Homocysteine and methylmalonic acid in Phenylketonuria patients.

Hyperhomocysteinemia and vitamin B12 deficiency have been reported in patients with phenylketonuria. In this study, total homocysteine (tHcy) and methylmalonic acid (MMA) levels were analyzed in samples from 25 phenylketonuria (PKU) patients. Comparisons were made between pre- and post-treatment values (n= 3); on treatment values, between periods with high and normal/low phenylalanine (Phe) levels (n= 20); and in women before, during and after pregnancy (n= 3). THcy levels decreased after treating PKU with metabolic formula (p=0.014). Except for a pregnant woman before pregnancy, none of the patients had tHcy values above the normal range. In fact, tHcy was < 5 µmol/L in 34% of the samples. We observed a decrease in Phe, tHcy, and tyrosine levels during pregnancy. MMA levels did not differ significantly, with values remaining in the normal range. These data indicate that there was no B12 deficiency in patients who adhere to the diet. In conclusion, in PKU patients treated with metabolic formula, tHcy is frequently not elevated, remaining even in the lower normal range in some patients. Thus, clinical follow-up and adherence to dietary treatment are crucial to prevent B12 deficiency.

Elevated holo-transcobalamin in Gaucher disease type II: A case report.

Gaucher disease (GD), one of the most common lysosomal disorders, is caused by deficiency of ß-glucocerebrosidase. Based on the presence and severity of neurological complications, GD is classified into types I, II (the most severe form), and III. Abnormalities in systemic markers of vitamin B12 (B12 ) metabolism have been reported in GD type I patients, suggesting a higher prevalence of B12 deficiency in these patients. A 2-month-old male with GD type II was admitted to the hospital presenting jaundice, hepatosplenomegaly, and ichthyosis. At admission, cholestasis and ascites, abnormal liver function enzymes, prolonged prothrombin time, and high levels of B12 were confirmed. Analysis of biomarkers of B12 status revealed elevated B12 and holo-transcobalamin (holo-TC) levels. The B12 profile found in our patient is the opposite to what is described for GD type I patients. Holo-TC may increase in inflammatory states or due to liver diseases. In GD, the accumulation of glucocerebroside may be a trigger that initiates a systemic inflammatory reaction, characterized by macrophage activation. We suggest higher levels of holo-TC could be associated with a more severe (neuronopathic) GD, and be a biomarker of GD type II.

Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC.

Thiolatocobalamins are a class of cobalamins comprised of naturally occurring and synthetic ligands. Glutathionylcobalamin (GSCbl) occurs naturally in mammalian cells, and also as an intermediate in the glutathione-dependent dealkylation of methylcobalamin (MeCbl) to form cob(I)alamin by pure recombinant CblC from C. elegans. Glutathione-driven deglutathionylation of GSCbl was demonstrated both in mammalian as well as in C. elegans CblC. Dethiolation is orders of magnitude faster than dealkylation of Co-C bonded cobalamins, which motivated us to investigate two synthetic thiolatocobalamins as substrates to repair the enzymatic activity of pathogenic CblC variants in humans. We report the synthesis and kinetic characterization of cysteaminylcobalamin (CyaCbl) and 2-mercaptopropionylglycinocobalamin (MpgCbl). Both CyaCbl and MpgCbl were obtained in high purity (90-95%) and yield (78-85%). UV-visible spectral properties agreed with those reported for other thiolatocobalamins with absorbance maxima observed at 372 nm and 532 nm. Both CyaCbl and MpgCbl bound to wild type human recombinant CblC inducing spectral blue-shifts characteristic of the respective base-on to base-off transitions. Addition of excess glutathione (GSH) resulted in rapid elimination of the ß-ligand to give aquacobalamin (H2OCbl) as the reaction product under aerobic conditions. Further, CyaCbl and MpgCbl underwent spontaneous dethiolation thereby repairing the loss of activity of pathogenic variants of human CblC, namely R161G and R161Q. We posit that thiolatocobalamins could be exploited therapeutically for the treatment of inborn errors of metabolism that impair processing of dietary and supplemental cobalamin forms. While these disorders are targets for newborn screening in some countries, there is currently no effective treatment available to patients.

DNMT3B deficiency alters mitochondrial biogenesis and α-ketoglutarate levels in human embryonic stem cells.

