Vitamin B12 Status and Supplementation in Plant-Based Diets.

Plant-based diets are increasingly popular worldwide. A well-planned plant-based diet lowers the risk of cardiovascular disease, type 2 diabetes and certain cancers. In contrast, a poorly planned plant-based diet increases the risk of certain micronutrient deficiencies, chiefly, vitamin B12 (B12). Because B12 is not present in plants or in unfortified plant-based foodstuffs, the safest way to prevent its deficiency in plant-based diets is to take an oral B12 supplement. Studies determining the dose and frequency of B12 to be taken by healthy individuals on a plant-based diet to support an adequate B12 status are scarce. Here, we summarize the natural sources, metabolic requirements, biomarker findings with and without supplementation with B12, and current recommendations to help prevent vitamin B12 deficiency in healthy individuals adhering or transitioning to plant-based diets. This review focuses on the prevention of vitamin B12 deficiency in healthy individuals adhering to plant-based diets. The information covered in this review does not apply to individuals suffering from autoimmune-based malabsorption of vitamin B12 resulting from pernicious anemia due to atrophic gastritis, other acquired causes of B12 malabsorption or to those with genetic disorders that impair vitamin B12 absorption, transport and utilization.

Plain language titleVitamin B₁₂ in Plant-Based DietsPlain language summaryPlant-based diets are increasingly popular worldwide. Because vitamin B₁₂ is not found in plants, individuals must acquire the micronutrient by consuming fortified foods or by taking an oral vitamin B₁₂ supplement. We review B₁₂ sources, required daily intake, and use of B₁₂ supplements among those on plant-based diets. The safest way to prevent B₁₂ deficiency in individuals adhering to plant-based diets is by using an oral B₁₂ supplement.

METHYLATION-ASSOCIATED PATHWAYS IN MACULAR TELANGIECTASIA TYPE 2 AND OPHTHALMOLOGIC FINDINGS IN PATIENTS WITH GENETIC METHYLATION DISORDERS.

PURPOSE: Serine (Ser) and glycine (Gly) levels were reported to differ between patients with macular telangiectasia type 2 (MacTel) compared with healthy controls. Because they are closely related to methylation metabolism, this report investigates methylation-associated metabolite levels in patients with MacTel and retinal changes in monogenetic methylation disorders. METHODS: Prospective, monocentric study on patients with MacTel and healthy controls underwent a standardized protocol including a blood draw. Methylation-associated metabolite levels in plasma were determined using targeted quantitative metabolomics. Furthermore, patient records of cystathionine beta-synthase, methylenetetrahydrofolate reductase, and methylmalonic aciduria and homocystinuria type C protein (MMACHC) deficiency were screened for reported retinal changes. RESULTS: In total, 29 patients with MacTel and 27 healthy controls were included. Patients with MacTel showed lower plasma Ser ( P = 0.02 and P = 0.01) and Gly ( P = 0.11 and P = 0.11) levels than controls. Principal component analyses revealed that methylation-associated metabolite, especially homocysteine, contributed to a distinct clustering of patients with MacTel. No retinal changes were seen in cystathionine beta-synthase (n = 1) and methylenetetrahydrofolate reductase (n = 2) deficiency, while two patients with MMACHC (n = 4) deficiency displayed extensive macular dystrophy. CONCLUSION: Patients with MacTel show distinct clustering of methylation-associated metabolite compared with controls. Of the three homocystinurias, only MMACHC resulted in macular dystrophy, possibly due to distinct compensatory pathways.

Spic regulates one-carbon metabolism and histone methylation in ground-state pluripotency.

Understanding mechanisms of epigenetic regulation in embryonic stem cells (ESCs) is of fundamental importance for stem cell and developmental biology. Here, we identify Spic, a member of the ETS family of transcription factors (TFs), as a marker of ground state pluripotency. We show that Spic is rapidly induced in ground state ESCs and in response to extracellular signal-regulated kinase (ERK) inhibition. We find that SPIC binds to enhancer elements and stabilizes NANOG binding to chromatin, particularly at genes involved in choline/one-carbon (1C) metabolism such as Bhmt, Bhmt2, and Dmgdh. Gain-of-function and loss-of-function experiments revealed that Spic controls 1C metabolism and the flux of S-adenosyl methionine to S-adenosyl-L-homocysteine (SAM-to-SAH), thereby, modulating the levels of H3R17me2 and H3K4me3 histone marks in ESCs. Our findings highlight betaine-dependent 1C metabolism as a hallmark of ground state pluripotency primarily activated by SPIC. These findings underscore the role of uncharacterized auxiliary TFs in linking cellular metabolism to epigenetic regulation in ESCs.

