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Clinica Chimica Acta 298 (2000) 55–68
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Carnitine-acylcarnitine translocase deficiency:
metabolic consequences of an impaired mitochondrial
carnitine cycle
a,
*, Ania C. Muntau a , Marinus Duran b ,
¨
Wulf Roschinger
b
Lambertus Dorland , Lodewijk IJlst c , Ronald J.A. Wanders c ,
Adelbert A. Roscher a
a
Department of Pediatrics, Ludwig-Maximilians-University Munich, D-80337 Munich, Germany
b
Department of Pediatrics, University Children’ s Hospital, Wilhelmina Kinderziekenhuis,
NL-3584 EA Utrecht, The Netherlands
c
Department of Pediatrics and Clinical Chemistry, University Hospital Amsterdam,
NL-1105 AZ Amsterdam, The Netherlands
Received 22 November 1999; received in revised form 21 February 2000; accepted 23 February 2000
Abstract
We describe a patient with carnitine-acylcarnitine translocase deficiency (MIM 212138), who
presented with neonatal generalized seizures, heart failure, and coma. Laboratory evaluation
revealed hypoglycemia, hyperammonemia, lactic acidemia, hyperuricemia, and mild dicarboxylic
aciduria. The fact that total plasma carnitine (7.1 mmol / l [20–30]) and free carnitine (1.9 mmol / l
[12–18]) were low together with a high acylcarnitine / free carnitine ratio of 2.7 [0.4–1.0]
prompted acylcarnitine analysis. This revealed the presence of large amounts of long-chain
derivatives including C 16:0 , C 16:1 , C 18:1 , C 18:2 . Based on these findings carnitine-acylcarnitine
translocase deficiency was suspected which was confirmed by enzyme studies in fibroblasts. The
underlying complex metabolic consequences of this defect are reviewed. Prenatal diagnosis was
performed in a subsequent pregnancy and a defect ruled out by measurement of carnitineacylcarnitine translocase activity in cultured chorionic villi cells. As the clinical recognition of a
life-threatening fatty acid oxidation disorder may be difficult, defects in this pathway should be
considered in any child with coma, an episode of a Reye-like syndrome, and cardiomyopathy.
Since routine laboratory tests often do not provide clues about potential disorders and profiles of
urinary organic acids may not be characteristic, we recommend to measure free carnitine and
*Corresponding author. Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital, Lind¨
wurmstr. 4, D-80337 Munchen,
Germany. Tel.: 1 49-89-5160-2811 or 3167; fax: 1 49-89-5160-3320.
¨
E-mail address: [email protected] (W. Roschinger)
0009-8981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0009-8981( 00 )00268-0
56
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et al. / Clinica Chimica Acta 298 (2000) 55 – 68
W. Roschinger
acylcarnitines in plasma in any child with hyperammonemia, hypo / hyperketotic hypoglycemia or
lactic acidemia for prompt treatment, proper genetic counseling, and potential prenatal diagnosis.
 2000 Elsevier Science B.V. All rights reserved.
Keywords: Carnitine-acylcarnitine translocase; Fatty acid oxidation; Hyperammonemia; Carnitine
deficiency; Neonatal coma; Prenatal diagnosis
1. Introduction
Glucose is the major source of energy for the fetus [1]. Immediately after
birth free fatty acids are mobilized from adipose tissue stores. A rapid increase
in the activity of carnitine palmitoyltransferase I and II and a rise in the capacity
to oxidize fatty acids is found in liver [2] and in heart [3] reflecting a prompt
adaptation to lipid as the essential metabolic fuel for the newborn [4]. Since
more than 40% of total calories in human milk and many formula diets are
derived from lipid, carnitine becomes an essential cofactor for energy production. In addition, carnitine plays a key role in the homeostasis of Coenzyme
A (CoA) related compounds. Apart from its primary function in the transport of
long chain fatty acids across the inner mitochondrial membrane [5], carnitine
also plays a central role in buffering the intramitochondrial pool of CoA by
exporting acylcarnitine esters from the intramitochondrial space to the cytosolic
compartment followed by its transport across the plasma membrane as known in
a variety of organic acidemias [6–8]. Furthermore, there is evidence suggesting
that peroxisomal b-oxidation is also dependent on carnitine [9].
The pathway of plasma membrane carnitine and fatty acid uptake, mitochondrial translocation of fatty acids, and intramitochondrial fatty acid boxidation plays also a major role in energy production during prolonged fasting.
Thirteen genetic defects in man have been recognized [10] with four disorders
affecting the carnitine cycle responsible for the plasma membrane carnitine
transport and shuttling long chain fatty acids from the cytosol into the
intramitochondrial space.
We report here a patient with complete deficiency of carnitine-acylcarnitine
translocase (CACT), a mitochondrial membrane carrier protein and component
of the carnitine cycle, that facilitates the transfer of acyl groups by catalyzing a
reversible exchange-diffusion of carnitine and acylcarnitines [11]. Human CACT
was cloned [12] and the gene was mapped to chromosome 3p21.31 [13]. The
structure and organization of the gene have been elucidated [14] and various
mutations have been described [12,15]. We also report prenatal exclusion of this
enzyme defect in a subsequent pregnancy. The clinical and biochemical
characteristics of our patient exemplified the severe metabolic consequences of
an impaired mitochondrial carnitine cycle that are delineated in this report.
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2. Case report
The female patient was born at term as the first child after an uneventful
pregnancy and normal delivery to consanguineous Turkish parents. The patient’s
birth weight (3180 g) and length (50 cm) were at the 50th percentile; head
circumference was 36 cm (90th percentile). She was breast-fed. At the age of 48
h she had a generalized seizure and respiratory distress associated with
tachycard arrhythmia. The blood glucose concentration was decreased (1.65
mmol / l [2.2–6.6]). The patient needed cardiopulmonary resuscitation for 2 h.
She was treated with intravenous glucose, epinephrine, calcium and mechanical
ventilation.
Initial laboratory evaluation revealed compensated metabolic acidosis (capillary blood: pH 7.49, BE 2 9, HCO 2
3 9.6 mmol / l, pCO 2 13 mmHg), hyperammonemia (188 mmol / l [ , 80]), lactic acidemia (4.4 mmol / l [0.5–1.4]),
hyperuricemia (798 mmol / l [119–417]), and normal glucose (3.5 mmol / l
[2.2–6.6]). At the age of 3 days activities of alanine aminotransferase (80 IU / l
[ , 26]), aspartate aminotransferase (130 IU / l [ , 26]), and creatine kinase
(5310 IU / l [10–80]) were elevated without an increased CK-MB fraction
(CK-MB-NAC 249 U / l,% CK-MB 4.7% [ , 6%]) or myoglobinuria. LDH was
normal (495 U / l [ , 1500]). Cerebrospinal fluid showed no pleocytosis,
increased protein (2.87 g / l [ , 1.5]), low glucose (1.8 mmol / l [1.5–4.4]).
After the initial crisis, the child was placed on a high-carbohydrate (18
g / kg 3 day), low-protein (1.1 g / kg 3 day), and low-fat diet (3.8 g / kg 3 day)
with medium chain triacylglycerol (MCT) and carnitine supplementation (100
mg / kg 3 day). Between 2 and 13 days of age, ammonia concentrations ranged
from 56 to 198 mmol / l with decompensation (495 mmol / l) following a stepwise
increase of protein intake exceeding 1.2 g / kg 3 day. The clinical condition
improved gradually and the patient was discharged from the intensive care unit
after 12 days. At 3 weeks of age the neurologic examination and developmental
assessment were normal. From the age of 6 weeks the patient developed
significant muscle weakness with persistent head lag at traction and absent deep
tendon reflexes. Gross motor development was severely delayed. Furthermore,
strabismus with weak ocular fixation was noted. Clinical follow up demonstrated
hepatomegaly and the development of progressive microcephaly with hydrocephalus e vacuo, ventricular enlargement, and cystic changes in the right
choroid plexus. During the initial metabolic crisis an electroencephalography
showed diffusely lowered amplitudes.
At 5 months of age, weight was 6500 g (50th percentile) and length was 60
cm (3rd percentile). Echocardiography showed left and right ventricular
hypertrophy, an atrial septal defect (0.7 cm) of the ostium secundum type with
demonstration of a left-to-right shunt, slight elevation of the pulmonary artery
pressure, and subtle pericardial effusion. Electrocardiography was normal.
Accidental fasting periods provoked episodes of severe hypoketotic hypo-
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glycemia, metabolic acidosis, hyperammonemia, and coma which responded to
intravenous glucose administration.
Increasing difficulty with poor oral intake, inadequate weight gain, intermittent vomiting, and recurrent aspiration pneumonia with focal infiltrates and
dystelectasis due to gastroesophageal reflux required a gastrostomy tube. In
addition, an impaired motility of the whole gastrointestinal tract was noted.
Since the age of 5 months-with an interruption of 1 week-the patient required
mechanical ventilation until she died at 7 months of age of pneumonia and
cardiorespiratory failure.
3. Materials and methods
Urinary organic acid analysis was done by a modification of the method of
Hoffmann et al. [16] by capillary gas chromatography of trimethylsilyl esters
and O-(2,3,4,5,6-pentafluorobenzyl)oximes of oxoacids formed after ethyl
acetate extraction. Metabolites were identified on the basis of their retention time
and confirmed by mass spectrometry.
Qualitative analysis of plasma long-chain acylcarnitines was performed by
Fast Atom Bombardment mass spectrometry (FAB-MS). Isolation of the
substances was carried out by solid-phase extraction [17]. Subsequently, butyl
esters were prepared and FAB-MS was done using methanol–glycerol–3nitrobenzylalcohol (2:1:1, v / v / v) as matrix.
Quantitative analysis of plasma acylcarnitines was performed by electrospray
tandem mass spectrometry (ESI-MS–MS) using a modification of the method of
Chace et al. [18]. A 3.0 mm-diameter disk was punched from a filter paper into a
96 well microtiter plate and spotted with 2 ml plasma. After addition of 50 ml of
an internal standard mixture containing free 2 H 3 -carnitine, 2 H 3 -butyrylcarnitine,
2
H 3 -octanoylcarnitine, and 2 H 3 -palmitoylcarnitine the spot was extracted with
150 ml methanol. The final concentrations of the stable isotope labelled internal
standards were 7.5 to 37.5 pmol / sample. The methanolic extract was transferred
to a second plate, evaporated to dryness in a vacuum concentrator (SpeedVac  )
at room temperature and derivatized with 50 ml of 3 mol / l HCl in n-butanol at
658C for 20 min. After evaporation of excess HCl–butanol the residue was
reconstituted with 100 ml of acetonitrile:water (1:1, v / v) containing 0.025%
formic acid and injected to the tandem mass spectrometer. Analysis was
performed on a tandem mass spectrometer PE-SCIEX API 365 LC–MS–MS
system equipped with a PE SCIEX Series 200 lp HPLC pump and a PE SCIEX
Series 200 autosampler for automated sample injection and solvent delivery (PE
SCIEX, Toronto, Canada).
Overall fatty acid b-oxidation in intact fibroblasts was measured essentially as
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59
described by Olpin et al. [19] using [9,10– 3 H] tetradecanoic acid and [9,10– 3 H]
hexadecanoic acid as substrates.
All enzymes were measured according to published procedures as reviewed
by Wanders et al. [20]. The activity of very-long-chain acyl-CoA dehydrogenase
(VLCAD) was measured spectrophotometrically using ferricenium [21]. EnoylCoA hydratase activity was measured spectrophotometrically at 340 nm using
crotonyl-CoA (C 4:1 -CoA) and dodecene-2-oyl-CoA (C 12:1 -CoA) as substrates as
described [22]. 3-Hydroxyacyl-CoA dehydrogenase activity was measured in the
backward reaction at 340 nm using acetoacetyl-CoA (C 4:0 ) and 3-oxohexadecanoyl-CoA (C 16:0 ) as substrates (see [22] for details). 3-Oxoacyl-CoA
thiolase activity was measured spectrophotometrically at 303 nm using 3oxohexadecanoyl-CoA as substrate [22]. Mitochondrial carnitine-acylcarnitine
translocase activity was measured according to a newly developed method
(Wanders et al., in preparation) based on measurement of the release of [ 14 C]
CO 2 from enzymatically prepared [ 14 C] acetylcarnitine in digitonin-permeabilized fibroblasts.
The long chain triacylglycerol loading test was performed after a 3 h fast
using a test dose of 1.5 g / kg body weight of sunflower oil. Blood glucose,
3-hydroxybutyrate, and free fatty acids were assayed before, 1 and 3 h after
loading. Organic acid analysis was performed 0–3 and 3–6 h after the load.
The 3-phenylpropionic loading test was performed by using a test dose of 25
mg / kg body weight and analyzing organic acids before, 0–6 and 6–12 h after
the load.
4. Results
The underlying complex metabolic consequences of an impaired mitochondrial carnitine cycle are summarized in Table 1 and reviewed in Section 5.
At 2 days of age the profile of organic acids excreted in the urine showed a
subtle elevation of dicarboxylic acids (C 6:0 , C 8:0 , C 10:0 , C 10:1 , C 12:0 ) and of
5-hydroxyhexanoic acid. A marked output of lactic and pyruvic acid was found
without elevation of orotic acid or ketones. Controls at 9 and 10 days of age
again revealed a mild dicarboxylic aciduria and increased output of
methylmalonic, methylcitric, and 3-hydroxypropionic acid but without any
elevation of propionylglycine, 3-hydroxyvaleric, or lactic acid. The profiles of
organic acids and fatty acids in the plasma were normal, whereas the total free
fatty acids–3-hydroxybutyrate ratio of 22.5 was elevated. Cis-4-decenoic acid
and cis-5-tetradecenoic acid, markers of medium-chain acyl-CoA dehydrogenase
and very long-chain acyl-CoA dehydrogenase deficiency were not increased.
The quantitation of carnitines in plasma at the age of 2 days revealed very low
concentrations for total plasma carnitine (7.1 [20–30]) and free carnitine (1.9
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Table 1
Suggested pathobiochemical basis for metabolic consequences of an impaired mitochondrial
carnitine cycle
Laboratory findings
Suggested pathobiochemical basis
Hypoglycemia
? Depletion of hepatic glycogen stores
? ATP, GTP, ITP ↓:
Pyruvate carboxylase ↓ (gluconeogenesis)
Phosphoenolpyruvate carboxykinase ↓ (gluconeogenesis)
? Acetyl-CoA ↓:
Pyruvate carboxylase ↓ (gluconeogenesis)
Pyruvate dehydrogenase ↑ (citric acid cycle or lipogenesis)
? Citrate ↓:
Acetyl-CoA carboxylase ↓ (lipogenesis)
6-Phosphofructose-1-kinase ↑ (glycolysis)
Fructose-1,6-bisphosphate ↑
Pyruvate kinase ↑ (glycolysis)
? Acetyl-CoA ↓, 3-Oxobutyryl-CoA ↓
starting molecules for ketogenesis
? Acetyl-CoA ↓:
N-acetylglutamate ↓
Carbamoylphosphate synthetase ↓ (urea cycle)
? Propionyl-CoA ↑:
N-acetylglutamate synthetase ↓ (urea cycle)
? Active v- and (v-1)-oxidation
Block of transmitochondrial
long chain fatty acid transport
Hypoketosis
Hyperammonemia
Dicarboxylic aciduria
Elevation of
acylcarnitines
[12–18]) with relatively elevated acylcarnitines (5.2 mmol / l [6–14]) reflected in
a high acylcarnitine-free carnitine ratio (2.7 [0.4–1.0]). On treatment with
L-carnitine (100 mg / kg 3 day) the plasma total carnitine level was 41 [40–54],
free carnitine 9.5 [29–42], and acylcarnitines 31.5 [9–15] mmol / l (normal range
related to the age of 3 months). A long chain triacylglycerol loading test resulted
in an increased excretion of dicarboxylic acids (C 6:0 , C 7:0 , C 8:0 , C 8:1 , C 9:0 , C 10:0 ,
C 10:1 , C 12:0 , C 12:1 ), 5-hydroxyhexanoic acid and 7-hydroxyoctanoic acid. Before
load, 1 h, and 3 h after load glucose was 4.7, 4.6, 4.2 mmol / l, 3-hydroxybutyrate was 0.24, 0.79, 0.36 mmol / l, total free fatty acids were 0.37, 0.88, 1.38
mmol / l, and ratios of total free fatty acids–3-hydroxybutyrate were 1.54, 1.11,
3.83. A loading test with 3-phenylpropionate was normal.
Qualitative analysis of long-chain acylcarnitines in the plasma by direct fast
atom bombardment-mass spectrometry revealed a profile of long-chain derivatives with large amounts of C 16:0 , C 16:1 , C 18:1 , and C 18:2 . A quantitative
acylcarnitine profile in the plasma generated by electrospray tandem mass
spectrometry is shown in Table 2.
At the age of 6 months enzymological studies in cultured skin fibroblasts from
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Table 2
Acylcarnitine profile and free carnitine in the plasma of our CACT-deficient patient. The profile
was determined using electrospray tandem mass spectrometry (ESI-MS–MS)a
Species
Free Carnitine
C 2 -Acylcarnitine
C 4:0 -Acylcarnitine
C 6:0 -Acylcarnitine
C 6:0 -Dicarboxylic–Acylcarnitine
C 8:0 -Acylcarnitine
C 10:0 -Acylcarnitine
C 10:1 -Acylcarnitine
C 10:2 -Acylcarnitine
C 12:0 -Acylcarnitine
C 12:0 -Dicarboxylic-Acylcarnitine
C 14:0 -Acylcarnitine
C 14:1 -Acylcarnitine
C 14:2 -Acylcarnitine
C 16:0 -Acylcarnitine
C 16:1 -Acylcarnitine
C 18:0 -Acylcarnitine
C 18:1 -Acylcarnitine
C 18:2 -Acylcarnitine
3-Hydroxy-C 14:0 -Acylcarnitine
3-Hydroxy-C 16:0 -Acylcarnitine
3-Hydroxy-C 16:1 –Acylcarnitine
3-Hydroxy-C 18:1 -Acylcarnitine
a
Acyl Group
Acetyl
Butyryl
Hexanoyl
Octanoyl
Decanoyl
Decenoyl
Decadienoyl
Dodecanoyl
Tetradecanoyl
Tetradecenoyl
Tetradecadienoyl
Hexadecanoyl
Hexadecenoyl
Octadecanoyl
Octadecenoyl
Octadecadienoyl
mmol / l
Range
19.0
4.40
1.03
1.22
0.47
0.55
0.72
0.22
0.03
1.26
0.30
1.32
0.67
0.15
18.7
1.94
2.00
3.50
0.71
0.06
0.16
0.16
0.11
11.8–60.5
0.47–6.73
0.14–0.86
0.11–0.77
0.00–0.08
0.04–0.34
0.01–0.17
0.02–0.17
0.01–0.10
0.03–0.32
0.01–0.18
0.03–0.25
0.02–0.23
0.01–0.14
0.39–2.70
0.01–0.16
0.15–1.23
0.14–1.19
0.03–0.42
0.00–0.05
0.00–0.04
0.00–0.09
0.00–0.04
Number of controls: 450
the patient revealed a complete deficiency of carnitine-acylcarnitine translocase
in the inner mitochondrial membrane, whereas all mitochondrial enzymes
involved in long-chain fatty acid b-oxidation showed normal activities (Table
3).
Post mortem liver biopsy showed normal lobular architecture with vesicular
transformation of hepatocytes reflecting cellular edema with extensive macrovesicular neutral fat accumulation. There were no signs of inflammatory
infiltrations, necrotic changes, cholestasis or fibrosis. Electron microscopy
revealed hepatocytes with severe accumulation of neutral lipids and small
amounts of non lysosomal bound glycogen. Mitochondria were normal in size
and shape. Myocardial biopsy revealed regular cellular nuclei with a normal
myofibrillar architecture. There were no inflammatory or necrotic changes.
Prenatal diagnosis was performed in a subsequent pregnancy after chorionic
villi biopsy at week 12 of gestation. We found both normal b-oxidation flux and
normal activity of mitochondrial CACT. Biochemical investigations with
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Table 3
b-oxidation flux and activities for mitochondrial enzymes involved in long-chain fatty acid
breakdown in cultured skin fibroblasts from our patient, her healthy brother, and from controls.
The values are expressed as means and standard deviations (mean6S.D.)
Overall b-oxidation
(nmol / h 3 mg protein)
nb
Patient
Brother
Controls
0.26 a
0.01 a
4.41
5.02
5.8162.27
8.1563.78
76
46
26 a
0.88
0.8560.35
43
Acyl-CoA dehydrogenase
Substrate: Hexadecanoyl-CoA (C 16:0 )
9.0
15.8
14.5365.83
74
Enoyl-CoA hydratase
Substrate: Dodecene-2-oyl-CoA (C 12:1 )
46
63
78625
59
118
52
99
67
99.5632.1
81.8622.8
105
102
0.44
0.68
0.8660.20
102
9.4
14.8
20.6467.79
47
0.01
23.3
7.93
49.8613.5
6.0861.30
8
8
Substrate:
[9,10– 3 H] Tetradecanoic acid (C 14:0 )
[9,10– 3 H] Hexadecanoic acid (C 16:0 )
Activity ratio:
[9,10– 3 H] C 14:0 / [9,10– 3 H] C 16:0
Enzyme activity measured
(nmol / min 3 mg protein)
3 -Hydroxyacyl-CoA dehydrogenase
Substrate:
3-Oxobutyryl-CoA, Acetoacetyl-CoA (C 4:0 )
3-Oxohexadecanoyl-CoA (C 16:0 )
Activity ratio:
3-Oxohexadecanoyl-CoA–3-Oxobutyryl-CoA
Long-Chain 3 -Oxoacyl-CoA thiolase
Substrate: 3-Oxohexadecanoyl-CoA (C 16:0 )
Carnitine-Acylcarnitine translocase
(pmol / min 3 mg protein)
CO 2 -synthesis from [ 14 C] Acetylcarnitine (C 2 )
ATP-synthesis from ADP 1 succinate / rotenon
a
b
Mean of three separate experiments.
n: Number of controls.
determination of glucose, lactate, and ammonia in the plasma, analysis of
organic acids in the urine and of acylcarnitines in the plasma were performed at
the first day of life and were repeated after 1 and 2 weeks. All values obtained
were within normal range. Enzyme analyses in cultured skin fibroblasts (Table
3) from the newborn child revealed normal activity of CACT confirming our
analysis in chorionic villi.
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5. Discussion
A neonatal generalized seizure with coma in a child of consanguineous
parents presenting with hyperammonemia prompted a metabolic assessment to
differentiate a urea cycle defect, an organic acidemia, or a transient hyperammonemia of the newborn. Although initial laboratory evaluation revealed
hypoglycemia, metabolic acidosis, an elevated anion gap, lactic acidemia, and
hyperuricemia suggesting an organic acidemia, all changes could also be
explained by transient anoxia during cardiopulmonary resuscitation. Neither
amino acid analysis in plasma and urine nor the analysis of organic acids in
urine allowed a distinct diagnosis.
Control investigations revealed a pattern of metabolites, typically seen in
patients with mild methylmalonic acidemia. This reflects the accumulation of
intramitochondrial propionyl CoA and methylmalonyl CoA and is possibly
aggravated by the accompanying carnitine deficiency that is expected to result in
impaired renal clearance of propionic and methylmalonic acid by decreased
formation of the acylcarnitine esters.
Initial hypoglycemia, low levels of ketones, dicarboxylic aciduria, and very
low concentrations of total plasma carnitine and free carnitine with a high
acylcarnitine–free carnitine ratio were suggestive of defects in the transport and
breakdown of fatty acids. We therefore performed a long chain triacylglycerol
and 3-phenylpropionic acid loading test. Marked excretion of dicarboxylic acids,
5-hydroxyhexanoic, and 7-hydroxyoctanoic acid combined with mobilized fatty
acids from adipose tissue stores ( . 1 mmol / l) and an elevated ratio of total free
fatty acids / 3-hydroxybutyrate (also noted without loading) reflected an active vand (v-1)-oxidation with impaired ketogenesis. Regular breakdown of the
odd-chain, ‘‘phenyl-labeled’’ fatty acid 3-phenylpropionic acid ruled out a
medium chain acyl CoA dehydrogenase deficiency.
Direct fast atom bombardment-mass spectrometry of plasma acylcarnitines to
specify the acyl moities attached to the carrier molecule carnitine revealed an
impressive profile of long-chain derivatives with large amounts of C 16:0 , C 16:1 ,
C 18:1 , and C 18:2 without an elevation of C 14:1 or C 14:2 – also not found as free
acids in the plasma-excluding a deficiency of very long-chain acyl CoA
dehydrogenase. Besides a peak at mass (m /z) 496, possibly referring to 3hydroxy-C 18:2 , no 3-hydroxy-C 18:1 was detected making a deficiency of longchain 3-hydroxy acyl CoA dehydrogenase unlikely.
The suggested differential diagnosis of carnitine palmitoyl transferase II or
carnitine-acylcarnitine translocase could be clarified by enzymologic investigations in fibroblasts showing a complete deficiency of the mitochondrial
carnitine-acylcarnitine carrier. In our metabolic workup — as in the patients of
Stanley and Ogier de Baulny [23,24] — we could not confirm absence of
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dicarboxylic aciduria as previously suggested being characteristic for disorders
of the carnitine cycle [25,26].
Dicarboxylic aciduria–in contrast to the acylcarnitines–only up to a chain
length of C 12 (C 6:0 , C 8:0 , C 10:0 , C 10:1 , C 12:0 ) with and without a long chain
triacylglycerol loading reflects observations about 60 years ago that had shown a
significant dicarboxylic aciduria derived from the corresponding chain length
C 9 –C 11 –monocarboxylic acids and a smaller output from C 8 and C 12 monocarboxylic acids after loading. In contrast, no dicarboxylic aciduria was detected
after administration of monocarboxylic acids of shorter or longer chain length
[27].
Elevated concentrations of acylcarnitines with a chain length of C 16 and C 18
demonstrate the block of transmitochondrial long chain fatty acid transport
preventing already the first round of b-oxidation initiated by the membranebound very long chain acyl CoA dehydrogenase.
Hypoglycemia under fasting is believed to be both the result of rapid
depletion of hepatic glycogen stores and hampered gluconeogenesis. The
inadequate rate of fatty acid oxidation is not able to produce considerable
quantities of ATP [28] required in the pyruvate carboxylase and (as GTP or ITP)
in the phosphoenolpyruvate carboxykinase reaction. In addition, accumulation of
long chain acyl-CoA may exert an inhibitory effect on mitochondrial adenin
nucleotide translocation in the intact liver cell [29]. The activation of pyruvate
carboxylase, key enzyme in the process of gluconeogenesis, and the inhibition of
pyruvate dehydrogenase are impaired by decreased acetyl CoA making the
sparing action of fatty acid oxidation on the oxidation of pyruvate and the
stimulation of gluconeogenesis impossible. In addition, insufficient formation of
citrate-derived from acetyl CoA channeled into the citric acid cycle [30]–both
inhibits acetyl-CoA carboxylase, the rate-limiting enzyme in the lipogenic
pathway, and stimulates 6-phosphofructose-1-kinase, the most important regulatory enzyme of glycolysis, producing fructose-1,6-bisphosphate, that–although
not proven in patients–activates pyruvate kinase and again slows down
gluconeogenesis at the level of phosphoenolpyruvate.
Hyperammonemia can arise from both elevated propionyl CoA [31] and low
concentrations of acetyl-CoA-as suggested for patients with organic acidurias
[32]-leading to limited availability of N-acetylglutamate, essential cofactor of
the urea cycle enzyme carbamoylphosphate synthase.
Hypoketosis is expected to result from the deficient fatty acid transport, since
the ketone bodies 3-hydroxybutyric and 3-oxobutyric (acetoacetic) acid are
derived from acetyl-CoA, the main product of this pathway. 3-oxobutyryl-CoA,
which is the starting material for ketogenesis, arises during the spiral of
b-oxidation or as a result of the condensation of 2 acetyl-CoA molecules.
Nevertheless, significant ketogenesis in liver is also known in b-oxidation
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defects [33] and may be explained by carnitine independent transport of
medium- and short-chain fatty acids across the mitochondrial membrane.
Clinically we observed a severe course with complete absence of psychomotor progress. Interestingly, in addition to a gastroesophageal reflux known in
patients with fatty acid oxidation defects [23,34], we noted an impaired motiliy
of the whole intestinal tract causing recurrent aspiration pneumonia.
Sixteen patients showing a severe phenotype have been described since 1992
[23,24,35–42]. One half died between 24 h and 3 weeks, and the other half
between 2 and 37 months of age. In addition, there have been reports of mild
phenotypes with normal growth and development in four children between 5
months and 4 years of age [40,41,43,44].
Inborn errors of metabolism can be rapidly lethal [39] and do often not cause
pathognomonic findings at autopsy. Since nonspecific symptoms like vomiting,
lethargy, and coma are not unusual in the immediate newborn period, pediatricians and neonatologists often consider more common conditions like sepsis,
toxic shock, or cardiac defects delaying an urgent metabolic workup. Since the
diagnosis of fatty acid oxidation defects is not straightforward and profiles of
urinary organic acids may not be characteristic, the assessment of the carnitine
status is critical to treat the patient promptly, to counsel the parents properly and
to be prepared for a possible prenatal diagnosis. That an increasing number of
screening centers have implemented an extended prospective newborn screening
program using tandem mass spectrometry will allow early detection of fatty acid
oxidation disorders and possibly even prevention of metabolic decompensation.
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