Embryonic stem cell renewal and differentiation is regulated by metabolites that serve as cofactors for epigenetic enzymes. An increase of α-ketoglutarate (α-KG), a cofactor for histone and DNA demethylases, triggers multilineage differentiation in human embryonic stem cells (hESCs). To gain further insight into how the metabolic fluxes in pluripotent stem cells can be influenced by inactivating mutations in epigenetic enzymes, we generated hESCs deficient for de novo DNA methyltransferases (DNMTs) 3A and 3B. Our data reveal a bidirectional dependence between DNMT3B and α-KG levels: a-KG is significantly upregulated in cells deficient for DNMT3B, while DNMT3B expression is downregulated in hESCs treated with α-KG. In addition, DNMT3B null hESCs exhibit a disturbed mitochondrial fission and fusion balance and a switch from glycolysis to oxidative phosphorylation. Taken together, our data reveal a novel link between DNMT3B and the metabolic flux of hESCs.

Optic Atrophy 1 Controls Human Neuronal Development by Preventing Aberrant Nuclear DNA Methylation.

Optic atrophy 1 (OPA1), a GTPase at the inner mitochondrial membrane involved in regulating mitochondrial fusion, stability, and energy output, is known to be crucial for neural development: Opa1 heterozygous mice show abnormal brain development, and inactivating mutations in OPA1 are linked to human neurological disorders. Here, we used genetically modified human embryonic and patient-derived induced pluripotent stem cells and reveal that OPA1 haploinsufficiency leads to aberrant nuclear DNA methylation and significantly alters the transcriptional circuitry in neural progenitor cells (NPCs). For instance, expression of the forkhead box G1 transcription factor, which is needed for GABAergic neuronal development, is repressed in OPA1+/- NPCs. Supporting this finding, OPA1+/- NPCs cannot give rise to GABAergic interneurons, whereas formation of glutamatergic neurons is not affected. Taken together, our data reveal that OPA1 controls nuclear DNA methylation and expression of key transcription factors needed for proper neural cell specification.

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.

d-Glycerate kinase deficiency in a neuropediatric patient.

d-Glyceric aciduria (DGA) due to d-glycerate kinase deficiency (DGKD) is a rare autosomal-recessive inborn error of metabolism that is usually linked to the metabolism of fructose and serine. We describe a Moroccan patient with DGKD whose metabolic defect has been characterized by metabolite studies, sequencing of genomic DNA and by studies on the RNA level. Since birth the index patient presented with severe muscular hypotonia, joint hypermobility and tremor. Enantioselective analysis showed elevated d-glyceric acid in the urine of the patient, but not in that of his parents. DNA analysis revealed homozygosity in the GLYCTK gene for c.517G>T [p.(Val173Leu)], the first mutation reported for exon 3 of this gene, as well as for the c.530-4A>G polymorphism. RNA studies suggest that none of these sequence variants affects splicing. The mother was heterozygous for both sequence variants, the father heterozygous for the first one and homozygous for the polymorphism, which further supports that c.517G>T is the functionally relevant nucleotide change. The conservation of GLYCTK throughout evolution suggests an important biological role of this enzyme, although it is not known yet how mutations are linked to clinical features. Future studies should investigate the molecular defect in a more general way and search for additional roles of GLYCTK beyond its established role in catabolism of serine and fructose.

Vitamin B12 Status Upon Short-Term Intervention with a Vegan Diet-A Randomized Controlled Trial in Healthy Participants.

Vegans are at an increased risk for certain micronutrient deficiencies, foremost of vitamin B12. Little is known about the short-term effects of dietary change to plant-based nutrition on vitamin B12 metabolism. Systemic biomarkers of vitamin B12 status, namely, serum vitamin B12 and holotranscobalamin, may respond quickly to a reduced intake of vitamin B12. To test this hypothesis, 53 healthy omnivore subjects were randomized to a controlled unsupplemented vegan diet (VD, n = 26) or meat-rich diet (MD, n = 27) for 4 weeks. Vitamin B12 status was examined by measurement of serum vitamin B12, holotranscobalamin (holo-TC), methylmalonic acid (MMA) and total plasma homocysteine (tHcy). Holo-TC decreased significantly in the VD compared to the MD group after four weeks of intervention, whereas metabolites MMA and tHcy were unaffected. Body weight remained stable in both groups. VD intervention led to a significant reduction of cholesterol intake, and adequate profiles of nutrient and micronutrient status. Lower intake of vitamin B12 was observed in VD, which was mirrored by a lower concentration of serum vitamin B12 and reduced holo-TC after 4 weeks. Plasma holo-TC may be a fast-responding biomarker to monitor adequate supply of vitamin B12 in plant-based individuals.

Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.

The emerging role of the mitochondrial fatty-acid synthase (mtFASII) in the regulation of energy metabolism.

