Elevation of 2-methylcitric acid I and II levels in serum, urine, and cerebrospinal fluid of patients with cobalamin deficiency

doi:10.1016/0026-0495(93)90010-L

Metabolism

Volume 42, Issue 8, August 1993, Pages 978-988

https://doi.org/10.1016/0026-0495(93)90010-L Get rights and content

Abstract

Citrate synthase catalyzes the condensation of acetyl-coenzyme A (CoA) and oxaloacetic acid to form citric acid. The enzyme also catalyzes the condensation of propionyl-CoA and oxaloacetic acid with a maximal reaction velocity (Vmax) approximately 10⁻⁴ times that of acetyl-CoA to form 2-methylcitric acid, which contains two asymmetric carbon atoms and exists as two pairs of related enantiomers designated as 2-methylcitric acid I and II. Cobalamin (Cbl) deficiency can lead to increases in intracellular levels of propionyl-CoA. To assess the magnitude of increased synthesis of 2-methylcitric acid in Cbl deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured 2-methylcitric acid levels in serum and cerebrospinal fluid (CSF) of normal subjects and patients with clinically confirmed Cbl deficiency. The normal range for 2-methylcitric acid level was 60 to 228 nmol/L for serum in 50 normal blood donors and 323 to 1,070 nmol/L for CSF in 19 normal subjects. In 50 patients with clinically confirmed Cbl deficiency, values for 2-methylcitric acid in serum ranged from 93 to 13,500 nmol/L; 44 (88%) had values above the normal range. In five patients with clinically confirmed Cbl deficiency, levels of the sum of 2-methylcitric acid I and II ranged from 1,370 to 16,300 nmol/L in CSF, and all five (100%) patients had levels above the normal range. We conclude that levels of 2-methylcitric acid are elevated in serum and CSF of most patients with Cbl deficiency.

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Samples were immediately centrifuged and plasma was collected, diluted in water, and frozen at −80°C until measurements were performed. Plasma methylmalonic acid and 2-methylcitrate levels were quantified by gas chromatography-mass spectrometry with stable isotopic internal calibration as previously described.50,51 Prism 9 version 9.2.0 by GraphPad was used to analyze all data.
Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of ∼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9-Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation.
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A discrepancy has been identified between numbers of expected and identified patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. Patients homozygous for the frequent c.833 T > C (p.I278T) are most often responsive to vitamin B₆, and can present with a total-homocysteine (tHcy) < 100 μM on a normal diet. In Denmark, patients with tHcy < 100 μM are not routinely sequenced for CBS² mutations. This study investigated the prevalence of CBS mutations and the common methylenetetrahydrofolate reductase (MTHFR) c.677C > T polymorphism in patients with tHcy ≥ 50 μM and the association with clinical manifestations. We studied a cohort of patients with intermediate and severe hyperhomocysteinemia (tHcy ≥ 50 μM) determined between 1996 and 2011. Among the 413 eligible patients, 184 (45%) patients agreed to participate in the present follow-up study. A MTHFR³ c.677TT genotype was found in 49% of the patients. Eight patients were found to have mutations in CBS². Of those, two were homozygous for c.833 T > C (p.I278T), and four were compound heterozygous for c.833 T > C. One c.833 T > C (p.I278T) compound heterozygote was identified by lowering the threshold for sequencing from tHcy at 100 μM to 50 μM. The most prominent clinical presentation among patients with a CBS² mutation was thrombosis presenting at a median age of 25 years. In case of arterial or venous thrombosis without any explanation in individuals below 40 years, tHcy should be part of the thrombophilia screening. When tHcy is between 50 and 100 μM genotyping for the MTHFR³ c.677TT is relevant, and when tHcy > 100 μM CBS should be genotyped.

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^☆: Supported by the Department of Health and Human Services, National Institutes of Health Grants No. DK-21365 (R.H.A.) and AG-09834 (S.P.S.) and The Chapman Research Foundation (S.P.S.).

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Elevation of 2-methylcitric acid I and II levels in serum, urine, and cerebrospinal fluid of patients with cobalamin deficiency☆

Abstract

J Biol Chem

Blood

Lancet

Metabolism

J Biol Chem

J Biol Chem

J Biol Chem

Clin Chim Acta

Anal Biochem

J Biol Chem

Megaloblastic anemias

Disorders of propionate and methylmalonate metabolism

Disorder of transsulfuration

Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis

N Engl J Med

Neurologic aspects of cobalamin deficiency

Medicine

Neuropsychiatric consequences of cobalamin deficiency

Abnormal fatty acid metabolism in peripheral nerves of patients with pernicious anemia

J Clin Invest

Methylation deficiency causes vitamin B12—associated neuropathy in the pig

J Neurochem

Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry

J Clin Invest

Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatography-mass spectrometry

J Clin Invest

Methylation deficiency causes vitamin B₁₂—associated neuropathy in the pig