Download Clinical Utility of Monoamine Neurotransmitter Metabolite Analysis in

Review
Clinical Chemistry 54:4
633–641 (2008)
Clinical Utility of Monoamine
Neurotransmitter Metabolite Analysis in
Cerebrospinal Fluid
Keith Hyland
BACKGROUND: Measurements of monoamine neurotransmitters and their metabolites in plasma and urine are
commonly used to aid in the detection and monitoring of
neuroblastoma and pheochromocytoma and the evaluation of hypotension or hypertension. Measurements of
these neurotransmitters and metabolites can also be helpful in the investigation of disorders that primarily affect
the central nervous system, but only when the measurements are made in cerebrospinal fluid (CSF).
CONTENT:
I describe CSF profiles of monoamine metabolites in the primary and secondary defects affecting
serotonin and catecholamine metabolism. I outline the
methods required to analyze these metabolites together
with details of specific sample handling requirements,
sample stability, and interfering compounds, and I emphasize a need for age-related reference intervals.
SUMMARY:
Measured values of monoamine metabolites
in CSF provide only a single-time snapshot of the overall turnover of the monoamine neurotransmitters
within the brain. Because these measurements reflect
the average concentrations accumulated from all brain
regions plus the regional changes that occur within the
spinal cord, they may miss subtle abnormalities in particular brain regions or changes that occur on a
minute-to-minute or diurnal basis. Clearly defined diagnosed disorders are currently limited to those affecting synthetic and catabolic pathways. In many cases,
abnormal monoamine metabolite concentrations are
found in CSF and an underlying etiology cannot be
found. Molecular screening of candidate genes related
to steps in the neurotransmission process, including
storage in presynaptic nerve vesicles, release, interaction with receptors, and reuptake, might be a fruitful
endeavor in these cases.
© 2008 American Association for Clinical Chemistry
Medical Neurogenetics, Atlanta, GA.
Address correspondence to the author at: Medical Neurogenetics, One Dunwoody
Park, Suite 250, Atlanta, GA 30338. Fax 678-507-5669, e-mail khyland@
medicalneurogenetics.com.
Received January 28, 2008; accepted January 29, 2008.
Previously published online at DOI: 10.1373/clinchem.2007.099986
Peripheral measurements of monoamine neurotransmitters (serotonin and the catecholamines, dopamine
and norepinephrine) and their metabolites in either
plasma or urine have been used most often to look at
mechanisms in hypotension or hypertension, and as
screening procedures to detect and monitor neuroblastoma and pheochromocytoma (1–3 ). Measurement of
these neurotransmitters and metabolites in peripheral
fluids, although useful for these disorders, rarely has
benefit for the investigation of disorders that primarily
affect the central nervous system (CNS).1 This review is
therefore not designed to cover the use of monoamine
and metabolite analysis in the periphery, but rather is
limited to the clinical utility of their measurement in
cerebrospinal fluid (CSF) as this relates to human disease within the CNS.
Pathways for the synthesis and catabolism of
serotonin and the catecholamines are shown in Fig. 1.
The major metabolites of these neurotransmitters
that appear in human CSF are homovanillic acid (HVA)
for dopamine, 5-hydroxyindoleacetic acid (5HIAA)
for serotonin, and 3-methoxy,4-hydroxyphenylglycol
(MHPG) for norepinephrine; the concentrations of
these are thought to reflect the overall turnover of the
neurotransmitters within the CNS (4 ). Measurement
of monoamine metabolites in CSF can therefore be
used to establish a “snapshot” of serotonin and catecholamine metabolism at one particular time that may
provide an indication of disease mechanisms that affect
the neurotransmitter pathways.
The monoamine neurotransmitters are involved
in the control of a wide variety of neuronal functions.
They regulate, among other things, psychomotor function (through involvement in the regulation of motor
coordination), reward-driven learning, arousal, processing of sensory input, memory, appetite, emotional
stability, sleep, mood, vomiting, sexual behavior, and
secretion of anterior pituitary and other hormones
Nonstandard abbreviations: CNS, central nervous system; CSF, cerebrospinal
fluid; HVA, homovanillic acid; 5HIAA, 5-hydroxyindoleacetic acid; MHPG, 3-methoxy,4-hydroxyphenylglycol; BH4, tetrahydrobiopterin; PLP, pyridoxal 5⬘-phosphate; AADC, aromatic L-amino acid decarboxylase; PNPO, pyridox(am)ine phosphate oxidase; EC, electrochemical.
633
Review
GTP
1
HVA, 5HIAA
(+/- 5HIAA in the dominant form)
NH2P3
2
HVA,
6PTP
HVA,
HVA
BH4
Norepinephrine
8 & 10
MHPG
6
qBH2
7
(B6 dependent)
11
Dopamine
Serotonin
(predicted)
8 &10
HVA
(predicted)
5HIAA (predicted)
5HTP
7
9
5HIAA
TRYP
5
4
L-dopa
HVA
HVA,
3
5HIAA
TYR
MHPG
5HIAA
HVA, 5HIAA, 3OMD,
L-dopa, 5HTP
HVA, 5HIAA, 3OMD,
threonine, glycine
8
5HIAA
Fig. 1. Disorders and associated CSF metabolite patterns found in the catecholamine and serotonin biosynthetic and
catabolic pathways.
1, GTP cyclohydrolase; 2, 6-pyruvoyltetrahydropterin synthase; 3, sepiapterin reductase; 4, tyrosine hydroxylase; 5, dihydropteridine reductase; 6, tryptophan hydroxylase; 7, aromatic L-amino acid decarboxylase; 8, monoamine oxidase; 9, dopamine
␤-hydroxylase; 10, catechol-O-methyltransferase; 11, pyridox(am)ine phosphate oxidase. NH2P3, dihydroneopterin triphosphate;
6PTP, 6-pyruvoyltetrahydropterin; qBH2, quinonoid dihydrobiopterin; TYR, tyrosine; TRYP, tryptophan; 5HTP, 5-hydroxytryptophan; 3OMD, 3-O-methyldopa. Solid lines, known inherited disorders; dashed line, site of a predicted inherited disorder; dashed
arrows, ⬎1 step is involved; 2, decreased levels; 1, increased levels.
(5, 6 ). In the field of neuropsychiatry, changes in
CSF monoamine metabolite concentration have been
linked to numerous disease phenotypes including depression (7 ), Alzheimer’s disease and Parkinson’s disease (8 ), obsessive compulsive disorder (9 ), and suicide (10 ). In these disorders, metabolite changes can
be subtle, and often only statistical analysis of large
populations allows differentiation from controls. For
this reason, the measurement of monoamine metabolites in CSF from these patient populations is not of
great clinical utility. Their measurement is of importance, however, for the diagnosis and monitoring of
inherited disorders that affect the synthesis, action, and
degradation of serotonin and the catecholamines. In
these conditions, changes in monoamine metabolites
are normally profound, particular patterns may be suggestive for individual conditions, and concentrations
634 Clinical Chemistry 54:4 (2008)
of metabolites can be used to monitor treatment outcomes (11 ).
PRIMARY INHERITED DISORDERS AFFECTING SEROTONIN AND
CATECHOLAMINE METABOLISM
The synthesis of serotonin and the catecholamines requires participation of 2 cofactors, tetrahydrobiopterin
(BH4) for the activity of tyrosine hydroxylase and tryptophan hydroxylase and pyridoxal 5⬘-phosphate (PLP)
for the activity of aromatic L-amino acid decarboxylase
(AADC) (Fig. 1). Tetrahydrobiopterin is formed in a
3-step pathway from GTP and involves the activities of
GTP cyclohydrolase, 6-pyruvoyltetrahydropterin synthase, and sepiapterin reductase. After its oxidation in
the 2 hydroxylase reactions, it is reduced back to the
active form by the action of pterin-4␣-carbinolamine
dehydratase and dihydropteridine reductase (Fig. 1)
Review
Monoamine Metabolites in CSF
Table 1. Metabolite patterns observed in CSF in the inherited disorders affecting catecholamine and serotonin
metabolism.
HVA
5-Hydroxyindoloeacteic
acid
MHPG
3-O-methyldopa
Disorders of BH4 synthesis (recessive)
2
2
2
N
GTP cyclohydrolase (dominant)
2
N
N
N
Tyrosine hydroxylase
2
N
2
N
Tryptophan hydroxylasea
N
2
N
N
AADC
2
2
2
1
PNPOb
2
2
2
1
Dopamine ␤-hydroxylase
1
N
2
N
Monoamine oxidase
2
2
2
N
N, normal; 2, decreased; 1, elevated.
a
Predicted.
b
This pattern is not always found. Elevations of threonine and glycine can also be seen in PNPO deficiency.
(12 ). Pathogenic mutations have been described in all
the known enzymes involved in BH4 metabolism (13 ).
As these mutations affect synthesis of both serotonin
and the catecholamines, concentrations of 5HIAA,
HVA, and MHPG are, in general, significantly decreased in CSF (14 ). An exception is found in dominantly inherited GTP cyclohydrolase deficiency (doparesponsive dystonia) (15 ); in this disorder, it appears
that the major impact is on the dopaminergic system,
as an isolated decrease in HVA is observed (16 ).
Primary defects affecting the AADC gene also lead
to decreases in all 3 CSF metabolites, as this enzyme is
likewise involved in both pathways (17 ). In addition,
there are elevations of L-dopa and 5-hydroxytrypophan, which are the substrates for the enzyme. L-Dopa
can also be methylated using S-adenosylmethionine as
the methyl group donor, and the product, 3-O-methyldopa, accumulates and provides a major indicator for
decreased activity of AADC (17 ). Pyridox(am)ine
phosphate oxidase (PNPO) is required for the maintenance of PLP levels within the CNS, and mutations in
this enzyme lead to a deficiency of PLP and consequently to decreased activity of AADC and a similar
metabolite pattern (18 ). Elevations of CSF threonine
and glycine can also be seen, as PLP is required for their
catabolism. In both primary AADC deficiency and
PNPO deficiency, the accumulating 3-O-methyldopa
can be further metabolized via transamination to form
vanillactic acid, which can be detected on a urine organic acid screen (17, 18 ).
Tyrosine hydroxylase deficiency leads to specific
decreases in HVA and MHPG but normal levels of
5HIAA, as serotonin synthesis is unaffected (19 ).
Mutations in tryptophan hydroxylase have yet to be
described, but an isolated decrease in 5HIAA can be
predicted. Formation of norepinephrine relies on the
activity of dopamine ␤-hydroxylase. This is a coppercontaining enzyme, and deficiency of dopamine ␤hydroxylase leads to increased HVA levels due to the
accumulation of dopamine and its subsequent conversion to this metabolite (20 ). Catabolism of serotonin
requires the activity of monoamine oxidase, and low
levels of 5HIAA are predicted in this disorder (21 ).
Catabolism of the catecholamines requires monoamine oxidase and catechol-O-methyltransferase
(Fig. 1), and again deficiencies of these enzymes likely
lead to low CSF concentrations of HVA and MHPG.
Monoamine metabolite profiles seen, or expected, in
these disorders are summarized in Table 1 and indicated in Fig. 1.