Malonyl-CoA synthetase (ACSF3) catalyzes the first step of the mitochondrial fatty acid biosynthesis (mtFASII). Mutations in ACSF3 cause CMAMMA a rare inborn error of metabolism. The clinical phenotype is very heterogeneous, with some patients presenting with neurologic manifestations. In some children, presenting symptoms such as coma, ketoacidosis and hypoglycemia are suggestive of an intermediary metabolic disorder. The overall pathophysiological mechanisms are not understood. In order to study the role of mtFASII in the regulation of energy metabolism we performed a comprehensive metabolic phenotyping with Seahorse technology proteomics in fibroblasts from healthy controls and ACSF3 patients. SILAC-based proteomics and lipidomic analysis were performed to investigate the effects of hypofunctional mtFASII on proteome and lipid homeostasis of complex lipids. Our data clearly confirmed an impaired mitochondrial flexibility characterized by reduced mitochondrial respiration and glycolytic flux due to a lower lipoylation degree. These findings were accompanied by the adaptational upregulation of ß-oxidation and by the reduction of anaplerotic amino acids as compensatory mechanism to address the required energy need. Finally, lipidomic analysis demonstrated that the content of the bioactive lipids sphingomyelins and cardiolipins was strongly increased. Our data clearly demonstrate the role of mtFASII in metabolic regulation. Moreover, we show that mtFASII acts as mediator in the lipid-mediated signaling processes in the regulation of energy homeostasis and metabolic flexibility.

Implementation of a fast method for the measurement of carnitine palmitoyltransferase 2 activity in lymphocytes by tandem mass spectrometry as confirmation for newborn screening.

Carnitine palmitoyltransferase II (CPT2) is a rare autosomal recessive inherited disorder affecting mitochondrial ß-oxidation. Confirmation diagnostics are mostly based on molecular sequencing of the CPT2 gene, especially to distinguish CPT2 and carnitine:aclycarnitine translocase deficiencies, which present with identical acylcarnitine profiles on newborn screening (NBS). In the past, different enzyme tests in muscle biopsies have been developed in order to study the functional effect in one of the main target organs. In this study, we implemented a method for measurement of CPT2 enzyme activity in human lymphocytes with detection of the reaction products via liquid chromatography mass spectrometry to enable the simultaneous evaluation of the functional impairment and the clear diagnosis of the disease. CPT2 activity was measured in samples collected from CPT2 patients (n = 11), heterozygous carriers (n = 6), and healthy individuals (n = 52). Seven patients out of 11 were homozygous for the common mutation c.338T>C and showed a residual activity with median values of 19.2 ± 3.7% of healthy controls. Heterozygous carriers showed a residual activity in the range of 42% to 75%. Four individuals carrying the heterozygous mutation c.338T>C showed a 2-fold higher residual activity as compared to homozygous individuals. Our optimized method for the measurement of CPT2 activity is able to clearly discriminate between patients and healthy individuals and offers the possibility to determine CPT2 activity in human lymphocytes avoiding the need of an invasive muscle biopsy. This method can be successfully used for confirmation diagnosis in case of positive NBS and would markedly reduce the time to define diagnosis.

A common pathomechanism in GMAP-210- and LBR-related diseases.

Biallelic loss-of-function mutations in TRIP11, encoding the golgin GMAP-210, cause the lethal human chondrodysplasia achondrogenesis 1A (ACG1A). We now find that a homozygous splice-site mutation of the lamin B receptor (LBR) gene results in the same phenotype. Intrigued by the genetic heterogeneity, we compared GMAP-210- and LBR-deficient primary cells to unravel how particular mutations in LBR cause a phenocopy of ACG1A. We could exclude a regulatory interaction between LBR and GMAP-210 in patients' cells. However, we discovered a common disruption of Golgi apparatus architecture that was accompanied by decreased secretory trafficking in both cases. Deficiency of Golgi-dependent glycan processing indicated a similar downstream effect of the disease-causing mutations upon Golgi function. Unexpectedly, our results thus point to a common pathogenic mechanism in GMAP-210- and LBR-related diseases attributable to defective secretory trafficking at the Golgi apparatus.

The diagnostic challenge in very-long chain acyl-CoA dehydrogenase deficiency (VLCADD).

