunteer (Figure 4a) and shows the
diagnostic metabolites 7DHC and 8DHC. The characteristic C18 H3 resonances
used for identification and quantification of cholesterol, 7DHC and 8DHC are
nicely resolved. The presence of 7DHC was confirmed by addition of the pure
compound to the sample and remeasuring the NMR spectrum. An addition
experiment could not be performed for 8DHC because this compound is not
commercially available. More certain identification of both metabolites can be
achieved when additional signals of these compounds are resolved. Unfortunately,
the current experimental conditions lack the required higher resolution.
Samples from two other SLOS patients showed a similar NMR spectrum. Ruan et al.
[248] and Xiong et al. [249], assigned 7DHC and 8DHC unequivocally in an 1HNMR spectrum. Our study for the first time quantifies both metabolites. The
quantitative data for 7DHC and 8DHC show discrepancies between NMR and GC
(Table 3). These may relate to the long time interval between GC and NMR
measurements (in some cases several years). It is known that the unsaturated
sterols decompose easily during storage, processing and/or analysis.
Accumulation of cholesta-5,7,9(11)-triene-3β-ol in the blood of SLOS patients has
been reported [248]. The C18 H3 protons of this compound has been reported to
resonate at 0.566 ppm [245]. However, we observed no resonance at this position
in the NMR spectra of SLOS patients. The concentration of this compound varies
considerably and may be in the low micromolar range in SLOS patients. Ruan et al.
reported concentrations from 0.8 to 79.4 µmol/l [248]. This may explain why we
were unable to detect this compound in our patient samples.
170
Inborn errors of lipid metabolism
Figure 4
Upfield regions of the 1-dimensional 1H-NMR spectra of patient plasma lipid extracts, plasma from a healthy control, and the
authentic ß-sitosterol and phytanic acid standards.
Methyl assignment for both authentic standards are shown (see Table 2 atom numbering). (A), control; (B), SLOS; (C), CTX; (D),
control; (E), sitosterolemia; (F), ß-sitosterol; (G), Refsum disease; (H), phytanic acid. Peaks in panels A–C correspond to the C-
18 H3 resonance of the indicated metabolite. The peak labeled
13C
in panel C represents the cholesterol C-18 H3
13C
satellite,
whereas the peak with unknown assignment is labeled with an X. The peak numbering in panel D is identical to the numbering
in Fig. 1 . Peaks labeled with an * in panels E and G correspond to ß-sitosterol (sito) and phytanic acid signals, respectively. The
C-20 H3 resonance of esterified phytanic acid is indicated by # in panel G. Chol (panel E), cholesterol.
Cerebrotendinous xanthomatosis (CTX; OMIM 213700)
The 1D lipid 1H-NMR spectrum of a 24-year-old male CTX patient is given in
Figure 4c. It shows an abnormally high concentration of the diagnostic metabolite
cholestanol (C18 H3 resonance at 0.645 ppm, close to the C18 H3 of cholesterol).
The NMR method revealed a cholestanol concentration of 0.15 mmol/l, which is
somewhat higher than the value determined by GC (0.11 mmol/l, Table 3). This
may be due to slight overlap with the tail of the cholesterol resonance.
Furthermore, the spectrum shows three other peaks: the 13C satellite of the
cholesterol C18 H3, the C18 H3 resonance of 7-lathosterol and one unknown
171
Chapter 4.1
metabolite (peak X). Although 7-lathosterol is also observed in healthy controls
(Figure 2), elevated levels were found in all investigated CTX patients (Table 3),
which corresponds with reference values determined by Wolthers et al. [265].
Interestingly, peak X at 0.61 ppm does not occur in control spectra, but was
present in the two other CTX patients as well (both female and before start of
therapy; 16 and 46 years old), although it was much weaker in the 16 year old
female. GC and GC-MS measurements also displayed an unknown peak in CTX
patients, which was tentatively identified as 8-lathosterol (Figure 2). Based on this
finding and substantial supporting evidence, peak X is tentatively assigned as the
C18 H3 resonance of 8-lathosterol. First, the relative peak area compared to
cholestanol was very similar in the gas chromatogram and the 1D 1H-NMR
spectrum of the same patient. Second, the lathosterol isomers show the same
structural difference as 7DHC compared to 8DHC, i.e. the position of the double
bond (Figure 2), and the frequency of peak X differs from 7-lathosterol in the
same way as 8DHC from 7DHC (cf. Figure 4b). Although the chemical shift
difference between the 7-lathosterol and 8-lathosterol and the chemical shift
difference between 7DHC and 8DHC is not identical (0.073 and 0.031 ppm
respectively), this does provide some tentative evidence. Third, our assignment is
strongly supported by corresponding chemical shift values found by Wilson et al.
for 7- and 8-lathosterol [245]. Finally, results described by Wolthers et al. confirm
the presence of 8-lathosterol in plasma of CTX patients [265] and the reported
concentration (60.0 µmol/l; n=1) corresponds very well with the average
concentration of 66.8 µmol/l determined by 1H-NMR (Table 3). Unfortunately, 8lathosterol is not commercially available for absolute confirmation of the
assignment. Furthermore, the current resolution does not allow identification of
additional 8-lathosterol signals.
Sitosterolemia (OMIM 210250)
Figure 4e shows the upfield region of the one-dimensional 1H-NMR spectrum of
the lipid extract of a sitosterolemic patient. For comparison, the same spectral
region is also shown for a healthy control and pure β-sitosterol in Figures 4d and
f, respectively. In the analysis, only β-sitosterol was taken into account, since this
is the major plant sterol, constituting about 65% of all absorbed plant sterols
[259]. Campesterol and stigmasterol contribute approximately 32% and 3%,
respectively. Unfortunately, β-sitosterol quantification was impossible under the
current experimental conditions, since the C18 H3 and C19 H3 resonances of β sitosterol and cholesterol overlap almost completely (Figure 4e) and other βsitosterol resonances were not resolved into individual doublets and triplets to
allow accurate quantification. Although the assignment of the C26 H3, C27 H3 and
C29 H3 resonances (0.80 – 0.85 ppm) is therefore not exactly known, their
presence in the sitosterolemia patient and absence in the healthy control is clear.
Peak positions in the authentic standard corresponded exactly with the observed
positions in the patient and repeated NMR measurement after addition of pure β -
172
Inborn errors of lipid metabolism
sitosterol confirmed the assignment. The weak resonances at approximately 0.75
to 0.80 ppm (at right of the β-sitosterol signals) likely result from campesterol and
dihydrobrassicasterol (24S stereo-isomer of campesterol), since corresponding
peak positions were found in the authentic standard of campesterol (data not
shown). Overlap with the campesterol signals provides an additional difficulty in
quantifying β-sitosterol. Therefore, higher resolution and precise knowledge of all
assignments is necessary to allow quantification of both β-sitosterol and
campesterol.
To support the diagnosis, a two-dimensional 1H-1H COSY spectrum can be
recorded. The resulting spectrum shows a characteristic cross peak at (1.68; 0.82)
ppm, originating from the long-range spin-spin coupling between C24 H and C26
H3, C27 H3 or C29 H3 of β-sitosterol (data not shown).