SECONDARY ABNORMALITIES OF SEROTONIN
AND CATECHOLAMINES
It is important to emphasize that changes in CSF serotonin and catecholamine metabolites can occur as a
consequence of problems in other areas of metabolism.
Secondary changes have been described in posthypoxia
(22 ), epilepsy (23 ), febrile convulsions (24 ), liver disease and viral infections (25 ), abnormalities of folate
metabolism (26, 27 ), Lesch Nyhan syndrome (28 ),
Down syndrome (29 ), urea cycle disorders (30 ), Rett
syndrome (31 ), Menkes disease (32 ), and deficiencies of arginase (33 ) and phenylalanine hydroxylase
(34 ). Isolated low concentrations of HVA have also
been found in many neurological conditions where
etiology is unclear (35, 36 ). This list is not comprehensive but does serve to demonstrate that other possibilities should be considered before making a diagnosis
of a primary disorder of neurotransmitter monoamine
Clinical Chemistry 54:4 (2008) 635
Review
metabolism when abnormal concentrations of CSF
metabolites have been found.
Because monoamine metabolite profiles can be
nonspecific, an absolute diagnosis of a primary defect
in monoamine metabolism requires further investigation. Pituitary prolactin secretion is regulated by
neurosecretory dopamine neurons in the hypothalamus that normally inhibit prolactin secretion. A finding of an increased serum prolactin can therefore
provide further evidence of a central dopamine deficiency when low CSF HVA concentrations have been
found (37 ). For tryptophan hydroxylase and tyrosine
hydroxylase deficiency, genomic sequencing is required for absolute diagnosis, as there is no easily accessible tissue that can be used to measure enzyme
activity. The CSF pattern seen in AADC deficiency is
almost always diagnostic but may not differentiate
between a primary AADC defect or secondary inhibition of the enzyme activity due to lack of PLP; PLP
measurement in CSF may allow distinction between
the two (18 ). AADC activity can be measured in
plasma, and a finding of low activity is diagnostic
for AADC deficiency. In the case of PNPO deficiency, plasma AADC activity can be increased, probably owing to upregulation of apoenzyme synthesis in
the absence of the PLP cofactor. Genomic sequencing
for AADC and PNPO may be used for final definitive
confirmation.
Confirmation of monoamine oxidase-A deficiency
is made by measurement of the enzyme activity in
dexamethasone-stimulated fibroblasts or by sequencing
(21 ). In patients with dopamine ␤-hydroxylase deficiency, activity of this enzyme is absent in plasma; however, there is genetically determined interindividual variation in plasma dopamine ␤-hydroxylase, with 3% to 4%
of the normal adult population having near-zero concentrations (38 ). The disorder can be confirmed by the evaluation of the norepinephrine-to-dopamine ratio in
plasma. Normally this ratio is around 10, but in patients
with dopamine ␤-hydroxylase deficiency it is reduced to
⬍0.1 (20 ). Genomic sequencing is also available (39, 40 ).
ANALYTICAL METHODS
Many different methods have been used to analyze serotonin, the catecholamines, and their metabolites in
biological fluids. These include capillary electrophoresis (41 ), HPLC/MS (42 ), GC/MS (43 ), and HPLC with
either fluorescence or electrochemical (EC) detection
(44, 45 ). The current methods used to analyze CSF
monoamines and metabolites in clinical and research
laboratories rely mostly on reversed-phase HPLC coupled with EC detection (19, 46 –52 ). These systems are
highly sensitive and selective. Electrochemical detectors are now extremely stable and metabolites can be
detected in the femtomole range. Selectivity is clearly
636 Clinical Chemistry 54:4 (2008)
demonstrated, as in many procedures CSF can be directly injected onto an HPLC column without prior
derivatization of the samples (47, 49, 50 ).
Methods in general have used isocratic conditions
and reversed-phase columns with or without the incorporation of ion pairing in the mobile phase (19, 46 –
51 ). Changes in pH and organic modifier (usually
methanol or acetonitrile) concentration markedly affect retention times of all metabolites and can be used
to obtain optimum separation of any desired components. Accurate measurement of HVA and 5HIAA is
easily obtained in all the systems described, but, MHPG
is present in relatively small concentrations in CSF, and
separation from other compounds can be challenging.
The difficulties of measuring this compound accurately were evident in the large interlaboratory variations noted in a pilot quality control program (53 ).
Partial purification of samples before analysis may alleviate this problem (19 ). Alternately, multielectrode
coulometric electrochemical cells in series, with or
without gradient elution, can be used to help resolve
coeluting compounds by their current/voltage characteristics (54 ).
As well as the measurement of HVA, 5HIAA, and
MHPG, there are occasions when other metabolite
analysis may become important. As previously described, blockage of AADC leads to accumulation 3-Omethyldopa, which is the primary accumulating metabolite and, as such, it acts as a marker for this disease.
In addition there is accumulation of 5-hydroxytryptophan and L-dopa (17 ). All of these compounds can be
resolved and measured using a simple isocratic system
(55 ) (Fig. 2).
When investigating CSF for a possible defect in
monoamine neurotransmitter metabolism, it is critical
that one also examine CSF concentrations of the cofactor BH4 and its precursor neopterin. This is also
achieved using HPLC with either fluorescence (14 ) or,
in series, EC and fluorescence detection (56 ). Normally
inherited defects in BH4 metabolism are detected at the
time of newborn screening, as BH4 is also required for
the activity of phenylalanine hydroxylase in the liver
and a deficiency of the cofactor leads to hyperphenylalaninemia (14 ). In dominantly inherited GTP cyclohydrolase deficiency and in defects affecting sepiapterin reductase (the third enzyme in the BH4
synthesis pathway), hyperphenylalaninemia is absent.