The impact of metabolic stressors on mitochondrial homeostasis in a renal epithelial cell model of methylmalonic aciduria.

Methylmalonic aciduria (MMA-uria) is caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). MUT deficiency hampers energy generation from specific amino acids, odd-chain fatty acids and cholesterol. Chronic kidney disease (CKD) is a well-known long-term complication. We exposed human renal epithelial cells from healthy controls and MMA-uria patients to different culture conditions (normal treatment (NT), high protein (HP) and isoleucine/valine (I/V)) to test the effect of metabolic stressors on renal mitochondrial energy metabolism. Creatinine levels were increased and antioxidant stress defense was severely comprised in MMA-uria cells. Alterations in mitochondrial homeostasis were observed. Changes in tricarboxylic acid cycle metabolites and impaired energy generation from fatty acid oxidation were detected. Methylcitrate as potentially toxic, disease-specific metabolite was increased by HP and I/V load. Mitophagy was disabled in MMA-uria cells, while autophagy was highly active particularly under HP and I/V conditions. Mitochondrial dynamics were shifted towards fission. Sirtuin1, a stress-resistance protein, was down-regulated by HP and I/V exposure in MMA-uria cells. Taken together, both interventions aggravated metabolic fingerprints observed in MMA-uria cells at baseline. The results point to protein toxicity in MMA-uria and lead to a better understanding, how the accumulating, potentially toxic organic acids might trigger CKD.

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome c in cells.

Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.

Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37.

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.

Methionine Adenosyltransferase I/III Deficiency Detected by Newborn Screening.

Methionine adenosyltransferase I/III deficiency is an inborn error of metabolism due to mutations in the MAT1A gene. It is the most common cause of hypermethioninemia in newborn screening. Heterozygotes are often asymptomatic. In contrast, homozygous or compound heterozygous individuals can develop severe neurological symptoms. Less than 70 cases with biallelic variants have been reported worldwide. A methionine-restricted diet is recommended if methionine levels are above 500−600 µmol/L. In this study, we report on a female patient identified with elevated methionine concentrations in a pilot newborn screening program. The patient carries a previously described variant c.1132G>A (p.Gly378Ser) in homozygosity. It is located at the C-terminus of MAT1A. In silico analysis suggests impaired protein stability by ß-turn disruption. On a methionine-restricted diet, her serum methionine concentration ranged between 49−605 µmol/L (median 358 µmol/L). Her clinical course was characterized by early-onset muscular hypotonia, mild developmental delay, delayed myelination and mild periventricular diffusion interference in MRI. At 21 months, the girl showed age-appropriate neurological development, but progressive diffusion disturbances in MRI. Little is known about the long-term outcome of this disorder and the necessity of treatment. Our case demonstrates that neurological symptoms can be transient and even patients with initial neurologic manifestations can show normal development under dietary management.

Analysis of S-Adenosylmethionine and S-Adenosylhomocysteine: Method Optimisation and Profiling in Healthy Adults upon Short-Term Dietary Intervention.

S-adenosylmethionine (SAM) is essential for methyl transfer reactions. All SAM is produced de novo via the methionine cycle. The demethylation of SAM produces S-adenosylhomocysteine (SAH), an inhibitor of methyltransferases and the precursor of homocysteine (Hcy). The measurement of SAM and SAH in plasma has value in the diagnosis of inborn errors of metabolism (IEM) and in research to assess methyl group homeostasis. The determination of SAM and SAH is complicated by the instability of SAM under neutral and alkaline conditions and the naturally low concentration of both SAM and SAH in plasma (nM range). Herein, we describe an optimised LC-MS/MS method for the determination of SAM and SAH in plasma, urine, and cells. The method is based on isotopic dilution and employs 20 µL of plasma or urine, or 500,000 cells, and has an instrumental running time of 5 min. The reference ranges for plasma SAM and SAH in a cohort of 33 healthy individuals (age: 19-60 years old; mean ± 2 SD) were 120 ± 36 nM and 21.5 ± 6.5 nM, respectively, in accordance with independent studies and diagnostic determinations. The method detected abnormal concentrations of SAM and SAH in patients with inborn errors of methyl group metabolism. Plasma and urinary SAM and SAH concentrations were determined for the first time in a randomised controlled trial of 53 healthy adult omnivores (age: 18-60 years old), before and after a 4 week intervention with a vegan or meat-rich diet, and revealed preserved variations of both metabolites and the SAM/SAH index.

Intracellular processing of vitamin B12 by MMACHC (CblC).

Vitamin B12 (cobalamin, Cbl, B12) is a water-soluble micronutrient synthesized exclusively by a group of microorganisms. Human beings are unable to make B12 and thus obtain the vitamin via intake of animal products, fermented plant-based foods or supplements. Vitamin B12 obtained from the diet comprises three major chemical forms, namely hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl). The most common form of B12 present in supplements is cyanocobalamin (CNCbl). Yet, these chemical forms cannot be utilized directly as they come, but instead, they undergo chemical processing by the MMACHC protein, also known as CblC. Processing of dietary B12 by CblC involves removal of the upper-axial ligand (beta-ligand) yielding the one-electron reduced intermediate cob(II)alamin. Newly formed cob(II)alamin undergoes trafficking and delivery to the two B12-dependent enzymes, cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MUT). The catalytic cycles of MS and MUT incorporate cob(II)alamin as a precursor to regenerate the coenzyme forms MeCbl and AdoCbl, respectively. Mutations and epimutations in the MMACHC gene result in cblC disease, the most common inborn error of B12 metabolism, which manifests with combined homocystinuria and methylmalonic aciduria. Elevation of metabolites homocysteine and methylmalonic acid occurs because the lack of an active CblC blocks formation of the indispensable precursor cob(II)alamin that is necessary to activate MS and MUT. Thus, in patients with cblC disease, vitamin B12 is absorbed and present in circulation in normal to high concentrations, yet, cells are unable to make use of it. Mutations in seemingly unrelated genes that modify MMACHC gene expression also result in clinical phenotypes that resemble cblC disease. We review current knowledge on structural and functional aspects of intracellular processing of vitamin B12 by the versatile protein CblC, its partners and possible regulators.

Biosynthesis, Quantification and Genetic Diseases of the Smallest Signaling Thiol Metabolite: Hydrogen Sulfide.

Hydrogen sulfide (H2S) is a gasotransmitter and the smallest signaling thiol metabolite with important roles in human health. The turnover of H2S in humans is mainly governed by enzymes of sulfur amino acid metabolism and also by the microbiome. As is the case with other small signaling molecules, disease-promoting effects of H2S largely depend on its concentration and compartmentalization. Genetic defects that impair the biogenesis and catabolism of H2S have been described; however, a gap in knowledge remains concerning physiological steady-state concentrations of H2S and their direct clinical implications. The small size and considerable reactivity of H2S renders its quantification in biological samples an experimental challenge. A compilation of methods currently employed to quantify H2S in biological specimens is provided in this review. Substantial discrepancy exists in the concentrations of H2S determined by different techniques. Available methodologies permit end-point measurement of H2S concentration, yet no definitive protocol exists for the continuous, real-time measurement of H2S produced by its enzymatic sources. We present a summary of available animal models, monogenic diseases that impair H2S metabolism in humans including structure-function relationships of pathogenic mutations, and discuss possible approaches to overcome current limitations of study.

Mitochondrial damage in renal epithelial cells is potentiated by protein exposure in propionic aciduria.

Propionic aciduria (PA) is caused by deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Due to inefficient propionate catabolism patients are endangered by life-threatening ketoacidotic crisis. Protein and amino acid restriction are major therapeutic pillars. However, long-term complications like neurological deterioration and cardiac abnormalities cannot be prevented. Chronic kidney disease (CKD), which is a well-known characteristic of methylmalonic aciduria two enzymatic steps downstream from PCC, has been recognized as a novel late-onset complication in PA. The pathophysiology of CKD in PA is unclear. We investigated mitochondrial structure and metabolism in human renal tubular cells of healthy controls and PA patients. The cells were exposed to either standard cell culture conditions (NT), high protein (HP) or high concentrations of isoleucine and valine (I/V). Mitochondrial morphology changed to condensed, fractured morphology in PA cells irrespective of the cell culture medium. HP and I/V exposure, however, potentiated oxidative stress in PA cells. Mitochondrial mass was enriched in PA cells, and further increased by HP and I/V exposure suggesting a need for compensation. Alterations in the tricarboxylic acid cycle intermediates and accumulation of medium- and long-chain acylcarnitines pointed to altered mitochondrial energy metabolism. Mitophagy was silenced while autophagy as cellular defense mechanisms was highly active in PA cells. The data demonstrate that PA is associated with renal mitochondrial damage which is aggravated by protein and I/V load. Preservation of mitochondrial energy homeostasis in renal cells may be a potential future therapeutic target.

Inhibition of metabotropic glutamate receptor III facilitates sensitization to alkylating chemotherapeutics in glioblastoma.

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.

