Cardiovascular manifestations of intermediate and major hyperhomocysteinemia due to vitamin B12 and folate deficiency and/or inherited disorders of one-carbon metabolism: a 3.5-year retrospective cross-sectional study of consecutive patients.

BACKGROUND: The association of moderate hyperhomocysteinemia (HHcy) (15-30 µmol/L) with cardiovascular diseases (CVD) has been challenged by the lack of benefit of vitamin supplementation to lowering homocysteine. Consequently, the results of interventional studies have confused the debate regarding the management of patients with intermediate/severe HHcy. OBJECTIVE: We sought to evaluate the association of intermediate (30-100 µmol/L) and severe (>100 µmol/L) HHcy related to vitamin deficiencies and/or inherited disorders with CVD outcomes. METHODS: We performed a retrospective cross-sectional study on consecutive patients who underwent a homocysteine assay in a French University Regional Hospital Center. Patients with CVD outcomes were assessed for vitamin B12, folate, Hcy, methylmalonic acid, and next-generation clinical exome sequencing. RESULTS: We evaluated 165 patients hospitalized for thromboembolic and other cardiovascular (CV) manifestations among 1006 patients consecutively recruited. Among them, 84% (138/165) had Hcy >30 µmol/L, 27% Hcy >50 µmol/L (44/165) and 3% Hcy >100 µmol/L (5/165). HHcy was related to vitamin B12 and/or folate deficiency in 55% (87/165), mutations in one or more genes of one-carbon and/or vitamin B12 metabolisms in 11% (19/165), and severe renal failure in 15% (21/141) of the studied patients. HHcy was the single vascular risk retrieved in almost 9% (15/165) of patients. Sixty % (101/165) of patients received a supplementation to treat HHcy, with a significant decrease in median Hcy from 41 to 17 µmol/L (IQR: 33.6-60.4 compared with 12.1-28). No recurrence of thromboembolic manifestations was observed after supplementation and antithrombotic treatment of patients who had HHcy as a single risk, after ∼4 y of follow-up. CONCLUSION: The high frequency of intermediate/severe HHcy differs from the frequent moderate HHcy reported in previous observational studies of patients with pre-existing CVD. Our study points out the importance of diagnosing and treating nutritional deficiencies and inherited disorders to reverse intermediate/severe HHcy associated with CVD outcomes.

Assessment of cellular cobalamin metabolism in Gaucher disease.

BACKGROUND: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. METHODS: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. RESULTS: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. CONCLUSIONS: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Genome and RNA sequencing in patients with methylmalonic aciduria of unknown cause.

PURPOSE: Our laboratory has classified patients with methylmalonic aciduria using somatic cell studies for over four decades. We have accumulated 127 fibroblast lines from patients with persistent elevated methylmalonic acid (MMA) levels in which no genetic cause could be identified. Cultured fibroblasts from 26 of these patients had low [14C]propionate incorporation into macromolecules, possibly reflecting decreased methylmalonyl-CoA mutase function. METHODS: Genome sequencing (GS), copy-number variation (CNV) analysis, and RNA sequencing were performed on genomic DNA and complementary DNA (cDNA) from these 26 patients. RESULTS: No patient had two pathogenic variants in any gene associated with cobalamin metabolism. Nine patients had heterozygous variants of unknown significance previously identified by a next-generation sequencing (NGS) panel targeting cobalamin metabolic genes. Three patients had pathogenic changes in genes not associated with cobalamin metabolism (PCCA, EPCAM, and a 17q12 duplication) that explain parts of their phenotypes other than elevated MMA. CONCLUSION: Genome and RNA sequencing did not detect any additional putative causal genetic defects in known cobalamin genes following somatic cell studies and the use of a targeted NGS panel. They did detect pathogenic variants in other genes in three patients that explained some aspects of their clinical presentation.

TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells.

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA.

Vitamin B12 deficiency.