These conditions can be detected only by abnormal
monoamine metabolite and pterin profiles in CSF (57 ).
Fig. 2 provides example chromatograms obtained
from the analysis of CSF from patients with deficiencies
of BH4, tyrosine hydroxylase, and aromatic L-amino
acid decarboxylase using a simple isocratic reversedphase HPLC system with coulometric EC detection.
This system has been in use for ⬎15 years in our clinical
Monoamine Metabolites in CSF
Review
Fig. 2. Example chromatograms.
(a), Patients with aromatic L-amino acid decarboxylase deficiency; note the elevations of 3-O-methyldopa, 5-hydroxytryptophan,
and L-dopa and the virtual absence of 5-hydroxyindoleacetic acid and homovanillic acid. (b), Patients with tyrosine hydroxylase
deficiency; note the normal concentration of 5-hydroxyindoleacetic acid and the virtual absence of homovanillic acid. (c),
Patients with tetrahydrobiopterin deficiency; note the absence of homovanillic acid and 5-hydroxyindoleacetic acid. (d), A
patient with normal concentrations of 5-hydroxyindoleacetic acid and homovanillic acid who is receiving acetaminophen (*).
(e), A mixture of standard compounds. 1, 3-methoxy-4-hydroxyphenylglycol; 2, L-dopa; 3, 5-hydroxyindoleacetic acid; 4,
homovanillic acid; 5, 3-O-methyldopa; 6, 5-hydroxytryptophan. Chromatographic conditions: 20 ␮L CSF was injected onto a 250
by 4.6 mm Phenomenex, SpheraClone 5 ␮m ODS (2 ) column using an ESA 542 autosampler cooled to 4 °C. Compounds were
eluted under isocratic conditions at a flow rate of 1.3 mL/min using an ESA 582 pump and mobile phase consisting of 0.05M
potassium phosphate buffer (pH 2.7) containing 1 mmol/L octyl sodium sulfate, 54 ␮mol/L EDTA, and 14% methanol.
Compounds were detected using an ESA Coulochem III dual electrode electrochemical detector. Electrode 1 was set at ⫺0.05
mV, and compounds were oxidized at electrode 2, which was set at ⫹400 mV.
Clinical Chemistry 54:4 (2008) 637
Review
a)
b)
1750
1500
1000
HVA (nmol/L)
5HIAA (nmol/L)
1250
750
500
250
0
1250
1000
750
500
250
0
4
8
12
0
16
0
4
Age (years)
1250
1250
1000
1000
750
500
250
0
12
16
d)
5HIAA (nmol/L)
5HIAA (nmol/L)
c)
0
8
Age (years)
250
500
750 1000 1250 1500 1750
HVA (nmol/L)
750
500
250
0
0
250
500
750 1000 1250 1500 1750
HVA (nmol/L)
Fig. 3. Relationships between homovanillic acid, 5-hydroxyindoleacetic acid, and age.
(a), The effect of age on 5-hydroxyindoleacetic acid (5HIAA) concentration. (b), The effect of age on homovanillic acid (HVA)
concentration. Note the rapid decrease in concentrations in the first few months of life, particularly for 5HIAA. (c), Linear
relationship between 5HIAA and HVA in the first 6 months of life (1/slope ⫽ 1.73; r 2 ⫽ 0.57; P ⬍ 0.0001). (d), Linear
relationship between 5HIAA and HVA from 6 months to 15 years of age (1/slope ⫽ 3.2; r 2 ⫽ 0.64; P ⬍ 0.0001). Note the
change in slope, indicating that the ratio of HVA to 5HIAA increases approximately 2-fold after the first 6 months of life.
laboratory. More than 1000 samples can be analyzed
without the need to change the analytical column. This
system does not allow detection of MHPG, but in most
instances where MHPG concentrations might be low,
specific measurement of this compound is not required, as changes in HVA concentration are always
also observed.
SAMPLE COLLECTION
Measurement of neurotransmitter metabolites in CSF
is of little value unless the method of collection and
sample handling are carefully controlled. Age, height,
diet, motility, concentration gradients, site of puncture, diurnal variations, and even season at time of
638 Clinical Chemistry 54:4 (2008)
birth have been suggested to affect measured concentrations (58, 59 ).
Obviously, in the clinical setting, it is not possible
to control for all of the above factors, but these confounding issues should be considered if large research
studies are being conducted. One of the most important factors in the clinical arena is the rostrocaudal
gradient for HVA and 5HIAA within the spinal cord
(11, 19, 60, 61 ). Values double with approximately every 5–10 mL of CSF drawn. For this reason, it is essential that patient data is compared to reference intervals
obtained using the same fraction of CSF. Values for
MHPG do not vary substantially with increased CSF
volume drawn, so they presumably reflect spinal cord
Review
Monoamine Metabolites in CSF
Table 2. Age-related reference intervals for monoamine metabolites in lumbar cerebrospinal fluid.
Age,
years
5-Hydroxyindoloeacteic
acid
HVA
3-O-methyldopa
L-Dopa
5-Hydroxytryptophan
MHPG
208–1159
337–1299
⬍300
⬍25
⬍10
95–274
0.2–0.5
179–711
450–1132
⬍300
⬍25
⬍10
52–136
0.5–2
129–520
294–1115
⬍300
⬍25
⬍10
41–71
2–5
74–345
233–928
⬍150
⬍25
⬍10
39–75
0–0.2
5–10
66–338
218–852
⬍100
⬍25
⬍10
37–69
10–15
67–189
167–563
⬍100
⬍25
⬍10
40–72
Adult
67–140
145–324
⬍100
⬍25
⬍10
35–65
All values are nmol/L. CSF was collected from the first drop, and the first 0.5-mL fraction was used for the analysis of monoamine metabolites.
concentrations rather than those within the brain (19 )
It is necessary that every laboratory define its own specific collection protocol and ensure that samples are
then collected appropriately.
It is also necessary to have age-related reference
intervals to which results can be compared, as metabolite concentrations are high in the newborn period,
rapidly drop during the first few months of life, and
then slowly decrease with age (Fig. 3, a and b; Table 2)
(47, 62 ). A clear linear relationship exists between
concentrations of 5HIAA and HVA in CSF (19, 63 ),
and this relationship does not change with the fraction
of CSF collected. There is, however, a change in slope of
the regression line for this relationship between data
obtained from individuals 0 to 6 months of age and
older than 6 months (Fig. 3, c and d) (19 ). This change
reflects a more rapid age-related drop in 5HIAA than
HVA in the first 6 months of life. Provided HVA/
5HIAA ratios are compared to the appropriate reference ratios, they can prove useful in detecting abnormalities in monoamine metabolism in situations where
CSF has not been collected in an optimal fashion (19 ).
SAMPLE STABILITY
Neurotransmitter metabolites are relatively stable in
CSF, but blood contamination during collection of
CSF can lead to oxidation of metabolites if the red
blood cells are allowed to hemolyze. Blood contaminated samples should, therefore, be centrifuged as soon
as possible after sample collection, and the clear CSF
should be transferred to a new container before freezing. Metabolite concentrations during analysis are
stable for at least 24 h if kept at 4 °C (19 ), and samples
can be frozen and thawed several times without
changes in 5HIAA and HVA (64 ). When stored at
⬍⫺70 °C, all the metabolites are stable for at least 5
years without a need for antioxidant addition (K.H.,
unpublished observation).
QUALITY
Commercial quality control materials are not available for use in the measurement of CSF monoamine
metabolites, but they can easily be prepared by dilution
or spiking of CSF. When preparing diluted specimens,
it is important to realize that the dilution process decreases the endogenous antioxidants in CSF, which can
lead to instability of metabolites. Preparation of quality
control materials with low metabolite values should
therefore be performed using artificial CSF containing
0.01 g/L ascorbic acid. In the clinical setting, suitable
internal standards have not been found to ensure injection integrity. Therefore the use of spiked samples is
recommended, where each sample is first run neat and
then diluted 50:50 with the external standard and
checked for appropriate recovery.
INTERFERING COMPOUNDS AND DRUG EFFECTS
Using the HPLC system defined in Fig. 2, I have analyzed ⬎10 000 CSF samples from patients of all ages
who have had a wide range of neurological and neuropsychiatric conditions. It is extremely rare to observe
other compounds that interfere with the measurement
of 5HIAA and HVA. None of the currently used anticonvulsants generate electrochemically active compounds that appear on the neurotransmitter metabolite chromatogram. Acetaminophen elutes at around
5 min but does not affect measurement of the desired
compounds (Fig. 2). Occasionally, small peaks that
elute close to 3-O-methyldopa may be observed, but
small changes in pH can be used to obtain clear separation if measurement of this compound is critical.
It is important to obtain a list of medications at the
time of CSF collection. Sinemet (L-dopa with carbidopa) therapy leads to a large accumulation of 3-Omethyldopa. Also, serotonin reuptake inhibitors and
certain antidepressants are known to influence CSF
metabolite concentrations (65, 66 ), and it is likely that
Clinical Chemistry 54:4 (2008) 639
Review
serotonin and catecholamine agonists and antagonists
will affect metabolite concentrations.
Conclusions
The measurement of monoamine metabolites in CSF is
not ideal, as it provides a only single snapshot of one
time point, and the measured values probably reflect
average concentrations accumulated from all brain regions together with the regional changes that occur
within the spinal cord. Thus, subtle abnormalities—in
particular brain regions or changes that occur on a
minute-to-minute or diurnal basis—may be missed.
This limits the clinical utility of metabolite measurement in CSF to situations where the monoamine pathways are affected in a global manner leading to large
changes in metabolite concentrations. Despite these
drawbacks, CSF monoamine neurotransmitter metabolite analysis has become fairly routine in many research laboratories and in specialized clinical laboratories that search for changes that might indicate
problems in this area of neurochemistry. Clearly de-
fined diagnosed disorders have been limited to those
affecting synthetic and catabolic pathways. The process
of neurotransmission, however, requires other steps
including storage in presynaptic nerve vesicles, release,
interaction with receptors, and reuptake. In most animal models where each of these steps has been knocked
out, the intracellular (and presumably extracellular)
concentrations of neurotransmitters are profoundly
changed (67–71 ). It is certain that these disorders occur in humans. In many cases, abnormal monoamine
metabolite concentrations are found in CSF and an underlying etiology cannot be found. These are likely candidates for which molecular screening of candidate
genes might be a fruitful endeavor.
Grant/Funding Support: This work was supported in
part by a grant from the Pediatric Neurotransmitter
Disease (PND) Association.
Financial Disclosures: K.H. is employed by Medical
Neurogenetics LLC, a company that performs neurotransmitter monoamine metabolite analyses in cerebrospinal fluid.
References
1. Kema IP, de Vries EG, Muskiet FA. Clinical chemistry of serotonin and metabolites. J Chromatogr
B Biomed Sci Appl 2000;747:33– 48.