Thus, β-sitosterol presence can be measured using our NMR method, although
quantification is still impossible.
Figure 5
Effect of plasmapheresis treatment on plasma
phytanic acid concentrations in a female Refsum
disease patient.
The duration of the plasmapheresis is indicated in
gray.
Refsum disease (OMIM 266500)
Figure 4g shows the 0.60 – 1.05 ppm region of the 1D 1H lipid NMR spectrum of a
29 year old female Refsum disease patient. Assignment of the methyl resonances
is given in the spectrum of pure phytanic acid (Figure 4h). The doublet at
0.84/0.85 ppm provides clear evidence for the presence of phytanic acid in the
Refsum sample (cf. Figure 4g). Remarkably, the C20 H3 protons, which resonate as
a doublet at 0.97/0.98 ppm in pure phytanic acid, appear absent in the patient
spectrum. However, due to esterification with glycerol [264], this peak has
probably shifted to 0.92/0.93 ppm, where a clear difference is seen between the
Refsum case and a healthy control. After addition of pure phytanic acid to the
patient sample, the doublet at 0.97/0.98 ppm was evident, confirming that
phytanic acid is not present in its free form in Refsum disease patients. Additional
computer simulations confirmed a shift towards lower ppm values upon
esterification with glycerol.
173
Chapter 4.1
Phytanic acid quantification by 1H-NMR (using half of the C16 H3/C17 H3 doublet
at 0.84 ppm) showed good correspondence with values determined by GC (Table
3). The effect of plasmapheresis on the phytanic acid concentration was followed
using NMR analysis. A rapid decrease in phytanic acid concentration of 50% was
observed. A return to its original value was found three days after the treatment
(Figure 5).
Table 3: Comparison of plasma lipid concentrations by 1H-NMR and gas chromatography for the
Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Refsum disease. All values are
reported in µmol/l, except for cholesterol, which is given in mmol/l. Reference values (Ref) were
taken from Blau et al. [140] , and for 7-lathosterol from Wolthers et al. [265] . No reference value
was available for 8-lathosterol.
Smith-Lemli-Opitz syndrome
7DHC
Cholesterol
NMR
GC
Ref
NMR
8DHC
GC
Ref
NMR
GC
Ref
1 (M, 5 m)
2.12
2.05
2.96-4.43
332
334
<0.02-0.57
363
225
<0.02
2 (M, 8 m)
0.39
0.26
2.96-4.43
153
353
<0.02-0.57
143
385
<0.02
3 (M, 7 y)
1.25
1.10
2.89-4.74
200
325
0.03-0.52
232
295
<0.02
Cerebrotendinous xanthomatosis
Cholesterol
Cholestanol
7-lathosterol
NMR
GC
Ref
NMR
GC
Ref
NMR
GC
1 (M, 24 y)
3.10
3.00
2.66-6.02
152
106
4-18
34
ND
2 (F, 16 y)
4.72
4.20
2.66-6.02
138
101
4-18
11
3 (F, 46 y)
4.30
3.90
2.66-6.02
127
125
4-18
39
Ref
NMR
8-lathosterol
Ref
NMR
GC
<10.6
75
ND
ND
<10.6
7
ND
ND
<10.6
118
ND
a
b
Refsum disease
Cholesterol
1 (F, 29 y)
NMR
1.90
GC
ND
Phytanic acid
2.66-6.02
GC
Ref
700
690
<10
2 (F, 29 y)
504
454
<10
3 (F, 29 y)
354
278
<10
4 (F, 29 y)
546
528
<10
5 (F, 29 y)
700
588
<10
6 (F, 29 y)
722
658
<10
ND: not determined
b: tentative assignment
a
Discussion
Many inherited metabolic diseases are caused by defects in lipid metabolism or
biosynthesis, often resulting in accumulation of unusual lipids in the blood and
tissues of affected patients. In this study, the successful use of 1H-NMR
spectroscopy of blood plasma lipid extracts to accurately identify and quantify
these lipids was demonstrated for four different inborn errors of lipid metabolism.
174
Inborn errors of lipid metabolism
Twenty-five lipid derived resonances could be assigned in the 1D spectra of
healthy controls, resulting in the identification of nine distinct molecular species.
This number increases to fourteen when also taking the investigated metabolic
diseases into account.
Although 1H-NMR spectroscopy of lipid extracts was used previously by Kriat et al.
[251] to study tumor induced effects, its application in clinical diagnosis has
hitherto been only described for the identification of unusual metabolites in the
blood plasma of Smith-Lemli-Opitz syndrome patients [248;249]. By using OMS
instead of the highly volatile TMS as a concentration and chemical shift reference,
we are for the first time able to accurately quantify metabolites in chloroform
solutions by proton NMR. A good correlation with conventional methods was
obtained for total cholesterol and triglyceride concentrations. This does not
exclude a possible less efficient extraction and hence worse correlation for other
metabolites. Based on the results obtained for cholesterol, a larger extraction
volume has to be used when high lipid concentrations are expected. Nevertheless,
the currently applied protocol will likely be sufficient for the quantification of
unusual lipids since their concentrations are normally low (<1 mmol/l) and
deviations are only seen at high concentrations (>6 mmol/l for cholesterol).
Since lipid 1H-NMR spectroscopy requires approximately one day of sample
preparation and only a limited number of samples can be worked up, it can not
compete with the automated enzymatic analyses of cholesterol and triglycerides in
blood. Automated extraction procedure may be a further solution. However, it
does provide an excellent alternative for the identification and quantification of
unusual lipids. For instance, cholestanol and cholesterol separation can be tedious
using conventional chromatographic methods, whereas both metabolites are
readily identified with 1H-NMR spectroscopy based on their different C18 H3
resonance. Similarly, the presence of 7DHC and 8DHC in the plasma of SLOS
patients is immediately evident from a simple 1D 1H-NMR spectrum.
Quantification of 8DHC may be more accurate using 1H-NMR, since
chromatographic measurements can not be calibrated correctly as the authentic
standard is not commercially available.
The diagnosis of SLOS, CTX and sitosterolemia can be made with almost a 100%
certainty based on NMR analysis. However, for Refsum disease, the total clinical
picture is required, since phytanic acid accumulation is also observed in the
Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and
rhizomelic chondrodysplasia punctata type 1 [264]. The investigation of these
diseases using 1H lipid NMR spectroscopy is a topic for future research.
A database containing the resonances of many more authentic standards of
molecules relevant in lipid metabolism has to be established, which will be helpful
to assign unknown signals. For NMR spectra of urine samples, an analogous
strategy has led to the identification of new inborn errors of metabolism
[33;36;42] and it is not unlikely that lipid NMR spectroscopy can also lead to the
discovery of currently unknown inherited disorders.
175
Chapter 4.1
Besides resonance positions, which can only provide clear information when there
is no overlap, peak ratios can be used to obtain additional important information.