Vitamin B12 (B12; also known as cobalamin) is a B vitamin that has an important role in cellular metabolism, especially in DNA synthesis, methylation and mitochondrial metabolism. Clinical B12 deficiency with classic haematological and neurological manifestations is relatively uncommon. However, subclinical deficiency affects between 2.5% and 26% of the general population depending on the definition used, although the clinical relevance is unclear. B12 deficiency can affect individuals at all ages, but most particularly elderly individuals. Infants, children, adolescents and women of reproductive age are also at high risk of deficiency in populations where dietary intake of B12-containing animal-derived foods is restricted. Deficiency is caused by either inadequate intake, inadequate bioavailability or malabsorption. Disruption of B12 transport in the blood, or impaired cellular uptake or metabolism causes an intracellular deficiency. Diagnostic biomarkers for B12 status include decreased levels of circulating total B12 and transcobalamin-bound B12, and abnormally increased levels of homocysteine and methylmalonic acid. However, the exact cut-offs to classify clinical and subclinical deficiency remain debated. Management depends on B12 supplementation, either via high-dose oral routes or via parenteral administration. This Primer describes the current knowledge surrounding B12 deficiency, and highlights improvements in diagnostic methods as well as shifting concepts about the prevalence, causes and manifestations of B12 deficiency.

Toxic synergism between quinolinic acid and organic acids accumulating in glutaric acidemia type I and in disorders of propionate metabolism in rat brain synaptosomes: Relevance for metabolic acidemias.

The brain of children affected by organic acidemias develop acute neurodegeneration linked to accumulation of endogenous toxic metabolites like glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids. Excitotoxic and oxidative events are involved in the toxic patterns elicited by these organic acids, although their single actions cannot explain the extent of brain damage observed in organic acidemias. The characterization of co-adjuvant factors involved in the magnification of early toxic processes evoked by these metabolites is essential to infer their actions in the human brain. Alterations in the kynurenine pathway (KP) - a metabolic route devoted to degrade tryptophan to form NAD(+) - produce increased levels of the excitotoxic metabolite quinolinic acid (QUIN), which has been involved in neurodegenerative disorders. Herein we investigated the effects of subtoxic concentrations of GA, 3-OHGA, MMA and PA, either alone or in combination with QUIN, on early toxic endpoints in rat brain synaptosomes. To establish specific mechanisms, we pre-incubated synaptosomes with different protective agents, including the endogenous N-methyl-d-aspartate (NMDA) receptor antagonist kynurenic acid (KA), the antioxidant S-allylcysteine (SAC) and the nitric oxide synthase (NOS) inhibitor nitro-l-arginine methyl ester (l-NAME). While the incubation of synaptosomes with toxic metabolites at subtoxic concentrations produced no effects, their co-incubation (QUIN+GA, +3-OHGA, +MMA or +PA) decreased the mitochondrial function and increased reactive oxygen species (ROS) formation and lipid peroxidation. For all cases, this effect was partially prevented by KA and l-NAME, and completely avoided by SAC. These findings suggest that early damaging events elicited by organic acids involved in metabolic acidemias can be magnified by toxic synergism with QUIN, and this process is mostly mediated by oxidative stress, and in a lesser extent by excitotoxicity and nitrosative stress. Therefore, QUIN can be hypothesized to contribute to the pathophysiology of brain degeneration in children with metabolic acidemias.

Pitfalls in the diagnostic evaluation of subacute combined degeneration.

We report a case of a 43-year-old man presenting with a 2-week history of painless ascending sensory disturbances, suspected to be suffering from acute inflammatory polyneuropathy. On clinical examination, deep tendon reflexes were preserved and muscle strength was 5/5 everywhere. Gait was ataxic with positive Romberg test. Lumbar puncture was normal and electroneurography demonstrated demyelination. With spinal cord involvement centred on the posterior tracts on MRI, differential diagnosis focused on cobalamin deficiency. Initial laboratory work up showed nearly normal holotranscobalamin (43 pmol/L, normal>50) suggesting no vitamin B12 deficiency. Surprisingly, further testing including methylmalonic acid (3732 nmol/L, normal<271) and homocysteine (48.5 µmol/L, normal<10) showed an impairment of vitamin B12-dependent metabolism leading to the diagnosis of subacute combined degeneration. Only after repeated history taking did the patient remember having taken tablets containing cobalamin for 3 days before hospitalisation. In case of B12 deficiency, holotranscobalamin can rapidly normalise during supplementation, whereas methylmalonic acid and homocysteine might help to detect B12 deficiency in patients who recently started supplementation.

Functional cobalamin (vitamin B12) deficiency: role of advanced age and disorders associated with increased oxidative stress.

BACKGROUND/OBJECTIVE: Functional cobalamin (Cbl; vitamin B12) deficiency (that is, high levels of the Cbl-dependent metabolites, methylmalonic acid (MMA) and homocysteine (HCys), despite normal serum Cbl values) is common in the elderly and is associated with neurocognitive abnormalities, but its cause is unknown. As only reduced Cbls are metabolically active, the possibility that functional Cbl deficiency is associated with disorders having biomarkers indicative of increased oxidative stress (oxidant risks) was considered. SUBJECTS/METHODS: A retrospective record review of community-dwelling adults evaluated over a 12-year period for Cbl deficiency in a primary care setting who had serum Cbl values ⩾400 pg/ml (n=170). RESULTS: When no oxidant risks were present, older subjects (⩾70 years) had higher metabolite values than younger individuals (<70 years). MMA values were even higher in the elderly when one oxidant risk was present and in younger subjects when two or more oxidant risks were present. Even at Cbl levels ⩾800 pg/ml, MMA values were increased in 73% of elderly subjects with at least one oxidant risk. HCys values were also higher in both age groups when at least two oxidant risks were present. Cyanocobalamin therapy decreased MMA and HCys values in 86 and 76% of subjects, respectively, with nonresponders more likely to have two or more oxidant risks. CONCLUSION: Functional Cbl deficiency is associated with disorders marked by increased oxidative stress particularly in the elderly; it occurs even when Cbl levels are high and is not consistently corrected with high-dose cyanocobalamin therapy. Thus, current approaches to recognizing and managing this disorder may be inadequate.

Novel c.2216T > C (p.I739T) mutation in exon 13 and c.1481T > A (p.L494X) mutation in exon 8 of MUT gene in a female with methylmalonic acidemia.

We report herein a 1.5-year-old girl with methylmalonic acidemia (MMA) in whom two missense mutations were found: a novel I739T mutation located in exon 13 and the L494X mutation in exon 8. The results of organic acid test showed a pronounced increase in methylmalonate excretion with increased methylcitrate and 3-OH-propionate excretion, leading to a diagnosis of MMA, and Vitamin B12 administration was started. Analysis of the mut gene confirmed a T-to-A substitution at nucleotide position 1481 in exon 8 and a T-to-C substitution at nucleotide position 2216 in exon 13, leading to the amino acid isoleucine at position 739 being changed to threonine, resulting in c.2216T > C (p.I739T). The patient has now been on high-dose oral administration of Vitamin B12 and carnitine therapy (900 mg of levocarnitine chloride) for 5 years without experiencing further attacks, and her cognitive and motor development is normal. Further tests on residual enzyme activity, as well as experience with more cases, may shed light on the relationship between gene mutations and phenotypes in MMA.

Renal dysfunction in methylmalonic acidurias: review for the pediatric nephrologist.

Methylmalonic acidurias are a heterogeneous group of inborn errors of branched-chain amino acid metabolism. Depending on the underlying etiology, acute or chronic renal disease constitutes major (long-term) complications. In recent decades, overall survival has improved due to optimized treatment strategies based on the use of standardized emergency protocols and dialysis techniques. The majority of these patients, especially those having mut°, cblB, and cblA deficiency, are at increased risk of developing chronic kidney disease secondary to tubulointerstitial nephritis to require hemo- or peritoneal dialysis. Kidney and/or liver transplantation, as organ replacement, or even gene therapy on a limited scale, are controversially discussed treatment options in methylmalonic acidurias. The pathophysiological basis of renal disease has not been clarified in detail until now, but a severe mitochondrial dysfunction and an impairment of tubular dicarboxylic acid transport due to accumulated toxic metabolic compounds has been recently proposed. Another severe renal complication of methylmalonic acidurias is the occurrence of cblC-associated infantile atypical hemolytic syndrome, which can result in acute kidney injury. Close collaboration between (pediatric) nephrologists and metabolic specialists is required for the long-term management of these patients.

Oxidative stress parameters in urine from patients with disorders of propionate metabolism: a beneficial effect of L:-carnitine supplementation.

Propionic (PA) and methylmalonic (MMA) acidurias are inherited disorders caused by deficiency of propionyl-CoA carboxylase and methylmalonyl-CoA mutase, respectively. Affected patients present acute metabolic crises in the neonatal period and long-term neurological deficits. Treatments of these diseases include a protein restricted diet and L: -carnitine supplementation. L: -Carnitine is widely used in the therapy of these diseases to prevent secondary L: -carnitine deficiency and promote detoxification, and several recent in vitro and in vivo studies have reported antioxidant and antiperoxidative effects of this compound. In this study, we evaluated the oxidative stress parameters, isoprostane and di-tyrosine levels, and the antioxidant capacity, in urine from patients with PA and MMA at the diagnosis, and during treatment with L: -carnitine and protein-restricted diet. We verified a significant increase of isoprostanes and di-tyrosine, as well as a significant reduction of the antioxidant capacity in urine from these patients at diagnosis, as compared to controls. Furthermore, treated patients presented a marked reduction of isoprostanes and di-tyrosine levels in relation to untreated patients. In addition, patients with higher levels of protein and lipid oxidative damage, determined by di-tyrosine and isoprostanes levels, also presented lower urinary concentrations of total and free L: -carnitine. In conclusion, the present results indicate that treatment with low protein diet and L: -carnitine significantly reduces urinary biomarkers of protein and lipid oxidative damage in patients with disorders of propionate metabolism and that L: -carnitine supplementation may be specially involved in this protection.

The MMACHC proteome: hallmarks of functional cobalamin deficiency in humans.

Cobalamin (Cbl, B(12)) is an essential micronutrient required to fulfill the enzymatic reactions of cytosolic methylcobalamin-dependent methionine synthase and mitochondrial adenosylcobalamin-dependent methylmalonyl-CoA mutase. Mutations in the MMACHC gene (cblC complementation group) disrupt processing of the upper-axial ligand of newly internalized cobalamins, leading to functional deficiency of the vitamin. Patients with cblC disease present with both hyperhomocysteinemia and methylmalonic acidemia, cognitive dysfunction, and megaloblastic anemia. In the present study we show that cultured skin fibroblasts from cblC patients export increased levels of both homocysteine and methylmalonic acid compared to control skin fibroblasts, and that they also have decreased levels of total intracellular folates. This is consistent with the clinical phenotype of functional cobalamin deficiency in vivo. The protein changes that accompany human functional Cbl deficiency are unknown. The proteome of control and cblC fibroblasts was quantitatively examined by two dimensional difference in-gel electrophoresis (2D-DIGE) and liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI/MS). Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, the neurological system and cell signaling. Pathway analysis of the differentially expressed proteins demonstrated strong associations with neurological disorders, muscular and skeletal disorders, and cardiovascular diseases in the cblC mutant cell lines. Supplementation of the cell cultures with hydroxocobalamin did not restore the cblC proteome to the patterns of expression observed in control cells. These results concur with the observed phenotype of patients with the cblC disorder and their sometimes poor response to treatment with hydroxocobalamin. Our findings could be valuable for designing alternative therapies to alleviate the clinical manifestation of the cblC disorder, as some of the protein changes detected in our study are common hallmarks of known pathologies such as Alzheimer's and Parkinson's diseases as well as muscular dystrophies.

Intact recovery from early 'acquired methylmalonic aciduria' secondary to maternal atrophic gastritis.

AIM: A 6-month-old infant with severe hyporegenerative anaemia, muscular hypotonia and developmental delay is reported, and the metabolic, diagnostic and therapeutic implications of this case are discussed. RESULTS: Diagnostic work-up disclosed vitamin B12 depletion with an elevated excretion of methylmalonic acid (MMA), but a normal plasma total homocysteine. MRI showed fronto-temporal atrophy and a delay in myelinization. The boy's disease was attributable to a maternal atrophic gastritis. After initiation of vitamin B12 supplementation, he quickly recovered regarding haematopoiesis and MMA excretion. His neurological development completely normalized during 18 months of follow-up including assessment by Bayley scores. CONCLUSION: As the majority of reported patients with this acquired form of methylmalonic aciduria show a persistent neurological deficit, early diagnosis of this condition is mandatory and should include sensitive markers of vitamin B12 depletion, namely MMA formation and plasma homocysteine.

Mechanism of vitamin B12-responsiveness in cblC methylmalonic aciduria with homocystinuria.

Patients with the cblC vitamin B(12) (cobalamin, cbl) disorder are defective in the intracellular synthesis of adenosylcobalamin and methylcobalamin and have combined homocystinuria and methylmalonic aciduria. While other vitamin B(12) disorders are treatable with high dose cyanocobalamin (CNCbl) or hydroxocobalamin (OHCbl), cblC patients respond well to OHCbl but not to CNCbl. Patient mutations were introduced into recombinant MMACHC (cblC) protein and the binding of CNCbl and OHCbl was examined. Three mutations were analyzed: G147D, associated with early onset, vitamin B(12) unresponsive disease; R161Q, associated with late onset disease that is highly responsive to OHCbl; and H122A, selected to test the hypothesis that H122 is central to a proposed vitamin B(12) binding motif on MMACHC. We report here that wild-type MMACHC binds both OHCbl and CNCbl with similar, tight affinity (K(d)=5.7 microM). We also report that MMACHC binds CNCbl in the base-off form, with the dimethylbenzimidazole (DMB) base of cobalamin displaced from coordination with the cobalt. In this form, wild-type MMACHC is able to reductively decyanate CNCbl to cob(II)alamin requiring only the presence of NADPH and FAD. We demonstrate that MMACHC with the G147D mutation is unable to bind either CNCbl or OHCbl, providing a straight forward explanation for the absence of response to either vitamin form. However, we show that MMACHC containing the R161Q mutation binds OHCbl with wild-type affinity, but is disturbed in binding CNCbl and has impaired decyanation. Finally, we show that H122A has reduced binding, but like R161Q, it binds OHCbl more tightly than CNCbl, suggesting that this histidine is not absolutely required for binding. These studies suggest that the ability of mutant MMACHC to respond to vitamin therapy depends on its ability to bind the vitamin with significant affinity, and for CNCbl, also on its ability to bind in the base-off form to facilitate reductive decyanation. These studies emphasize the continued use of OHCbl with cblC patients for maximum therapeutic effect.

Vitamin B12 absorption and metabolism in milk-fed lambs.

AIM: To quantify aspects of absorption and retention of vitamin B12 from milk in pre-ruminant lambs, and the possible effect of activation of dependent metabolic pathways (propionate-succinate) on vitamin B12 concentrations in tissues. METHODS: In Experiment 1, two groups of eight newborn lambs each received either milk substitute alone, or milk substitute with added propionate for 4 weeks. Half of the lambs in each group received vitamin B12 injections twice weekly. Blood and liver samples were taken on Day -1 and at the end of the trial, and blood was also collected twice weekly, for measurement in plasma of vitamin B12, methylmalonic acid (MMA), and blood-binder transcobalamin II/haptocorrin. The lambs were weighed at 0, 2 and 4 weeks. In Experiment 2, 12 milk-fed newborn lambs (n=2 or 3/group) were injected I/M with 0.2, 0.4, 0.8, 1.6 or 3.2 microg vitamin B12 and a further 12 were supplemented orally with 2, 4, 8, 16 or 32 microg vitamin B12, daily for 2 weeks. Three lambs remained as untreated controls. Blood and liver samples were taken at the start and end of the trial, and blood was also collected twice weekly, for measurement of vitamin B12 and MMA concentrations in plasma. The lambs were weighed on Days 0, 7 and 14. In Experiment 3, 14 lambs from Experiment 2 were used to repeat the highest I/M and oral rates of supplementation used in Experiment 2. Blood samples were taken at frequent intervals for 24 hours, and analysed for concentrations of vitamin B12 in plasma. RESULTS: Propionate supplementation was associated with increased concentrations of vitamin B12 in plasma in lambs supplemented with vitamin B12 but not in those not supplemented (p=0.047), but had no detectable effect on concentrations of vitamin B12 in liver (Experiment 1). Lambs with concentrations of vitamin B12 in plasma and liver in the marginal reference range were able to metabolise propionate and maintain normal concentrations of MMA at a rate of intake of propionate close to that which would depress appetite. Close to 100% of the vitamin B12 administered by I/M injection appeared in plasma in the first hour but 85% was removed from the circulation within 16 hours. The maximum liver concentration was achieved at 900 nmol/kg (=1,200 microg) fresh tissue. There were no significant differences in liveweight gain (LWG) regardless of treatment. CONCLUSIONS: Lambs with concentrations of vitamin B12 in plasma and liver in the marginal reference range were able to metabolise propionate and maintain normal concentrations of MMA in plasma at a rate of absorption of propionate close to that which would depress appetite. The coefficient of absorption of oral vitamin B12 in milk-fed lambs was low (<10%), and the ability of lambs to retain the vitamin even when tissues had physiologically low concentrations was poor. CLINICAL RELEVANCE: Pre-ruminant lambs had a low requirement for vitamin B12, and even at low tissue concentrations had poor ability to absorb and retain the vitamin. However, if supplementation is required, a method that provides a modest but continual supply may be most effective in protection against deficiency.

The use of blood concentrations of vitamins and their respective functional indicators to define folate and vitamin B12 status.

In recent years there has been growing interest in the vitamins folic acid and vitamin B12 because of the realization that the status of these vitamins in populations is less than adequate, and that such inadequacy may be linked to adverse public health outcomes. This concern has prompted the United States, Canada, and other countries to fortify grain products with folic acid, while additional countries are considering doing so in the near future. This presentation provides a new approach which relies on the combination of the concentrations in blood of vitamins and their respective functional indicators to establish cutoff points for assessing folate and vitamin B12 status in populations. The premise is based on the fact that the relationship between plasma vitamin concentrations and their respective functional indicators is inverse and biphasic, with a steep slope at low concentrations of the vitamin and a more moderate slope at higher plasma vitamin concentrations. We propose that the intersection of these two slopes be used as a guideline for assessing the status of these vitamins and the adequacy of fortification programs. The cutoff would be 10 nmol/L for serum folate and 340 nmol/L for red blood cell folate, based on lowest plasma homocysteine. For serum vitamin B12, the cutoff would be 150 pmol/L based on lowest methylmalonic acid and 300 pmol/L based on lowest homocysteine.

Nutrition therapy of organic acidaemias with amino acid-based formulas: emphasis on methylmalonic and propionic acidaemia.

Failure to thrive has been described in patients with organic acidaemias due to a variety of causes, both organic and inorganic. Failure to thrive in patients with methylmalonic acidaemia (MMA) and propionic acidaemia (PA) may be related to inadequate protein and energy intake rather than pathology of disease. Inadequate protein intake can also result in decreased resting energy expenditure, clinical signs and symptoms of amino acid deficiency, increased risk of infection, and developmental delay. Amino acid-based formulas (also referred to as 'medical foods') provide a key source of nitrogen, energy, vitamins and minerals which, when prescribed appropriately, can promote anabolism and growth. Although protein requirements in patients with organic acidaemias have not been elucidated, providing an adequate balance of protein, energy and other nutrients will help promote growth.

Executive dysfunction in hyperhomocystinemia responds to homocysteine-lowering treatment.

An elevated serum homocysteine level is a risk factor for the development of cognitive impairment. Reported is a late-onset case of hyperhomocystinemia due to a vitamin B12 metabolic deficit (cobalamin C) with cognitive impairment, primarily in frontal/executive function. After homocysteine-lowering therapy, the patient's functional and neuropsychological status improved in conjunction with a decrease in leukoariosis on his MRI scan. These findings suggest that homocysteine-related cognitive impairment may be partially reversible.

Vitamin B12 and folate depletion in cognition: a review.

In cross-sectional studies, low levels of folate and B12 have been shown to be associated with cognitive decline and dementia Evidence for the putative role of folate, vitamin B12 in neurocognitive and other neurological functions comes from reported cases of severe vitamin deficiencies, particularly pernicious anemia, and homozygous defects in genes that encode for enzymes of one-carbon metabolism. The neurological alterations seen in these cases allow for a biological role of vitamins in neurophysiology. Results are quite controversial and there is an open debate in literature, considering that the potential and differential role of folate and B12 vitamin in memory acquisition and cognitive development is not completely understood or accepted. What is not clear is the fact that vitamin B12 and folate deficiency deteriorate a pre-existing not overt pathological situation or can be dangerous even in normal subjects. Even more intriguing is the interaction between B12 and folate, and their role in developing hyperhomocysteinemia. The approach to the rehabilitation of the deficiency with adequate vitamin supplementation is very confusing. Some authors suggest it, even in chronic situations, others deny any possible role. Starting from these quite confusing perspectives, the aim of this review is to report and categorize the data obtained from the literature. Despite the plausible biochemical mechanism, further studies, based on clinical, neuropsychological, laboratory and (lastly) pathological features will be necessary to better understand this fascinating biochemical riddle.

[Diagnosis and treatment of methylmalonic acidemia in 14 cases].

OBJECTIVE: Methylmalonic acidemia (MMA) is one of the most common disorders of congenital organic acid metabolism. This study aimed at exploring the clinical characteristics and treatment of the disease to help improve our understanding of it. METHODS: The clinical data of 14 patients with MMA admitted to our hospital between January 2002 and July 2003 were analyzed and the diagnoses were confirmed by gas chromatography/mass spectrometry (GC/MS). The patients consisted of 4 males and 10 females, whose age of onset ranged from birth to 9 years with 7 cases younger than 1 month (50%) and 10 cases younger than 1 year (71%). RESULTS: The main clinical manifestations were lethargy (6 cases), developmental retardation or regradation (7 cases), convulsion (6 cases), recurrent vomiting (4 cases), difficulty with feeding (4 cases), muscular dystonia (5 cases with hypotonia, 3 with hypertonia) and yellowish hair (4 cases), etc. Some cases were also presented with hair loss, hepatomegaly, ataxic or stiff gait, and motor weakness with muscular atrophy. The laboratory findings showed metabolic acidosis in 6 cases, hyperammonemia in 5 cases, ketonuria in 4 cases and remarkable elevation of urinary methylmalonic acid concentration in all cases. Some abnormalities in globus pallidus and cerebral white matter as well as diffuse cerebral atrophy were noted by the brain CT and MRI in 5 respective cases, while 4 cases did not receive neuroradiological examinations. Peripheral neuropathies were found by electromyography in 2 patients and bilateral optic nerve atrophy was detected by eyeground examination in 1 child. Three patients died before the diagnoses were made. Of the 11 survivals, 10 children have received therapy of vitamin B12 (VitB12) and supplementation of L-carnitine with restricted-protein diet. The follow-up for a period ranging from 3 months to 1.5 year (mean 8.5 months) of 7 cases with medical therapy showed a favorable outcome without any symptoms in 1 case and apparent improvement in 4 cases (the diffuse cerebral atrophy in MRI completely recovered in one case), however, 2 patients died from severe metabolic acidosis. CONCLUSIONS: The main clinical features of MMA include lethargy, developmental retardation or regradation, convulsion, recurrent vomiting, difficulty with feeding, muscular dystonia, yellowish hair, metabolic acidosis, hyperammonemia and ketonuria, etc. Urine organic acids analysis with GC/MS is critical to the early diagnosis of MMA. Early diagnosis and appropriate long-term treatment are essential to improve the prognosis of the disease.