2. Monsaingeon M, Perel Y, Simonnet G, Corcuff JB.
Comparative values of catecholamines and metabolites for the diagnosis of neuroblastoma. Eur
J Pediatr 2003;162:397– 402.
3. Reisch N, Peczkowska M, Januszewicz A, Neumann HP. Pheochromocytoma: presentation, diagnosis and treatment. J Hypertens 2006;24:
2331–9.
4. Wester P, Bergstrom U, Eriksson A, Gezelius C,
Hardy J, Winblad B. Ventricular cerebrospinal
fluid monoamine transmitter and metabolite concentrations reflect human brain neurochemistry in
autopsy cases. J Neurochem 1990;54:1148 –56.
5. Walther DJ, Bader M. A unique central tryptophan hydroxylase isoform. Biochem Pharmacol
2003;66:1673– 80.
6. Hyland K. Neurochemistry and defects of biogenic
amine neurotransmitter metabolism. J Inherit
Metab Dis 1999;22:353– 63.
7. Bottiglieri T, Laundy M, Crellin R, Toone BK,
Carney MW, Reynolds EH. Homocysteine, folate,
methylation, and monoamine metabolism in depression. J Neurol Neurosurg Psychiatry 2000;69:
228 –32.
8. Hartikainen P, Reinikainen KJ, Soininen H, Sirvio
J, Soikkeli R, Riekkinen PJ. Neurochemical markers in the cerebrospinal fluid of patients with
Alzheimer’s disease, Parkinson’s disease and
amyotrophic lateral sclerosis and normal controls.
J Neural Transm Park Dis Dement Sect 1992;4:
53– 68.
9. Baumgarten HG, Grozdanovic Z. Role of serotonin in obsessive-compulsive disorder. Br J Psychiatry Suppl 1998;(35):13–20.
10. Jokinen J, Nordstrom AL, Nordstrom P. The relationship between CSF HVA/5-HIAA ratio and sui-
640 Clinical Chemistry 54:4 (2008)
11.
12.
13.
14.
15.
16.
17.
18.
cide intent in suicide attempters. Arch Suicide Res
2007;11:187–92.
Hyland K. The lumbar puncture for diagnosis of
pediatric neurotransmitter diseases. Ann Neurol
2003;54 (Suppl 6):S13–7.
Thony B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration and functions.
Biochem J 2000;347 (Pt 1):1–16.
Thony B, Blau N. Mutations in the BH4-metabolizing genes GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase, sepiapterin reductase, carbinolamine-4a-dehydratase, and
dihydropteridine reductase. Hum Mutat 2006;
27:870 – 8.
Blau N, Thony B, Cotton RG, Hyland K. Disorders
of tetrahydrobiopterin and related biogenic
amines. In: Scriver CR, Beaudet AL, Sly WS, Valle
D, eds. The Metabolic and Molecular Bases of
Inherited Disease. New York: McGraw-Hill, 2001;
1725–76.
Ichinose H, Ohye T, Takahashi E, Seki N, Hori T,
Segawa M, et al. Hereditary progressive dystonia
with marked diurnal fluctuation caused by mutations in the GTP cyclohydrolase I gene. Nat Genet
1994;8:236 – 42.
Furukawa Y. Genetics and biochemistry of doparesponsive dystonia: significance of striatal tyrosine hydroxylase protein loss. Adv Neurol 2003;
91:401–10.
Hyland K, Surtees RA, Rodeck C, Clayton PT.
Aromatic L-amino acid decarboxylase deficiency:
clinical features, diagnosis, and treatment of a
new inborn error of neurotransmitter amine synthesis. Neurology 1992;42:1980 – 8.
Mills PB, Surtees RA, Champion MP, Beesley CE,
Dalton N, Scambler PJ, et al. Neonatal epileptic
encephalopathy caused by mutations in the PNPO
gene encoding pyridox(am)ine 5⬘-phosphate oxidase. Hum Mol Genet 2005;14:1077– 86.
19. Brautigam C, Wevers RA, Jansen RJ, Smeitink JA,
de Rijk-van Andel JF, Gabreels FJ, Hoffmann GF.
Biochemical hallmarks of tyrosine hydroxylase
deficiency. Clin Chem 1998;44:1897–904.
20. Robertson D, Haile V, Perry SE, Robertson RM,
Phillips JA, Biaggioni I. Dopamine beta-hydroxylase deficiency: a genetic disorder of cardiovascular regulation. Hypertension 1991;18:1– 8.
21. Brunner HG, Nelen M, Breakefield XO, Ropers
HH, van Oost BA. Abnormal behavior associated
with a point mutation in the structural gene for
monoamine oxidase A. Science (Wash DC) 1993;
262:578 – 80.
22. Hyland K, Peterschmitt MJ, Soull JS, Korsen MS,
Gascon G, Gibson JB, et al. Confusing CSF neurochemical picture suggesting 6-pyruvoyltetrahydropterin synthase deficiency in neonates with
probable hypoxic-ischemic encephalopathy. J Inherit Metab Dis 1999;22:18.
23. Devinsky O, Emoto S, Goldstein DS, Stull R, Porter
RJ, Theodore WH, Nadi NS. Cerebrospinal fluid
and serum levels of dopa, catechols, and monoamine metabolites in patients with epilepsy.
Epilepsia 1992;33:263–70.
24. Giroud M, Dumas R, Dauvergne M, D’Athis P,
Rochette L, Beley A, Bralet J. 5-Hydroxyindoleacetic acid and homovanillic acid in cerebrospinal
fluid of children with febrile convulsions. Epilepsia 1990;31:178 – 81.
25. O’Kusky JR, Boyes BE, Walker DG, McGeer EG.
Cytomegalovirus infection of the developing
brain alters catecholamine and indoleamine metabolism. Brain Res 1991;559:322–30.
