Serum vitamin B12 not reflecting vitamin B12 status in patients with type 2 diabetes

Contradictory results for concentrations of vitamin B12, holotranscobalamin (holoTC), and methylmalonic acid (MMA) have been reported. We tested the hypothesis that the extracellular vitamin B12 markers are not reflecting the intracellular vitamin B12-dependent biochemical reactions in individuals with type 2 diabetes. The study included 92 patients with diabetes and 72 controls with similar age and sex distribution. We measured vitamin B12 markers [MMA, total serum vitamin B12, holoTC, total homocysteine (tHcy)], red blood cell (RBC)-B12, and the plasma concentrations of the methylation markers [S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)]. In comparison to controls, diabetic patients showed significantly higher concentrations of plasma SAH (median 15.1 vs. 11.8 nmol/L; p < 0.001) and lower SAM/SAH ratio (9.1 vs. 8.2; p = 0.006). Concentrations of total vitamin B12 and holoTC did not differ significantly between the groups, but plasma MMA concentrations were significantly higher in diabetics (250 vs. 206 nmol/L). However, RBC-B12 was lower in diabetics compared to controls (median 230 vs. 260 pmol/L; p = 0.001). The inverse correlation between MMA and RBC-B12 was stronger in the controls compared to that in the patients (correlation coefficient in controls R = −0.446, p = 0.001; in patients R = −0.289, p = 0.022). Metformin treatment was associated with a lower total serum vitamin B12, but a comparable RBC-B12 and a slightly lower MMA and better methylation index. In conclusion, patients with type 2 diabetes showed normal extracellular vitamin B12, but disturbed intracellular B12-dependent biochemical reactions. Metformin treatment was associated with low serum vitamin B12 and improved intracellular vitamin B12 metabolism despite low serum vitamin B12.     

Protection and reactivation of human methylmalonyl-CoA mutase by MMAA protein

Previous studies have reported that some adenosylcobalamin-dependent enzymes suffer inactivation during catalysis due to the oxidation of cobalamin. In addition, the protection or reactivation of their catalytic activities by proteins called “protectases” or reactivases is well known in bacteria. In this study, we examined the influence of human MMAA protein on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase (MCM) by testing both purified recombinant proteins in vitro. Our results showed that MMAA plays dual roles in MCM activity. When it was added at the beginning of the reaction, it prevents inactivation by guarding MCM. After 60 min of reaction, when MCM is inactive, the addition of MMAA increases the enzymatic activity through GTP hydrolysis, indicating reactivation of MCM by exchange of the damaged cofactor. Interaction between MCM and MMAA observed in vitro was confirmed in vivo by yeast two-hybrid system.     

Cloning of genomic and cDNA for mouse isovaleryl-CoA dehydrogenase (IVD) and evolutionary comparison to other known IVDs

Isovaleryl-CoA dehydrogenase (IVD) is an intramitochondrial homotetrameric flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. Deficiency of IVD in humans causes isovaleric acidemia, which shows tremendous clinical variability for reasons that are unknown. To help better understand this disorder, we have cloned and sequenced the mouse IVD genomic and cDNAs. The mouse IVD gene spans approximately 17 kb and contains 12 coding exons organized identically to the human gene. It maps to mouse chromosome 2 in the area of band 2E4-E5, corresponding to the syntenic region of human chromosome 15. Mouse IVD predicted amino acid sequences are 95.8 and 89.6% identical to that of the rat and human sequences, respectively, with conservation of key functional residues. We have now identified IVD sequences from seven species. Comparison of these sequences shows that the rat and mouse proteins are the most closely related, both of which, in turn, share highest homology to human. All of the mammalian enzymes appear to be more closely related than any of the IVDs on other branches of the phylogram, while the fly and worm IVDs are the most divergent. The invertebrate IVDs are more closely related to the mammalian enzymes than to those from two plant species.     

Age and Brain Structural Related Effects of Glutaric and 3-Hydroxyglutaric Acids on Glutamate Binding to Plasma Membranes During Rat Brain Development

(1) In the present study we determined the effects of glutaric (GA, 0.01–1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0–100 μM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na⁺-independent and Na⁺-dependent [³H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na⁺-independent [³H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na⁺-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [³H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na⁺-independent [³H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na⁺-dependent [³H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.     

Optimization and performance of a rapid gas chromatography–mass spectrometry analysis for methylmalonic acid determination in serum and plasma

We have developed a rapid and sensitive GC–MS assay for methylmalonic acid determination in serum and plasma utilizing an anion exchange solid-phase extraction and trimethylsilyl derivatization. Each step of the procedure was optimized by the experimental design methods to assure the assay reliable performance. The limit of detection and limit of quantitation were 0.025 and 0.1 μmol/l. The total coefficient of variation for the method was 9.8, 4.4, and 4.6% at the concentration of 0.2, 3.1, and 6.2 μmol/l methylmalonic acid concentration, respectively. The assay are linear up to 9.0 μmol/l, and showed good correlation with a reference method. The method has proven to be reliable in routine production, producing clean chromatography, unique ion fragments, and consistent ion mass ratio.     

Two novel isovaleryl-CoA dehydrogenase gene mutations in a Chinese infant

Isovaleric acidemia (IVA) is a rare inherited metabolic disease caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). Newborn screening with tandem mass spectrometry leads to early identification of individuals with risk of IVA. The family specific mutations are useful for prenatal diagnosis. Molecular genetic analysis helps to further confirm the clinical diagnosis of IVA. We describe here the clinical and metabolic features of a Chinese infant with early onset IVA. Sequence analysis of the IVD gene identifies compound heterozygous mutations in this patient, c.39G > A (p.W13X) nonsense mutation and c.597C > G (p.I199M) missense mutation, both of which are previously unreported. Structural analyses suggest that the p.I199M missense mutation may destabilize the IVD monomer structure and affect the interaction between IVD and flavin adenine dinucleotide. Both the clinical and genetic features of this patient help to further expand our knowledge of IVA.     

Antioxidants successfully reduce ROS production in propionic acidemia fibroblasts

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients’ cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls’ fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA.     

Molecular genetic study of glutaric aciduria,type I: Identification of a novel mutation

Glutaric acidemia type I (GA-1) is an inborn error of metabolism due to deficiency of glutaryl-CoA dehydrogenase (GCDH), which catalyzes the conversion of glutaryl-CoA to crotonyl-CoA. GA-1 occurs in about 1 in 100 000 infants worldwide. The GCDH gene is on human chromosome 19p13.2, spans about 7 kb and comprises 11 exons and 10 introns. Tandem mass spectrometry (MS/MS) was used for clinical diagnosis in a proband from Iran with GA-1. Sanger sequencing was performed using primers specific for coding exons and exon-intron flanking regions of the GCDH gene in the proband. Cosegregation analysis and in silico assessment were performed to confirm the pathogenicity of the candidate variant. A novel homozygous missense variant c.1147C > A (p.Arg383Ser) in exon 11 of GCDH was identified. Examination of variant through in silico software tools determines its deleterious effect on protein in terms of function and stability. The variant cosegregates with the disease in family. In this study, the clinical and molecular aspects of GA-1 were investigated, which showed one novel mutation in the GCDH gene in an Iranian patient. The variant is categorized as pathogenic according to the the guideline of the American College of Medical Genetics and Genomics (ACMG) for variant interpretation. This mutation c.1147C > A (p.Arg383Ser) may also be prevalent among Iranian populations.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.     

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.