For example, the CH2:CH3 ratio (peaks 9 and 4 in Figure 2, respectively) can
provide evidence on the presence of branched chain fatty acids since this will
lower this ratio. For healthy individuals (n=3), we found a CH2:CH3 peak ratio of
6.61 ± 0.07 (note that the CH3 integral has to be corrected for overlap with
cholesterol resonances). This corresponds very well with a ratio of 6.67 calculated
for a theoretical 1:1:1 mixture of palmitic, oleic and linoleic acid, which together
constitute approximately 75% of all plasma fatty acids. As expected, a much lower
ratio of 3.87 was found for our Refsum disease patient, which is fully explained by
the branching of the phytanic acid and its concentration compared to other fatty
acids in the sample. Although the presence of phytanic acid can be more easily
ascertained based on detection of the C16 H3/C17 H3 resonance (Figure 1), it is
not unlikely that peak ratios can be helpful to detect abnormalities in spectra
without any clear unusual signals.
In conclusion, we have presented a new method to identify (un)usual lipids in
blood plasma or serum using 1H-NMR spectroscopy. Furthermore, metabolite
concentrations can be accurately determined by using the non-volatile OMS as a
chemical shift and concentration reference compound. The technique is well suited
for the diagnosis and follow up of inborn errors of lipid metabolism, which was
demonstrated for four different inherited diseases.
Acknowledgements
All 1H-NMR spectra were recorded at the Dutch hf-NMR facility at the Department
of Biophysical Chemistry, Radboud University Nijmegen, The Netherlands (head
Prof. Dr. S.S. Wijmenga). We thank Jos Joordens for his technical assistance and
Fokje Zijlstra and Arno van Rooy for sterol analysis with conventional GC and GCMS techniques. We also thank the department of clinical chemistry (head Prof. Dr.
J.L. Willems) for sample selection and for the enzymatic determination of
cholesterol and triglyceride concentrations. Finally, we thank Prof. Dr. A.F.H.
Stalenhoef, Dr. E. Morava, Dr. G. Drost, Dr. A. Verrips, Dr. P. Jira and Dr. L.J.H. van
Tits for referring relevant patient samples.
176
FIVE
SUMMARY AND FUTURE PERSPECTIVES
177
178
CHAPTER
5.1
Summary
179
180
Summary
Summary
This thesis describes the use of proton nuclear magnetic resonance (1H-NMR)
spectroscopy as a tool for diagnosing inborn errors of metabolism in body fluids.
The technique yields an overview of proton-containing metabolites, is rapid and
nondestructive, and requires minimal sample pretreatment. Moreover, structural
information from metabolites can be obtained from NMR spectra. Our studies
demonstrate that NMR spectroscopy of body fluids may be considered as an
alternative analytical approach for diagnosing known, but also as yet unknown,
inborn errors of metabolism. In this thesis we describe the NMR spectroscopy
contributions to the unraveling of two novel inborn errors of metabolism.
Chapter 1
The aim of our study is described in chapter 1.1 and current knowledge about
NMR spectroscopy in the field of inborn errors of metabolism is reviewed in
chapter 1.3.
Chapter 2
The second chapter describes the NMR findings for a group of compounds that
normally will not be detected in a metabolic laboratory. These compounds contain
an N-acetyl group (Figure 1).
Figure 1
General
structure
of
an
N-acetylated
compound. The three equivalent protons
from thr N-acetyl group resonate as a singlet
in a small part of the 1H-NMR spectrum.
In chapter 2.1 13 inborn errors of metabolism that involve N-acetylated
compounds are studied using 1H-NMR spectroscopy. Urine samples were
measured from patients suffering from Salla disease, French-type sialuria, αmannosidosis, β-mannosidosis, fucosidosis, aspartylglucosaminuria, GM1gangliosidosis, GM2-gangliosidosis, sialidosis, Canavan disease, citrullinemia,
tyrosinemia type I and tyrosinemia type II. Diagnostic important N-acetylated
metabolites could be detected in the urine 1D and 2D NMR spectra of patients of
almost all inborn errors that were measured. No abnormalities were observed in
the spectra of urine from patients with GM1- or GM2-gangliosidosis. Chapters 2.2
and 2.3 demonstrate the diagnostic importance of N-acetylated metabolites. In
181
Chapter 5.1
Chapter 2.2, in vivo and in vitro NMR spectroscopic investigations are described in
two patients. The patients are two unrelated girls with almost absent CNS myelin,
intractable epilepsy, nystagmus, progressive microcephaly, and severe
psychomotor retardation. Moreover, in the 1H-NMR spectra of cerebrospinal fluid
of the patients, high concentrations of N-acetylaspartylglutamate (NAAG) could be
observed. Elevated NAAG concentration in CSF may be the biochemical hallmark
for a novel neurometabolic disorder and may provide a key for understanding the
molecular defect of this disease.
Chapter 2.3 describes the in vitro NMR
spectroscopic investigations at the metabolite level on eight unrelated patients
with aminoacylase 1 deficiency; a novel disease whereby NMR spectroscopy was
involved in the elucidation of the molecular defect. The 1D 1H-NMR urine spectra
from these patients show a characteristic profile, mainly due to the N-acetyl
singlets deriving from N-acetylated amino acids. Using a combination of several
1D and 2D NMR experiments, resonances of eleven N-acetylated amino acids
could be assigned and quantified.
Chapter 3
In this chapter we used both in vitro and in vitro NMR spectroscopic techniques to
identify unusually high resonances in the brain and body fluids. In chapter 3.1,
several NMR experiments are described to identify an unknown peak in NMR
spectra of human cerebrospinal fluid and blood plasma. The peak was assigned to
dimethyl sulfone (DMSO2). In plasma and CSF from healthy controls, the
concentration of DMSO2 ranged between 0 and 25 µmol/l. For the first time we
desribe the presence of DMSO2 human CSF. Furthermore, we document an
increased concentration of DMSO2 in plasma from three out of four patients with
methionine adenosyltransferase I/III (MAT I/III) deficiency.
Medication, food or ‘innocent’ supplements can cause unusual resonances in NMR
spectra that may be misinterpreted as diagnostic resonances. Chapter 3.2 reports
the detection of DMSO2 (also known as methylsulfonylmethane (MSM) or
sulfonylbismethane) in the brain and CSF of a 4-year-old girl using in vivo and in
vitro NMR spectroscopy. The presence of this exogenous metabolite resulted from
ingestion of a dietary supplement containing DMSO2. The concentration of this
compound amounted to 1.2 mmol/l in brain tissue and 1.7 mmol/l in cerebrospinal fluid.
Chapter 3.3 describes in vivo and in vitro NMR spectroscopic investigations on a
61-year-old woman with a 3-methylglutaconyl-CoA hydratase (3MGH) deficiency
or 3-methylglutaconic aciduria type I. MR imaging of her brain revealed a
leukoencephalopathy.
Increased concentrations of 3-hydroxyisovaleric acid (3HIVA), 3-methylglutaconic
acid (3MGA) (cis and trans) and 3-methylglutaric acid (3MG) were observed in the
NMR spectra of the patient’s urine. In the CSF, the 3HIVA concentration was 10
times higher than in the plasma of the patient and only the cis isomer of 3MGA
182
Summary
was observed. In vivo brain MRSI showed an abnormal resonance at 1.28 ppm that
may be caused by 3HIVA. The brain 3HIVA was calculated to be 0.9 mmol/l, about
five times the concentration in CSF. Furthermore, four other diseases involving
leucine metabolism are described. Urine samples were measured from patients
suffering from maple syrup urine disease, isovaleric academia, 3-methylcrotonyl
CoA carboxylase deficiency and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.