26. Clayton PT, Smith I, Harding B, Hyland K, Leonard
JV, Leeming RJ. Subacute combined degeneration
of the cord, dementia and parkinsonism due to an
inborn error of folate metabolism. J Neurol Neurosurg Psychiatry 1986;49:920 –7.
27. Ramaekers VT, Hausler M, Opladen T, Heimann
Review
Monoamine Metabolites in CSF
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
G, Blau N. Psychomotor retardation, spastic paraplegia, cerebellar ataxia and dyskinesia associated with low 5-methyltetrahydrofolate in cerebrospinal fluid: a novel neurometabolic condition
responding to folinic acid substitution. Neuropediatrics 2002;33:301– 8.
Hyland K, Kasim S, Egami K, Arnold LA, Jinnah
HA. Tetrahydrobiopterin deficiency and dopamine loss in a genetic mouse model of LeschNyhan disease. J Inherit Metab Dis 2004;27:
165–78.
Mann DM, Lincoln J, Yates PO, Brennan CM.
Monoamine metabolism in Down syndrome. Lancet 1980;2:1366 –7.
Hyman SL, Porter CA, Page TJ, Iwata BA, Kissel R,
Batshaw ML. Behavior management of feeding
disturbances in urea cycle and organic acid disorders. J Pediatr 1987;111:558 – 62.
Wenk GL, Naidu S, Casanova MF, Kitt CA, Moser
H. Altered neurochemical markers in Rett’s syndrome. Neurology 1991;41:1753– 6.
Kaler SG, Goldstein DS, Holmes C, Salerno JA,
Gahl WA. Plasma and cerebrospinal fluid neurochemical pattern in Menkes disease. Ann Neurol
1993;33:171–5.
Hyland K, Smith I, Clayton PT, Leonard JV. Impaired neurotransmitter amine metabolism in arginase deficiency. J Neurol Neurosurg Psychiatry
1985;48:1188 –9.
Guttler F, Lou H. Dietary problems of phenylketonuria: effect on CNS transmitters and their
possible role in behaviour and neuropsychological function. J Inherit Metab Dis 1986;9 Suppl
2:169 –77.
Van Der Heyden JC, Rotteveel JJ, Wevers RA.
Decreased homovanillic acid concentrations in
cerebrospinal fluid in children without a known
defect in dopamine metabolism. Eur J Paediatr
Neurol 2003;7:31–7.
Garcia-Cazorla A, Serrano M, Perez-Duenas B,
Gonzalez V, Ormazabal A, Pineda M, et al. Secondary abnormalities of neurotransmitters in infants with neurological disorders. Dev Med Child
Neurol 2007;49:740 – 4.
Spada M, Ferraris S, Ferrero GB, Sartore M, Lanza
C, Perfetto F, et al. Monitoring treatment in tetrahydrobiopterin deficiency by serum prolactin.
J Inherit Metab Dis 1996;19:231–3.
Dunnette J, Weinshilboum R. Inheritance of low
immunoreactive human plasma dopamine-betahydroxylase: radioimmunoassay studies. J Clin
Invest 1977;60:1080 –7.
Garland EM, Hahn MK, Ketch TP, Keller NR, Kim
CH, Kim KS, et al. Genetic basis of clinical catecholamine disorders. Ann N Y Acad Sci 2002;971:
506 –14.
Timmers HJ, Deinum J, Wevers RA, Lenders JW.
Congenital dopamine-beta-hydroxylase deficiency
in humans. Ann N Y Acad Sci 2004;1018:520 –3.
Chiu TC, Lin YW, Huang YF, Chang HT. Analysis
of biologically active amines by CE. Electrophoresis 2006;27:4792– 807.
Manini P, Andreoli R, Cavazzini S, Bergamaschi E,
Mutti A, Niessen WM. Liquid chromatographyelectrospray tandem mass spectrometry of acidic
monoamine metabolites. J Chromatogr B Biomed
Sci Appl 2000;744:423–31.
Parnetti L, Gottfries J, Karlsson I, Langstrom G,
Gottfries CG, Svennerholm L. Monoamines and
their metabolites in cerebrospinal fluid of patients
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
with senile dementia of Alzheimer type using
high performance liquid chromatography and gas
chromatography-mass spectrometry. Acta Psychiatr Scand 1987;75:542– 8.
Tsunoda M. Recent advances in methods for the
analysis of catecholamines and their metabolites.
Anal Bioanal Chem 2006;386:506 –14.
Peaston RT, Weinkove C. Measurement of catecholamines and their metabolites. Ann Clin
Biochem 2004;41:17–38.
Hyland K, Smith I, Howells DW, Clayton PT, Leonard JV. The determination of pterins, biogenic
amine metabolites and aromatic amino acids in
cerebrospinal fluid using isocratic reverse phase
liquid chromatography with in series dual cell
coulometric electrochemical and fluorescence
detection: use in the study of inborn errors of
dihydropteridine reductase and 5,10-methylenetetrahydrofolatye reductase. In: Wachter H,
Curtius HCh, Pfleiderer W, eds. Biochemical and
Clinical Aspects of Pteridines. Vol. 4. New York:
Gruyter. 1985;85–99.
Hyland K, Surtees RA, Heales SJ, Bowron A, Howells DW, Smith I. Cerebrospinal fluid concentrations of pterins and metabolites of serotonin and
dopamine in a pediatric reference population.
Pediatr Res 1993;34:10 – 4.
Scheinin M, Chang WH, Jimerson DC, Linnoila M.
Measurement of 3-methoxy-4-hydroxyphenylglycol in human plasma with high-performance liquid chromatography using electrochemical detection. Anal Biochem 1983;132:165–70.
Ormazabal A, Garcia-Cazorla A, Fernandez Y,
Fernandez-Alvarez E, Campistol J, Artuch R. HPLC
with electrochemical and fluorescence detection
procedures for the diagnosis of inborn errors of
biogenic amines and pterins. J Neurosci Methods
2005;142:153– 8.
Candito M, Nagatsu T, Chambon P, Chatel M.
High-performance liquid chromatographic measurement of cerebrospinal fluid tetrahydrobiopterin, neopterin, homovanillic acid and 5-hydroxindoleacetic acid in neurological diseases.
J Chromatogr B Biomed Appl 1994;657:61– 6.
Yamamoto H. Changes in CSF neurotransmitters
during the first year of life. Pediatr Neurol 1991;
7:406 –10.
Baig S, Halawa I, Qureshi GA. High performance
liquid chromatography as a tool in the definition
of abnormalities in monoamine and tryptophan
metabolites in cerebrospinal fluid from patients
with neurological disorders. Biomed Chromatogr
1991;5:108 –12.
Brautigam C, Weykamp C, Hoffmann GF, Wevers
RA. Neurotransmitter metabolites in CSF: an external quality control scheme. J Inherit Metab Dis
2002;25:287–98.
Matson WR, Langlais P, Volicer L, Gamache PH,
Bird E, Mark KA. n-Electrode three-dimensional
liquid chromatography with electrochemical detection for determination of neurotransmitters.
Clin Chem 1984;30:1477– 88.
Hyland K, Clayton PT. Aromatic L-amino acid decarboxylase deficiency: diagnostic methodology.
Clin Chem 1992;38:2405–10.
Howells DW, Hyland K. Direct analysis of tetrahydrobiopterin in cerebrospinal fluid by high-performance liquid chromatography with redox
electrochemistry: prevention of autoxidation during storage and analysis. Clin Chim Acta 1987;
167:23–30.
57. Blau N, Bonafe L, Thony B. Tetrahydrobiopterin
deficiencies without hyperphenylalaninemia: diagnosis and genetics of dopa-responsive dystonia
and sepiapterin reductase deficiency. Mol Genet
Metab 2001;74:172– 85.
58. Bertilsson L, Asberg M. Amine metabolites in
the cerebrospinal fluid as a measure of central
neurotransmitter function: methodological aspects. Adv Biochem Psychopharmacol 1984;39:
27–34.
59. Chotai J, Murphy DL, Constantino JN. Cerebrospinal fluid monoamine metabolite levels in human newborn infants born in winter differ from
those born in summer. Psychiatry Res 2006;145:
189 –97.
60. Blennow K, Wallin A, Gottfries CG, Mansson JE,
Svennerholm L. Concentration gradients for
monoamine metabolites in lumbar cerebrospinal
fluid. J Neural Transm Park Dis Dement Sect
1993;5:5–15.
61. Kruesi MJ, Swedo SE, Hamburger SD, Potter WZ,
Rapoport JL. Concentration gradient of CSF
monoamine metabolites in children and adolescents. Biol Psychiatry 1988;24:507–14.
62. Blennow K, Wallin A, Gottfries CG, Karlsson I,
Mansson JE, Skoog I, et al. Cerebrospinal fluid
monoamine metabolites in 114 healthy individuals 18 – 88 years of age. Eur Neuropsychopharmacol 1993;3:55– 61.
63. Hyland K. Abnormalities of biogenic amine metabolism. J Inherit Metab Dis 1993;16:676 –90.
64. Strawn JR, Ekhator NN, Geracioti TD Jr. In-use
stability of monoamine metabolites in human
cerebrospinal fluid. J Chromatogr B Biomed Sci
Appl 2001;760:301– 6.
65. Sheline Y, Bardgett ME, Csernansky JG. Correlated reductions in cerebrospinal fluid 5-HIAA
and MHPG concentrations after treatment with
selective serotonin reuptake inhibitors. J Clin Psychopharmacol 1997;17:11– 4.
66. Bowden CL, Koslow SH, Hanin I, Maas JW, Davis
JM, Robins E. Effects of amitriptyline and imipramine on brain amine neurotransmitter metabolites in cerebrospinal fluid. Clin Pharmacol Ther
1985;37:316 –24.
67. Giros B, Jaber M, Jones SR, Wightman RM,
Caron MG. Hyperlocomotion and indifference
to cocaine and amphetamine in mice lacking
the dopamine transporter. Nature (Lond) 1996;
379:606 –12.
68. Xu F, Gainetdinov RR, Wetsel WC, Jones SR,
Bohn LM, Miller GW, et al. Mice lacking the
norepinephrine transporter are supersensitive
to psychostimulants. Nat Neurosci 2000;3:465–
71.
69. Bengel D, Murphy DL, Andrews AM, Wichems
CH, Feltner D, Heils A, et al. Altered brain serotonin homeostasis and locomotor insensitivity to
3,4-methylenedioxymethamphetamine (“Ecstasy”)
in serotonin transporter-deficient mice. Mol Pharmacol 1998;53:649 –55.
70. Wang YM, Gainetdinov RR, Fumagalli F, Xu F,
Jones SR, Bock CB, et al. Knockout of the vesicular monoamine transporter 2 gene results in
neonatal death and supersensitivity to cocaine
and amphetamine. Neuron 1997;19:1285–96.
71. Chen L, Zhuang X. Transgenic mouse models of
dopamine deficiency. Ann Neurol 2003;54 (Suppl
6):S91–102.
Clinical Chemistry 54:4 (2008) 641