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is the most common defect of mitochondrial ß-oxidation of long-chain fatty acids. However, the unambiguous diagnosis of true VLCADD patients may be challenging, and a high rate of false positive individuals identified by newborn screening undergo confirmation diagnostics. In this study, we show the outcome of enzyme testing in lymphocytes as a confirmatory tool in newborns identified by screening, and the correlation with molecular sequencing of the ACADVL gene. From April 2013 to March 2017, in 403 individuals with characteristic acylcarnitine profiles indicative of VLCADD, palmitoyl-CoA oxidation was measured followed by molecular genetic analysis in most of the patients with residual activity (RA) <50%. In almost 50% of the samples (209/403) the RA was >50%, one-third of the individuals (125/403) displayed a RA of 30-50% and 69/403 individuals showed a residual activity of 0-30%. Sequencing of the ACADVL gene revealed that all individuals with activities below 24% were true VLCADD patients, individuals with residual activities between 24 and 27% carried either one or two mutations. Twenty new mutations could be identified and functionally classified based on their effect on enzyme function. Finally, we observed an up-regulation of MCAD-activity in many patients. However, this did not correlate with the degree of VLCAD RA. Although the likely clinical phenotype cannot be fully foreseen by genetic and functional tests as it depends on many factors, our data demonstrate the strength of this functional enzyme test in lymphocytes as a quick and reliable method for confirmation diagnostics of VLCADD.

Mimicking Ketonuria in the Ketogenesis Defect 3-Hydroxy-3-MethylglutarylCoenzyme A Lyase Deficiency: An Artefact in the Analysis of Urinary Organic Acids

Abstract 3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HMGCL, HMGCL) deficiency is a rare inborn error of ketogenesis. Even if the ketogenic enzyme is fully disrupted, an elevated signal for the ketone body acetoacetic acid is a frequent observation in the analysis of urinary organic acids, at least if derivatization is performed by methylation. We provide an explanation for this phenomenon and trace it back to degradation of the derivatized 3-hydroxy-3-methylglutaric acid and high temperature of the injector of the gas chromatograph.

Corrigendum: Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.

[This corrects the article on p. 27 in vol. 3, PMID: 27446930.].

Triheptanoin: long-term effects in the very long-chain acyl-CoA dehydrogenase-deficient mouse.

A rather new approach in the treatment of long-chain fatty acid oxidation disorders is represented by triheptanoin, a triglyceride with three medium-odd-chain heptanoic acids (C7), due to its anaplerotic potential. We here investigate the effects of a 1-year triheptanoin-based diet on the clinical phenotype of very long-chain-acyl-CoA-dehydrogenase-deficient (VLCAD-/-) mice. The cardiac function was assessed in VLCAD-/- mice by in vivo MRI. Metabolic adaptations were identified by the expression of genes regulating energy metabolism and anaplerotic processes using real-time PCR, and the results were correlated with the measurement of the glycolytic enzymes pyruvate dehydrogenase and pyruvate kinase. Finally, the intrahepatic lipid accumulation and oxidative stress in response to the long-term triheptanoin diet were assessed. Triheptanoin was not able to prevent the development of systolic dysfunction in VLCAD-/- mice despite an upregulation of cardiac glucose oxidation. Strikingly, the anaplerotic effects of triheptanoin were restricted to the liver. Despite this, the hepatic lipic content was increased upon triheptanoin supplementation. Our data demonstrate that the concept of anaplerosis does not apply to all tissues equally.

Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.

Vitamin B12 (cobalamin, Cbl, B12) is an indispensable water-soluble micronutrient that serves as a coenzyme for cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MCM). Deficiency of Cbl, whether nutritional or due to inborn errors of Cbl metabolism, inactivate MS and MCM leading to the accumulation of homocysteine (Hcy) and methylmalonic acid (MMA), respectively. In conjunction with total B12 and its bioactive protein-bound form, holo-transcobalamin (holo-TC), Hcy, and MMA are the preferred serum biomarkers utilized to determine B12 status. Clinically, vitamin B12 deficiency leads to neurological deterioration and megaloblastic anemia, and, if left untreated, to death. Subclinical vitamin B12 deficiency (usually defined as a total serum B12 of <200 pmol/L) presents asymptomatically or with rather subtle generic symptoms that oftentimes are mistakenly ascribed to unrelated disorders. Numerous studies have now established that serum vitamin B12 has limited diagnostic value as a stand-alone marker. Low serum levels of vitamin B12 not always represent deficiency, and likewise, severe functional deficiency of the micronutrient has been documented in the presence of normal and even high levels of serum vitamin B12. This review discusses the usefulness and limitations of current biomarkers of B12 status in newborn screening, infant and adult diagnostics, the algorithms utilized to diagnose B12 deficiency and unusual findings of vitamin B12 status in various human disorders.

De novo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1 year of supplementation as compared with a control diet. The de novo biosynthesis and elongation of fatty acids, and peroxisomal ß-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced de novo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected de novo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal ß-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing.