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Creatine metabolism in combined methylmalonic aciduria and homocystinuria.

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Creatine Metabolism in
Combined Methylmalonic
Aciduria and
Olaf A. Bodamer, MD,1,2 Trilochan Sahoo, MD,2
Arthur L. Beaudet, MD,2 William E. O’Brien, PhD,2
Teodoro Bottiglieri, PhD,3 Sylvia Stöckler-Ipsiroglu, MD,1
Conrad Wagner, PhD,4 and Fernando Scaglia, MD2
Methylation is an important aspect of many fundamental
biological processes including creatine biosynthesis. We
studied five patients with an inborn error of cobalamin
metabolism to characterize the relation between homocysteine and creatine metabolism. Plasma guanidinoacetate concentrations were increased, 14.9 ⴞ 4.8␮mol/L
(p < 0.0001), whereas plasma creatine concentrations
were in the low reference range, 43.8 ⴞ 20.7␮mol/L
(p ⴝ not significant). Individuals with combined methylmalonic aciduria and homocystinuria have a functional
impairment of the creatine synthetic pathway probably
secondary to a relative depletion of labile methyl groups.
The neurotoxic effects of guanidinoacetate may be partly
responsible for the observed neurological phenotype.
Ann Neurol 2005;57:557–560
(Trans-) Methylation contributes in decisive biological
processes including control of gene expression and
plays an essential role in cellular physiology.1 Labile
methyl groups are generated through a series of reactions that convert methionine to homocysteine (Fig).2
Homocysteine either is remethylated to form methionine in a cobalamin and folate or betaine-dependent
reaction, or it is degraded to ultimately yield sulfate.2
Most labile methyl groups are used for synthesis of creatine from guanidinoacetate (GAA), a reaction that is
catalyzed by the enzyme guanidinoacetate methyltransferase (GAMT) (see Fig).2
From the 1Unit of Biochemical Genetics, Department of Pediatrics,
University of Vienna Children’s Hospital, Vienna, Austria; 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston; 3Mass Spectrometry Unit, Baylor University Medical
Center, Dallas, TX; and 4Department of Biochemistry, Vanderbilt
University School of Medicine, Nashville, TN.
Received Jun 8, 2004, and in revised form Jan 17, 2005. Accepted
for publication Jan 17, 2005.
Published online Mar 28, 2005, in Wiley InterScience
( DOI: 10.1002/ana.20419
Address correspondence to Dr Bodamer, Unit of Biochemical Genetics, Department of Pediatrics, University of Vienna Children’s
Hospital, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
The importance of a functional creatine synthetic
pathway for normal neurological development was underscored by the identification of GAMT-deficient patients who have a highly variable spectrum of neurological and muscular symptoms.3 This condition is
characterized biochemically by significant increases of
GAA and low creatine levels predominantly in central
nervous system and skeletal muscle. Neurological
symptoms are alleviated and psychomotor development
may be improved by use of creatine supplementation.3
Combined methylmalonic aciduria and homocystinuria
(cblC) is caused by either impaired release of cobalamin from lysosomes (complementation group F
[cblF], Mendelian Inheritance in Man (MIM)
#277380) or by defects in intracellular cobalamin processing to form adenosylcobalamin and methylcobalamin (complementation group C [cblC], MIM
#277400; and complementation group D [cblD],
MIM #277410).4,5 CblC is caused by a yet undefined
molecular defect of intracellular cobalamin metabolism.4 It is the most common of the inborn errors of
cobalamin metabolism.5 This defect leads to impaired
remethylation of homocysteine, resulting in low methionine and high homocysteine concentrations, and to
neurological symptoms that include developmental delay, muscular hypotonia, seizures, and nystagmus.4,5
We hypothesized that the lack of methyl groups for
the methylation reaction mediated by GAMT might be
responsible for a clinical presentation in cblC similar to
that of GAMT-deficient patients. This hypothesis
prompted us to study a group of patients with cblC to
evaluate the integrity of their creatine synthesis by
measuring different metabolites of the creatine biosynthesis pathway.
Subjects and Methods
The clinical characteristics of the subjects with cblC in this
study have been reported previously.4,6 Anthropometric and
relevant clinical data are listed in Table 1. CblC was confirmed by complementation analysis in fibroblast cultures of
all patients (Dr D. Rosenblatt, McGill University). The clinical research study was performed in the General Clinical Research Center of Texas Children’s Hospital. The study protocol received prior approval by the Institutional Review
Board of Baylor College of Medicine (#11560).
On admission, the parents and, where appropriate, the patients themselves gave their informed consent. The patients
were admitted at 8 AM after an overnight fast. A cannula was
inserted and blood was drawn before and 1 hour after a meal
prepared according to the patient’s usual daily protein intake
at 8 AM, 12 PM, and 5 PM, respectively.
Subjects with cblC were on a low-protein diet (0.7–
1.5gm/kg/day) without supplementation of an amino acid
mixture. Patients were treated with 97 ⫾ 16mg L-carnitine
per kilogram per day, 288 ⫾ 14mg betaine per kilogram per
day, and a daily intramuscular injection of 1mg hydroxocobalamin. Subjects continued their diet and pharmacotherapy
throughout the study (see Table 1).
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Fig. Methionine, homocysteine, and creatine metabolism. Enzymes marked with an asterisk are those with known deficiencies.
(dashed line) Feedback control. AGAT ⫽ arginine-guanidinoacetate amidino methyltransferase; cbl I ⫽ cobalamin in redox state
I; cbl II ⫽ cobalamin in redox state II; GAMT ⫽ guanidinoacetate methyltransferase; THF ⫽ tetrahydrofolate.
Plasma GAA and creatine concentrations were analyzed
using liquid chromatography tandem mass spectrometry
(Waters 2690 LC System; Waters, Milford, MA; Quattro
LC; Micromass, Beverly, MA), as reported previously.7
Plasma amino acids and homocysteine concentrations were
analyzed using an amino acid analyzer and tandem mass
spectrometry (Quattro LC), respectively. Plasma methylmalonic acid was determined by isotope dilution using gas chromatography and mass spectrometry (Agilent System; Agilent
Technologies, Palo Alto, CA). Plasma S-adenosylmethionine
(SAM) and S-adenosylhomocysteine (SAH) concentrations
were analyzed as their fluorescent isoindoles by highperformance liquid chromatography (Waters 626 LC System), as described previously.8 Fluorescence was measured
with a Perkin-Elmer 650-40 instrument (excitation (ex) 400,
emmission (em) 490; Perkin-Elmer, Oak Brook, IL).8
The brain level of GAA and creatine was assessed using in
vivo proton magnetic resonance spectroscopy at the Department of Diagnostic Imaging, Texas Children’s Hospital, on a
1.5T whole body clinical magnetic resonance imaging scanner (Gyroscan Intera 1.5 T, software release 9; Philips Medical System, Best, The Netherlands) in four of the five patients (data not shown).
Mean and standard deviation were calculated using standard mathematical formulae. Statistical analysis was done using paired and unpaired Student’s t test. Significance was
assumed for p ⬍ 0.05.
Plasma concentrations of methylmalonic acid, methionine, homocysteine, SAM, SAH, GAA, and creatine
are listed in Table 2. There was no appreciable difference between preabsorptive and postabsorptive values
Table 1. Anthropometric and Clinical Data of Subjects with cblC
Patient No.
Age (yr)
Protein Intake
(g/mkg bw)
Early onset
Early onset
Early onset
Early onset
Late onset
Clinical presentation: early onset, severe clinical phenotype, presentation within the first year of life; late onset, mild clinical phenotype,
presentation after 15 years of life.
GFR ⫽ glomerular filtration rate.
Annals of Neurology
Vol 57
No 4
April 2005
Table 2. Plasma Concentrations (mean ⫾ SD) of
Methionine, Homocysteine, SAM, SAH, GAA,
Creatine and MMA
1.09 ⫾ 0.8 ␮mol/L
21.59 ⫾ 7 ␮mol/L
homocysteine 46.4 ⫾ 15.4 ␮mol/L
117.6 ⫾ 37.4 nmol/L
73.6 ⫾ 98.7 nmol/L
3.9 ⫾ 2.6
14.9 ⫾ 4.8 ␮mol/L
43.8 ⫾ 20.7 ␮mol/L
⬍ 0.5
SD ⫽ standard deviation; SAM ⫽ S-adenosylmethionine; SAH ⫽
S-adenosylhomocysteine; GAA ⫽ guanidinoacetate; MMA ⫽ methylmalonic aciduria; NS ⫽ not significant.
for the measured compounds in all five patients; therefore, they were combined. To estimate their significance,
we compared the means of these combined values with
the upper limit of the given reference range (for GAA,
SAH, homocysteine, and methylmalonic acid) and with
the lower limit of the given reference range (for creatine,
methionine, SAM, and SAM/SAH) by using a onesample t test (see Table 2). Plasma GAA concentrations
were significantly increased in all subjects with cblC,
whereas plasma creatine concentrations were within normal limits. Although plasma SAH concentrations were
not significantly increased, there was a trend toward
greater concentrations, whereas SAM concentrations
were essentially within the reference range (see Table 2).
Between 75 and 80% of the labile methyl groups generated through the remethylation pathway are used for
methylation of GAA by GAMT to yield creatine,
whereas the remainder is used for methylation of other
biomolecules including nucleic acids and histones (see
Fig).1,9,10 Therefore, it is conceivable that reduced supply of labile methyl groups for adequate methylation
may cause a wide range of predominantly neurological
symptoms, as observed in patients with cblC.10 In fact,
individuals with cblC have a broad spectrum of neurological symptoms including seizures and psychomotor
retardation that shows some overlap with that of patients with GAMT deficiency.3,4 However, we expect
that the pathomechanism of neurological disease in patients with cblC would be different than the one observed in GAMT deficiency because increased availability of labile methyl groups in GAMT deficiency should
not lead to impairment of global methylation reactions.
Deficient remethylation of homocysteine in individuals
with cblC and the reduced availability of labile methyl
groups with its likely impact on creatine synthesis led
us to study creatine and homocysteine metabolism in
five patients with cblC.4,6
Plasma GAA concentrations were significantly increased in all subjects with cblC to levels typically
found in patients with GAMT deficiency,3 whereas
creatine concentrations remained in the low-reference
to reference range. No diurnal fluctuation of plasma
GAA and creatine concentrations was observed, and no
effect could be attributed to dietary intake. Because
there was no decreased renal function as judged by the
observed glomerular filtration rates, the increased GAA
concentrations could be explained by partial inhibition
of GAMT through increased tissue SAH concentrations.11 In fact, Baric and colleagues12 recently reported a child with biochemically and molecularly
proven SAH hydrolase deficiency and significant increases of plasma SAH and GAA concentrations.
Prominent increases in plasma SAH concentrations
were found in most samples independently from dietary intake. Previous reports suggest that the concentration of SAH is a linear function of plasma homocysteine concentrations,10 which confirms our data. In
addition, substantial differences in tissue SAH distribution and concentration would preclude us to reach any
conclusions about SAH concentrations in the central
nervous system.10 In this respect, analysis of methylated compounds in cerebrospinal fluid in our patients
might have assisted in explaining some of our findings,
but cerebrospinal fluid sampling was not done because
of the structure of the research protocol.
Residual methylation of GAA and possibly adequate
dietary creatine intake were apparently sufficient to
keep plasma creatine concentrations within the lowreference to reference range. Magnetic resonance spectroscopy showed normal intracerebral creatine levels in
the four patients studied. One individual with cblC
demonstrated a peak at the position of GAA. The neurological phenotype of patients with cblC may be explained in part by the neurotoxic effects of GAA in
conjunction with the reduced activities of methyltransferases involved in neurotransmitter metabolism such
as catechol O-methyltransferase and phenylethanolamine N-methyltransferase.10,13 The neurotoxic effects
of GAA on the developing brain may be explained by
the interaction of GAA with GABAA receptors at a
molecular level.14 In addition, the neurological phenotype could also be mediated by a direct neurotoxic effect of homocysteine. In a recently published study of
113 patients with coronary heart disease, it was found
that increased levels of total plasma homocysteine were
associated with low concentrations of cerebral N-acetyl
aspartate and creatine, an effect that may be explained
by activation of the N-methyl-D-aspartate receptors.13,15
These findings lent further support to the hypothesis
that homocysteine could be neurotoxic, because it was
previously reported in a 10-month-old infant with
methylenetetrahydrofolate reductase deficiency, for
Bodamer et al: Creatine Metabolism in cblC
whom high levels of homocysteine were associated with
a decreased cerebral N-acetylaspartate concentration.16
We anticipate that GAA levels may fluctuate depending on the metabolic control and may therefore
constitute a useful marker of metabolic control in these
individuals. Supplementation with creatine monohydrate at a dose of 200mg/kg/day may be particularly
beneficial because synthesis of GAA is tightly regulated
through feedback inhibition. This novel therapeutic
approach could be used in conjunction with currently
available therapies such as GAA-decreasing therapy, as
reported in GAMT-deficient patients.17,18
This study was supported by grants from the NIH (National Institute
of Diabetes and Digestive and Kidney Diseases; DH15289;
5P30DH26557) and the United States Department of Veterans Affairs.
We are indebted to the families and patients for their continued
support. We acknowledge the support of the Baylor College of
Medicine Mental Retardation Research Center and the Clinical Nutrition Research Unit of Vanderbilt University School of Medicine.
We thank the excellent nursing staff at the Texas Children’s Hospital General Clinical Research Center.
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characteristics and diagnostic clues in inborn errors of creatine
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methylmalonic aciduria and homocystinuria. J Child Neurol
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(cblC). Neurology 2001;56:1113.
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proton MR spectroscopy of the brain in hyperhomocysteinemia
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guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation. Mol Genet Metab 2001;74:413– 419.
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and sequencing of rat kidney L-arginine:glycine amidinotransferase. Studies on the mechanism of regulation by growth hormone and creatine. J Biol Chem 1994;269:17556 –17560.
Fright Is a Provoking
Factor in Vanishing White
Matter Disease
Gerre Vermeulen, MD,1 Rainer Seidl, MD,2
Saadet Mercimek-Mahmutoglu, MD,2
Jan J. Rotteveel, MD, PhD,3 Gert C. Scheper, PhD,1
and Marjo S. van der Knaap, MD, PhD1
Leukoencephalopathy with vanishing white matter is an
inherited disorder with a chronic progressive disease
course and additional episodes of rapid neurological deterioration. These episodes typically are provoked by febrile infections or minor head trauma. We report on two
patients who experienced an episode of rapid neurological deterioration after a fright.
Ann Neurol 2005;57:560 –563
Leukoencephalopathy with vanishing white matter
(VWM), also known as childhood ataxia with central
From the 1Department of Pediatrics/Child Neurology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands; 2Department of Neuropediatrics, Vienna University, Vienna, Austria; and
Department of Pediatric Neurology, University Medical Center St
Radboud, Nijmegen, The Netherlands.
Received Nov 3, 2004, and in revised form Jan 7, 2005. Accepted
for publication Jan 17, 2005.
Published online Mar 28, 2005, in Wiley InterScience
( DOI: 10.1002/ana.20418
Address correspondence to Dr van der Knaap, Department of Pediatrics/Child Neurology, Vrije Universiteit Medical Center, P.O.
Box 7057, 1007 MB Amsterdam, The Netherlands.
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
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combined, creating, methylmalonic, metabolico, homocystinuria, aciduria
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