Diagnostic metabolites could be detected in the urine NMR spectra of all patients
of these inborn errors of metabolism. Chapter 3.4 is a case report on the 61-yearold woman, described in chapter 3.3. This case demonstrates four features of 3methylglutaconic aciduria type I. The patient is unique in terms of age of
presentation and perhaps of onset of her disease; 3-methylglutaconic aciduria
type I may present as a leukoencephalopathy; in vivo MR spectroscopy may
demonstrate increased brain tissue levels of 3HIVA. Furthermore we describe two
novel apparently mild mutations.
Chapter 4
Chapter 4 investigates the potential use of 1H-NMR spectroscopy to identify and
quantify lipids present in the blood of patients with different inborn errors of lipid
metabolism. We extracted blood plasma or serum lipids in chloroform–methanol
(2:1 by volume). After addition of the nonvolatile chemical shift and concentration
reference compound octamethyl-cyclotetrasiloxane (OMS), we performed 1H-NMR
measurements. Blood plasma or serum lipid extracts were measured from controls
and patients suffering from Smith–Lemli–Opitz syndrome, cerebrotendinous
xanthomatosis, sitosterolemia, and Refsum disease. NMR analysis led to a correct
diagnosis for all 4 diseases, whereas the concentration of the diagnostic
metabolite could be determined for 3. Simultaneous lipid quantification was
possible for the first time.
183
Chapter 5.1
Samenvatting
De Nederlandse titel van mijn proefschrift is: NMR-spectroscopie van lichaams-
vloeistoffen; een metabolomics benadering van erfelijke stofwisselingsziekten. In
deze titel staan een aantal wetenschappelijke termen die voor de niet-
wetenschapper mogelijk moeilijk te begrijpen zijn. In onderstaande tekst zal ik de
titel en mijn onderzoek nader toelichten.
Er bestaan meer dan vijfhonderd verschillende erfelijke stofwisselingsziekten die
allemaal moeilijke namen hebben. Op pagina 44 in tabel 2 van dit proefschrift
staan enkele voorbeelden van deze ziekten. Elke stofwisselingsziekte afzonderlijk
is zeldzaam; gezamenlijk vormen ze een klinisch belangrijke categorie ziekten.
Stofwisselingsziekten komen voor in alle leeftijdsgroepen, maar het meest bij
kinderen. Vanaf 1 januari 2007 wordt het bloed van pasgeboren baby´s in
Nederland onderzocht op de aanwezigheid van veertien stofwisselingsziekten (de
hielprik). Vroege opsporing en behandeling van deze ziekten kan blijvende schade
voorkomen.
Wat is een stofwisselingsziekte?
Bij patiënten met een erfelijke stofwisselingsziekte is de aanmaak of verwerking
van één of meerdere stoffen (metabolieten) verstoord. Meestal wordt dit
veroorzaakt doordat één onderdeeltje van alle cellen, een enzym, niet (goed)
werkt. Het gevolg van die verstoring is dat er ergens in het lichaam een teveel of
tekort aan een bepaalde metaboliet ontstaat. Ook in de lichaamsvloeistoffen
(urine, bloed of hersenvloeistof) van deze patiënten kan een te laag of juist te
hoog gehalte (concentratie) van een bepaalde metaboliet aanwezig zijn.
Hoe kunnen we deze ziekten opsporen?
Bij een afwijkende metabolietenconcentratie in een lichaamsvloeistof kan er dus
sprake zijn van een stofwisselingsziekte. Het kan daarbij om te hoge of te lage
concentraties van een bepaalde metaboliet gaan. Metabolomics is het opsporen en
bepalen
van
concentraties
van
alle
metabolieten
in
een
cel
of
een
lichaamsvloeistof. Een ziekenhuislaboratorium dat gespecialiseerd is in het
diagnosticeren (opsporen) van erfelijke stofwisselingsziekten gebruikt verschillende technieken voor het routinematig bepalen van metabolieten in lichaamsvloeistoffen. Metabolieten kan men onderverdelen in groepen op basis van hun
chemische eigenschappen. Zo zijn er bijvoorbeeld suikers, aminozuren of
organische zuren. Voor de bepaling van de diverse groepen metabolieten heb je in
een laboratorium verschillende technieken nodig.
184
Samenvatting
In dit proefschrift gebruik ik een nieuwe analysetechniek die in tegenstelling tot de
andere technieken vrijwel alle metabolieten tegelijk kan aantonen. Deze techniek
noemen we NMR-spectroscopie.
NMR-spectroscopie van lichaamsvloeistoffen
Metabolieten hebben met elkaar gemeen dat ze allemaal waterstofatomen
bevatten. In de scheikunde gebruiken we hiervoor de afkorting 1H. Met een sterke
magneet (Figuur 1, bladzijde 19) en radiogolven kunnen we deze waterstofatomen
zichtbaar maken; we noemen dit een 1H-NMR-experiment. Een urine-1H-NMRexperiment duurt ongeveer dertig minuten. Het resultaat van een
1H-NMR-
experiment zijn patronen van pieken; dit noemen we een 1H-NMR-spectrum. Een
1H-NMR-spectrum
van een urine van een gezonde persoon bestaat uit meer dan
driehonderd pieken; elke piek wordt veroorzaakt door één bepaalde metaboliet
(Figuur 4, bladzijde 41). Urine-1H-NMR-spectra van patiënten met een erfelijke
stofwisselingsziekte tonen vaak een afwijkend piekenpatroon. Het kan daarbij
gaan om de aanwezigheid van abnormaal hoge concentratie van een bepaalde stof
of juist de afwezigheid van een bepaalde stof. Met een 1H-NMR-spectrum kunnen
we dus iets zeggen over de mate van aan- of afwezigheid van bepaalde
metabolieten in een lichaamsvloeistof. Deze informatie gebruiken we voor het
opsporen van een stofwisselingsziekte.
Wat symboliseert de omslag van dit boek?
Op de omslag van mijn proefschrift staan ongeveer tweehonderd metabolieten die
voorkomen in de mens. Al deze metabolieten kunnen we met
spectroscopie eenvoudig meten. In
1H-NMR-spectra
1H-NMR-
van patiënten met een
stofwisselingsziekte zijn soms heel hoge concentraties van een bepaalde
metaboliet te meten (een hoge piek in het NMR-spectrum). Soms is de hoeveelheid
van een bepaalde metaboliet nauwelijks zichtbaar (een kleine piek in het NMRspectrum). In hoeverre we de concentratie van een bepaalde metaboliet kunnen
vaststellen, verschilt dus per stofwisselingsziekte.
Het proefschrift
In dit proefschrift heb ik de mogelijkheden van NMR-spectroscopie beschreven om
in lichaamsvloeistoffen erfelijke stofwisselingsziekten op te sporen.
In hoofdstuk 2 heb ik één groep metabolieten onderzocht die binnen een normaal
ziekenhuislaboratorium niet worden geanalyseerd (N-acetylmetabolieten). In dit
hoofdstuk
beschrijf
ik
de
NMR-resultaten
van
dertien
bekende
erfelijke
stofwisselingsziekten (hoofdstuk 2.1) en twee ziekten die nog niet eerder
beschreven zijn (hoofdstuk 2.2 en 2.3). In de urine- of hersenvocht-1H-NMRspectra van deze patiënten werden steeds afwijkende pieken gevonden die
185
Chapter 5.1
veroorzaakt werden door één of meerdere N-acetylmetabolieten. Aan de hand van
deze metabolieten konden we de betreffende ziekten vaststellen.
Met NMR is het ook mogelijk om metabolieten te meten binnen organen van het
menselijke lichaam. Bij deze experimenten wordt de patiënt in een grote speciale
magneet geschoven. Met de magneet en radiogolven wordt eerst een beeld
gemaakt van bijvoorbeeld de hersenen. Daar kunnen we van een klein volume een
1H-NMR-spectrum
hoofdstuk
3
opnemen. We noemen dit in vivo MR-spectroscopie (MRS). In
gebruiken
we
deze
techniek
in
combinatie
met
1H-NMR-
spectroscopie van lichaamsvloeistoffen om ongewone pieken te identificeren.
Hoofdstuk 3.1 en 3.2 beschrijft de ontdekking van de stof dimethyl sulfone in
hersenvocht, bloed en de hersenen. Hoofdstuk 3.3 en 3.4 gaat over een patient
met
een
zeldzame
stofwisselingsziekte.
In
de
1H-NMR-spectra
van
de
lichaamsvloeistoffen en de hersenen vonden we ongewone pieken. Deze konden
we allemaal identificeren met NMR.
Hoofdstuk 4 staat in het teken van lipiden. Zij vormen een speciale groep
metabolieten. Lipiden zijn vetten en vetachtige stoffen die in de stofwisseling van
de mens een belangrijke rol spelen. Enkele voorbeelden hiervan zijn cholesterol,
fosfolipiden en triglyceriden. Het is bekend dat een aantal lipiden een rol spelen in
erfelijke stofwisselingsziekten. In dit hoofdstuk hebben we de mogelijkheden van
NMR-spectroscopie onderzocht om lipiden te meten in bloedplasma. Door
bloedplasma op een speciale manier te behandelen konden we de lipiden
zichtbaar maken in een 1H-NMR-spectrum. Bloed van gezonde vrijwilligers hebben
we vergeleken met patiënten. Vier verschillende erfelijke stofwisselingsziekten
werden onderzocht. Bekend was dat bij deze ziekten lipiden betrokken waren.
NMR-spectroscopie gaf een correcte diagnose voor de vier ziekten. Dit is de eerste
studie die laat zien dat het mogelijk is met NMR-spectroscopie diverse lipiden aan
te tonen en te kwantificeren in biologische monsters.
186
CHAPTER
5.2
Future perspectives
187
Chapter 5.2
188
Future perspectives
Future perspectives
Although many inborn errors of metabolism can be readily diagnosed with NMR,
the technique is not yet routinely available in the majority of hospital laboratories.
This is likely due to the high cost of the equipment and the fact that spectrum
interpretation is usually not straightforward, requiring highly trained personnel.
Several ongoing developments, which may make the application of body fluid NMR
in diagnosing inborn errors of metabolism more successful and hopefully also
more accessible, will be discussed in this chapter. The first three paragraphs will
deal with advances made in NMR hardware technology, while the final two
paragraphs will cover some of the progress in spectral analysis software and pulse
sequence design.
DETECTION OF OTHER NUCLEI
Protons are highly abundant in many metabolites and have a high sensitivity,
making them the most widely used nuclei in the study of inborn errors of
metabolism. However, several other NMR active nuclei, which can provide
important additional information, are present as well. Although 19F and 31P have a
favorable natural abundance and relative sensitivity, there are few phosphorous or
fluorine-containing metabolites present in body fluids. On the contrary, 13C and
15N are present in many metabolites, but suffer from their low inherent sensitivity
and natural abundance. These disadvantages may be overcome with the use of
cryogenic probe technology, which was first presented by Styles et al. in 1984
[266]. By cooling the receiver coil and preamplifier to approximately 20 K with
liquid helium, the resistance of the coil is significantly reduced. Consequently, the
thermal noise is decreased by approximately a factor 4. This leads to a
corresponding increase in the signal-to-noise ratio (S/N) per scan or, for the same
S/N, a 16-fold reduction in acquisition time when compared to a normal probe.
The improved sensitivity is such that 13C spectra with good S/N can be obtained
for body fluid samples with acceptable acquisition times [267]. The higher
sensitivity obtained by using cryoprobe technology can be advantageous for
proton NMR as well. With the resulting decrease in detection limit it is likely that
NMR can diagnose many additional inborn errors of metabolism, where relatively
low amounts of metabolites accumulate. It furthermore may facilitate the discovery
of new diseases.
The low natural abundance of several nuclei can also be used as an advantage in
metabolic investigations, since their weak background signal allows the study of
the metabolism of labeled compounds. An example of this technique was already
given in 1975 by Tanaka et al. [268], who elucidated the metabolic pathway of
13C-labelled
valine in a boy suffering from methylmalonic acidemia by
administering the patient orally with DL-[α-13C]-valine and DL-[α, β-13C]-valine.
189
Chapter 5.2
LINKING NMR WITH OTHER ANALYTICAL TECHNIQUES
On one hand, NMR has the advantage that many metabolites can be detected
simultaneously and that no preselection is required. On the other hand, this is also
a disadvantage, since it can cause highly complex spectra with a considerable
amount of overlap. By linking NMR to HPLC it is possible to separate and directly
identify the compounds present in complex mixtures. The first example employing
coupled 500 MHz HPLC-NMR was presented in 1992 by Spraul et al. [269], who
investigated the metabolism of the drug ibuprofen in urine samples. Both
stopped-flow and continuous-flow experiments were performed. In 1995, the first
set up combining NMR with both HPLC and MS was presented [270] and the
stopped-flow method with parallel NMR and MS detection has now become
standard [271]. In body fluid research, HPLC-NMR and HPLC-NMR-MS have mainly
been used to investigate the metabolic fate of different drugs and potential drug
candidates [28;269;272-275]. Both methods have proven to be valuable tools to
obtain structural and pharmaceutical data of metabolites present in complex
matrices with a single experiment. In the field of inborn errors of metabolism, it is
expected that the combination of NMR with liquid chromatography and/or mass
spectrometry can also be helpful in elucidating (partially) unknown pathways.
Furthermore, the detection of new metabolites and their associated diseases may
become less complicated.
MICROPROBES
Over the years, NMR has undergone some major developments, but the
conventional 5 mm sample tube used today is still essentially the same as in the
1960s. Historically, relatively large amounts of sample were needed to compensate
for the low inherent NMR sensitivity. Nowadays, ultra-high-field spectrometers
equipped with cryoprobe technology are becoming available, making
measurement of mass-limited samples theoretically possible. The sensitivity of
these samples can be further enhanced by using microprobes. With decreasing
diameter d of the detector coil, the NMR mass sensitivity (S/N per mole) increases
to a first approximation with 1/d [276;277]. A five fold increase in mass
sensitivity, which corresponds to a 25-fold reduction in measurement time
compared to a conventional 5 mm probe, was reported for a 1 mm highresolution probe with 2.5 μl active sample volume [278;279]. Other advantages of
using low-volume probes are a reduction of approximately a factor 100 in the use
of expensive deuterated solvents and a decreased residual solvent signal [278].
Furthermore, microprobes are very well suited for hyphenation with several
separation techniques, including HPLC and capillary electrophoresis [280].
In body fluid NMR, the application of microprobes is very valuable when only a
minor amount of fluid is available. Its effective use in studying a few microliters of
rodent CSF and blood plasma has been demonstrated [278;279]. An additional
190
Future perspectives
advantage when studying metabolic disorders in small animal models (e.g. in
“knock-out” animals) is that enough body fluid material for microprobe NMR
investigations can be obtained without sacrificing the animal. Although
microprobes have not yet been applied to study inborn errors of metabolism in
man, their enhanced sensitivity and small sample volumes can be promising in this
field as well.
DIFFUSION EDITED NMR SPECTROSCOPY
The presence of macromolecules in blood plasma, serum and CSF can hamper
NMR analysis and they are therefore often removed prior to NMR measurement, as
described previously. Unfortunately, this can result in loss of metabolites and
furthermore inhibit the study of many possible interactions. Diffusion edited
spectroscopy applied to intact samples provides an alternative for spectral
simplification [281;282]. Under normal conditions, molecular diffusion is not
proportional to observed spectral intensities. However, when pulsed field
gradients are applied during the experiment, diffusion can cause changes in signal
intensities. When compared to a conventional spectrum, fast diffusing molecules
such as low molecular weight metabolites will show a much larger intensity
difference than slowly diffusing molecules such as lipoproteins. Diffusion edited
spectroscopy can therefore effectively suppress small molecule resonances by a
factor 100 or more, making it possible to directly study macromolecules
[282;283]. A spectrum displaying only low molecular weight metabolites is
obtained by subtracting the macromolecular spectrum from a total spectrum
recorded without gradients. For a plasma sample, the resulting spectrum is highly
similar to a spectrum obtained after plasma deproteinization by ultrafiltration
[282]. Since diffusion edited NMR does not require any sample preparation, the
experimental time required can be significantly reduced (up to 30 minutes in the
case of ultrafiltration), making the use of NMR as a diagnostic tool in clinical
chemistry more attractive. The application of diffusion edited spectra to aid
resonance assignment in studies of diseases has also been suggested [281;284].
Additionally, a combination of diffusion edited NMR spectroscopy with
chemometric methods in toxicological investigations has been reported [285].
In contrast to spin-echo sequences, which can be applied to filter out
macromolecule resonances as well, metabolites can be readily quantified with
diffusion edited NMR spectroscopy. However, TSP-d4 cannot be used as a
concentration reference in this case, since it is partially invisible due to its
interaction with proteins. Formate can for instance be used as an alternative.
191
Chapter 5.2
AUTOMATION
In complex mixtures like body fluids, each component generates its own NMR
spectrum, which possibly overlaps with other compounds in the mixture and
makes analysis often difficult. Besides using liquid chromatography coupled to
NMR as discussed in paragraph 4.2, this problem can also be solved with
automated signal acquisition and special analysis software combined with spectral
databases, which can greatly facilitate spectral interpretation and diagnosis of
inborn errors of metabolism [286]. Automated NMR measurements require that
the apparatus is equipped with a sample changer and that it is capable of self
locking and shimming. Nowadays, probes that also perform automatic tuning and
matching are becoming available as well. Next, the computer has to start
acquisition of the appropriate experiments. In one suggested automation scheme,
first a 1D 1H spectrum is recorded and processed, which is then used to detect
deviations from normal spectra by principal component analysis incorporating a
database with control spectra [286]. Subsequently, a two-dimensional COSY is
recorded to identify metabolites that are indicative of the possible inborn errors of
metabolism. The success of this approach was demonstrated by correct diagnosis
of a few inherited metabolic diseases [286]. A somewhat different set-up used
only 1D 1H spectra to directly identify compounds by chemical shift and database
comparison [287]. Specialized deconvolution software performs the automatic
peak assignment and subsequent metabolite quantification based on peak areas.
In total, 1000 urine samples, including 69 samples from patients with 13 different
inborn errors of metabolism, were analyzed in less than 2 minutes per sample. All
healthy controls were classified as normal, whereas 66 of the pathological samples
were correctly diagnosed [287]. A third chemometric method capable of
automated analysis is a neural network trained to distinguish 1D 1H spectra of
patients with a specific disorder from healthy individuals [59;288]. A neural
network is created by supplying data for which the outcome is already known and
can include many different diseases. A diagnostic accuracy of almost 100% has
been accomplished in the case of the metabolic disorder cystinuria [59].
Automated spectral acquisition and analysis can greatly accelerate spectrum
interpretation and facilitate clinical diagnosis by providing clear “yes or no”
answers. A second advantage is that it permits NMR measurements to be
performed by non-specialists. Therefore, it is likely that further development of
these methods can make NMR spectroscopy more attractive as a routine
diagnostic tool in hospital laboratories.
Although all new developments discussed above show promising results, it is
expected that especially automated data acquisition and analysis will provide a
large step forward in making NMR spectroscopy a suitable method for standard
clinical analysis. It is also likely that a combination of two or more of the described
192
Future perspectives
techniques will be employed within several years, incorporating each of their
separate advantages.
193
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Dankwoord
Dankwoord
Ik wil graag iedereen bedanken die op welke wijze dan ook heeft bijgedragen aan
de totstandkoming van dit proefschrift.
Mijn promotor, prof. R. Wevers, drijvende kracht achter dit NMR onderzoek. Beste
Ron, ik heb veel aan je te danken; jij hebt mij de mogelijkheid gegeven om dit
promotieonderzoek uit te voeren. Heel veel dank voor je vasthoudendheid, je
vertrouwen en je begeleiding! Ik hoop dat we nog lang zullen samenwerken, met
ooit een eigen magneet binnen ons laboratorium.
Mijn copromotor, dr. M. Willemsen. Beste Michèl, bedankt voor de inspirerende
avonden bij jou thuis waar jij, Ron en ik ideeën en de voortgang van mijn promotie
bespraken. Als kinderarts toon je altijd veel belangstelling voor het biochemische
onderzoek. Jouw enthousiasme motiveert enorm!
En verder een ‘dankjewel’ aan: mijn paranimfen Fokje en Leon, alle coauteurs,
vooral Marlies Oostendorp en Marinette van der Graaf, mijn collega’s van het
laboratorium Kindergeneeskunde en Neurologie - met een speciale dank aan
Sandra Hoenderop -, Jos Joordens, Esther Hermans, Bruker Germany (Manfred
Spraul, Hartmut Schäfer and Peter Neidig), Bruker Switzerland (Detlef Moskau and
Sandra Loss), alle studenten en arts-assistenten die bij mij stage hebben gelopen.
Een bijzonder ‘dankjewel’ aan mijn familie.
Mijn schoonouders. Ad en Nelly Hulsken, jullie hebben altijd veel belangstelling
voor mijn werkzaamheden in het ziekenhuis. Bedankt voor alles wat jullie voor ons
hebben gedaan en doen; als ouders en als opa en oma van Dagmar en Kai.
Mijn ouders. Manfred en Marian Engelke. Pa en ma, jullie hebben me altijd de
vrijheid gegeven in de keuzes die ik maakte. Bedankt voor dit vertrouwen en voor
alles wat jullie voor ons hebben gedaan en doen. Ik kan altijd op jullie rekenen!
Robert, bedankt voor de gave omslag van dit boek!
En ten slotte dank aan de drie belangrijkste mensen in mijn leven: Astrid, Dagmar
en Kai. Lieve Astrid, bedankt voor alles wat was, is en wat nog komen zal … ik hou
van je!
211
212
Curriculum Vitae
Curriculum Vitae
Udo Engelke wordt op 28 april geboren te Kevelaer in Duitsland. Op 7-jarige
leeftijd verhuist hij met zijn ouders, broertje Robert en zusje Corina naar
Nederland. Hij groeit op in Venray waar hij in 1983 het Mavo-diploma behaalt aan
de katholieke Mavo “de Kolk”. Datzelfde jaar begint hij aan de opleiding
Middelbaar Laboratoriumonderwijs (MLO) te Venlo die hij in 1987 succesvol
afrondt. In 1987 start Udo met de opleiding Hoger Laboratoriumonderwijs (HLO;
differentiatie chemie) aan de Hogeschool Eindhoven. Op 23 januari 1992 studeert
hij af.
Sinds 1 februari 1992 is Udo werkzaam als research-analist op de afdeling
laboratorium Kindergeneeskunde & Neurologie van het Universitair Medisch
Centrum Nijmegen St Radboud. Het aandachtgebied van Udo is NMRspectroscopie van lichaamsvloeistoffen bij patiënten met erfelijke stofwisselingsziekten.
Op 19 december 1997 trouwt Udo met Astrid Hulsken. Twee jaar later verhuizen
ze vanuit Nijmegen naar Molenhoek, waar ze samen met hun kinderen, Dagmar
(1999) en Kai (2003) nog steeds wonen.
213
214
List of publications
List of publications
•
Engelke UF, Sass JO, van Coster RN, Gerlo E, Olbrich H, Krywawych S, Calvin J, Hart C,
Omran H and Wevers RA
NMR Spectroscopy of aminoacylase 1 deficiency, a novel inborn error of metabolism
NMR Biomed 2007: in press
•
Willemsen MA, Engelke UF, van der Graaf M and Wevers RA
Methylsulfonylmethane (MSM) ingestion causes a significant resonance in proton
magnetic resonance spectra of brain and cerebrospinal fluid
Neuropediatrics 2006; 37: 312-314
•
Engelke UF, Moolenaar SH, Hoenderop SM, Morava E, van der Graaf M, Heerschap A
and Wevers RA
Handbook of 1H-NMR spectroscopy in inborn errors of metabolism: body fluid NMR
spectroscopy and in vivo MR spectroscopy
2nd edition; 2006 SPS Verlagsgesellschaft mbH, Heilbronn, In press
•
Engelke UF, Oostendorp M, Wevers RA
NMR spectroscopy of body fluids as a metabolomics approach to inborn errors of
metabolism
IN: The Handbook of Metabonomics and Metabolomics 2007: (14) 373 – 410
Editors: John C. Lindon, Jeremy K. Nicholson and Elaine Holmes
Published by Elsevier B.V.
•
Burlina AP, Schmitt B, Engelke UF, Wevers RA, Burlina AB, Boltshauser E.
Hypoacetylaspartia: clinical and biochemical follow-up of a patient.
Adv Exp Med Biol. 2006; 576:283-7; discussion 361-3.
•
Engelke UF, Oostendorp M , Willemsen MA, Wevers RA.
Diagnosing inborn errors of lipid metabolism with proton nuclear magnetic
resonance spectroscopy.
Clin Chem. 2006;52(7):1395-405.
215
List of publications
•
Engelke UF, Kremer B, Kluijtmans LA, van der Graaf M, Morava E, Loupatty FJ,
Wanders RJ, Moskau D, Loss S, van den Bergh E, Wevers RA.
NMR spectroscopic studies on the late onset form of 3-methylglutaconic aciduria
type I and other defects in leucine metabolism.
NMR Biomed. 2006; 19(2): 271-8.
•
Wortmann S, Rodenburg RJ, Huizing M, Loupatty FJ, de Koning T, Kluijtmans LA,
Engelke UF, Wevers R, Smeitink JA, Morava E.
Association of 3-methylglutaconic aciduria with sensori-neural deafness,
encephalopathy, and Leigh-like syndrome (MEGDEL association) in four patients with
a disorder of the oxidative phosphorylation.
Mol Genet Metab. 2006; 88(1):47-52.
•
Sass JO, Mohr V, Olbrich H, Engelke UF, Horvath J, Fliegauf M, Loges NT, SchweitzerKrantz S, Moebus R, Weiler P, Kispert A, Superti-Furga A, Wevers RA, Omran H.
Mutations in ACY1, the gene encoding aminoacylase 1, cause a novel inborn error of
metabolism.
Am J Hum Genet. 2006;78(3):401-9.
•
Van Coster RN, Gerlo EA, Giardina TG, Engelke UF, Smet JE, De Praeter CM,
Meersschaut VA, De Meirleir LJ, Seneca SH, Devreese B, Leroy JG, Herga S, Perrier JP,
Wevers RA, Lissens W.
Aminoacylase I deficiency: a novel inborn error of metabolism.
Biochem Biophys Res Commun. 2005;338(3):1322-6.
•
Engelke UF, Tangerman A, Willemsen MA, Moskau D, Loss S, Mudd SH, Wevers RA.
Dimethyl sulfone in human cerebrospinal fluid and blood plasma confirmed by one-
dimensional (1)H and two-dimensional (1)H-(13)C NMR.
NMR Biomed. 2005;18(5):331-6.
•
Boltshauser E, Schmitt B, Wevers RA, Engelke UF, Burlina AB, Burlina AP.
Follow-up of a child with hypoacetylaspartia.
Neuropediatrics. 2004;35(4):255-8.
•
Wolf NI, Haas D, Hoffmann GF, Jakobs C, Salomons GS, Wevers RA, Engelke UF, Rating
D.
Sedation with 4-hydroxybutyric acid: a potential pitfall in the diagnosis of SSADH
deficiency.
J Inherit Metab Dis. 2004;27(2):291-3.
216
List of publications
•
Wolf NI, Willemsen MA, Engelke UF, van der Knaap MS, Pouwels PJ, Harting I,
Zschocke J, Sistermans EA, Rating D, Wevers RA.
Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate
in CSF.
Neurology. 2004;62(9):1503-8.
•
Groenen PM, Engelke UF, Wevers RA, Hendriks JC, Eskes TK, Merkus HM, SteegersTheunissen RP.
High-resolution 1H NMR spectroscopy of amniotic fluids from spina bifida fetuses
and controls.
Eur J Obstet Gynecol Reprod Biol. 2004;112(1):16-23.
•
Engelke UF, Liebrand-van Sambeek ML, de Jong JG, Leroy JG, Morava E, Smeitink JA,
Wevers RA.
N-acetylated metabolites in urine: proton nuclear magnetic resonance spectroscopic
study on patients with inborn errors of metabolism.
Clin Chem. 2004;50(1):58-66.
•
Mazon Ramos A, Gil-Setas A, Berrade Zubiri S, Bandres Echeverri T, Wevers R, Engelke
UF, Zschocke J.
Primary trimethylaminuria or fish odor syndrome. A novel mutation in the first
documented case in Spain
Med Clin (Barc). 2003;120(6):219-21. Spanish.
•
Wevers R.A., Engelke U.F.H., Moolenaar, S.H.
Proton NMR spectroscopy on body fluids
In: Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases
Editors: Blau N, Duran M, Blaskovics ME, Gibson KM.
Springer-Verlag Berlin Heidelberg 2003
•
Morava E, Kosztolanyi G, Engelke UF, Wevers RA.
Reversal of clinical symptoms and radiographic abnormalities with protein restriction
and ascorbic acid in alkaptonuria.
Ann Clin Biochem. 2003;40(Pt 1):108-11.
•
Ruitenbeek W, Kobayashi K, Iijima M, Smeitink JA, Engelke UF, De Abreu RA, Kwast
HT, Saheki T, Boelen CA, De Jong JG, Wevers RA.
Moderate citrullinaemia without hyperammonaemia in a child with mutated and
deficient argininosuccinate synthetase.
Ann Clin Biochem. 2003;40(Pt 1):102-7.
217
List of publications
•
Moolenaar SH, Engelke UF, Wevers RA.
Proton nuclear magnetic resonance spectroscopy of body fluids in the field of inborn
errors of metabolism.
Ann Clin Biochem. 2003;40(Pt 1):16-24. Review.
•
Van Cappellen Van Walsum AM, Jongsma HW, Wevers RA, Nijhuis JG, Crevels J,
Engelke UF, De Abreu RA, Moolenaar SH, Oeseburg B, Nijland R.
1H-NMR spectroscopy of cerebrospinal fluid of fetal sheep during hypoxia-induced
acidemia and recovery.
Pediatr Res. 2002;52(1):56-63.
•
Moolenaar SH, Engelke UF, Abeling NG, Mandel H, Duran M, Wevers RA.
Prolidase deficiency diagnosed by 1H NMR spectroscopy of urine.
J Inherit Metab Dis. 2001;24(8):843-50.
•
Moolenaar SH, Göhlich-Ratmann G, Engelke UF, Spraul M, Humpfer E, Dvortsak P,
Voit T, Hoffmann GF, Bräutigam C, van Kuilenburg AB, van Gennip A, Vreken P,
Wevers RA.
beta-Ureidopropionase deficiency: a novel inborn error of metabolism discovered
using NMR spectroscopy on urine.
Magn Reson Med. 2001;46(5):1014-7.
•
Moolenaar SH, van der Knaap MS, Engelke UF, Pouwels PJ, Janssen-Zijlstra FS,
Verhoeven NM, Jakobs C, Wevers RA.
In vivo and in vitro NMR spectroscopy reveal a putative novel inborn error involving
polyol metabolism.
NMR Biomed. 2001;14(3):167-76.
•
van Cappellen van Walsum AM, Jongsma HW, Wevers RA, Nijhuis JG, Crevels J, Engelke
UF, Moolenaar SH, Oeseburg B, Nijland R.
Hypoxia in fetal lambs: a study with (1)H-MNR spectroscopy of cerebrospinal fluid.
Pediatr Res. 2001;49(5):698-704.
•
Binzak BA, Wevers RA, Moolenaar SH, Lee YM, Hwu WL, Poggi-Bach J, Engelke UF,
Hoard HM, Vockley JG, Vockley J.
Cloning of dimethylglycine dehydrogenase and a new human inborn error of
metabolism, dimethylglycine dehydrogenase deficiency.
Am J Hum Genet. 2001;68(4):839-47.
218
List of publications
•
Boss EA, Moolenaar SH, Massuger LF, Boonstra H, Engelke UF, de Jong JG, Wevers RA.
High-resolution proton nuclear magnetic resonance spectroscopy of ovarian cyst
fluid.
NMR Biomed. 2000;13(5):297-305.
•
van der Knaap MS, Wevers RA, Struys EA, Verhoeven NM, Pouwels PJ, Engelke
UF, Feikema W, Valk J, Jakobs C.
Leukoencephalopathy associated with a disturbance in the metabolism of polyols.
Ann Neurol. 1999;46(6):925-8.
•
Lehnert W, Stogmann W, Engelke UF, Wevers RA, van den Berg GB.
Long-term follow up of a new case of hawkinsinuria.
Eur J Pediatr. 1999;158(7):578-82.
•
Wevers RA, Engelke UF, Moolenaar SH, Bräutigam C, de Jong JG, Duran R, de
Abreu RA, van Gennip AH.
1H-NMR spectroscopy of body fluids: inborn errors of purine and pyrimidine
metabolism.
Clin Chem. 1999;45(4):539-48.
•
Moolenaar SH, Poggi-Bach J, Engelke UF, Corstiaensen JM, Heerschap A, de
Jong JG, Binzak BA, Vockley J, Wevers RA.
Defect in dimethylglycine dehydrogenase, a new inborn error of metabolism: NMR
spectroscopy study.
Clin Chem. 1999;45(4):459-64.
•
Massuger LF, van Vierzen PB, Engelke UF, Heerschap A, Wevers R.
1H-magnetic resonance spectroscopy: a new technique to discriminate benign from
malignant ovarian tumors.
Cancer. 1998;82(9):1726-30.
•
Wevers RA, Engelke UF, Rotteveel JJ, Heerschap A, De Jong JG, Abeling NG, van Gennip
AH, de Abreu RA.
1H NMR spectroscopy of body fluids in patients with inborn errors of purine and
pyrimidine metabolism.
J Inherit Metab Dis. 1997;20(3):345-50.
•
Wevers RA, Engelke UF, Wendel U, de Jong JG, Gabreëls FJ, Heerschap A.
Standardized method for high-resolution 1H-NMR of cerebrospinal fluid.
Clin Chem. 1995;41(5):744-51.
219
List of publications
•
Abeling NG, van Gennip AH, Bakker HD, Heerschap A, Engelke UF, Wevers RA.
Diagnosis of a new case of trimethylaminuria using direct proton NMR spectroscopy
of urine.
J Inherit Metab Dis. 1995;18(2):182-4.
•
Wevers RA, Engelke UF, Heerschap A.
High-resolution 1H-NMR spectroscopy of blood plasma for metabolic studies.
Clin Chem. 1994;40(7 Pt 1):1245-50.
220