Defective lysosomal storage in Fabry disease modifies mitochondrial structure, metabolism and turnover in renal epithelial cells.

Fabry disease (FD) is an X-linked lysosomal storage disorder. Deficiency of the lysosomal enzyme alpha-galactosidase (GLA) leads to accumulation of potentially toxic globotriaosylceramide (Gb3) on a multisystem level. Cardiac and cerebrovascular abnormalities as well as progressive renal failure are severe, life-threatening long-term complications. The complete pathophysiology of chronic kidney disease (CKD) in FD and the role of tubular involvement for its progression are unclear. We established human renal tubular epithelial cell lines from the urine of male FD patients and male controls. The renal tubular system is rich in mitochondria and involved in transport processes at high-energy costs. Our studies revealed fragmented mitochondria with disrupted cristae structure in FD patient cells. Oxidative stress levels were elevated and oxidative phosphorylation was upregulated in FD pointing at enhanced energetic needs. Mitochondrial homeostasis and energy metabolism revealed major changes as evidenced by differences in mitochondrial number, energy production and fuel consumption. The changes were accompanied by activation of the autophagy machinery in FD. Sirtuin1, an important sensor of (renal) metabolic stress and modifier of different defense pathways, was highly expressed in FD. Our data show that lysosomal FD impairs mitochondrial function and results in severe disturbance of mitochondrial energy metabolism in renal cells. This insight on a tissue-specific level points to new therapeutic targets which might enhance treatment efficacy.

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.

Metabolic Profiling in Human Fibroblasts Enables Subtype Clustering in Glycogen Storage Disease.

Glycogen storage disease subtypes I and III (GSD I and GSD III) are monogenic inherited disorders of metabolism that disrupt glycogen metabolism. Unavailability of glucose in GSD I and induction of gluconeogenesis in GSD III modify energy sources and possibly, mitochondrial function. Abnormal mitochondrial structure and function were described in mice with GSD Ia, yet significantly less research is available in human cells and ketotic forms of the disease. We hypothesized that impaired glycogen storage results in distinct metabolic phenotypes in the extra- and intracellular compartments that may contribute to pathogenesis. Herein, we examined mitochondrial organization in live cells by spinning-disk confocal microscopy and profiled extra- and intracellular metabolites by targeted LC-MS/MS in cultured fibroblasts from healthy controls and from patients with GSD Ia, GSD Ib, and GSD III. Results from live imaging revealed that mitochondrial content and network morphology of GSD cells are comparable to that of healthy controls. Likewise, healthy controls and GSD cells exhibited comparable basal oxygen consumption rates. Targeted metabolomics followed by principal component analysis (PCA) and hierarchical clustering (HC) uncovered metabolically distinct poises of healthy controls and GSD subtypes. Assessment of individual metabolites recapitulated dysfunctional energy production (glycolysis, Krebs cycle, succinate), reduced creatinine export in GSD Ia and GSD III, and reduced antioxidant defense of the cysteine and glutathione systems. Our study serves as proof-of-concept that extra- and intracellular metabolite profiles distinguish glycogen storage disease subtypes from healthy controls. We posit that metabolite profiles provide hints to disease mechanisms as well as to nutritional and pharmacological elements that may optimize current treatment strategies.

Vegan diet reduces neutrophils, monocytes and platelets related to branched-chain amino acids - A randomized, controlled trial.

BACKGROUND: Vegan diet (VD) has improved inflammatory activity in patients with rheumatoid arthritis (RA) in several small controlled trials. The underlying mechanism remains widely unclear. We investigated the effect of a VD in comparison to a meat-rich diet (MD) on markers of inflammation (which have been shown to be relevant in patients with RA) in healthy volunteers. METHODS: 53 healthy, omnivore subjects were randomized to a controlled VD (n = 26) or MD (n = 27) for 4 weeks following a pre-treatment phase of a one week controlled mixed diet. Primary parameters of interest were sialylation of immunoglobulins, percentage of regulatory T-cells and level of interleukin 10 (IL10). Usual care immune parameters used in patients with RA and amino acid serum levels as well as granulocytes and monocytes colony stimulating factor (GM-CSF) serum levels were secondary parameters. RESULTS: In the VD group, total leukocyte, neutrophil, monocyte and platelet counts decreased and after four weeks they were significantly lower compared to the MD group (ANCOVA: leukocytes p = 0.003, neutrophils p = 0.001, monocytes p = 0.032, platelets p = 0.004). Leukocytes, neutrophils, monocytes, and platelets correlated with each other and likewise conform with serum levels of branched-chain amino acids, which were significantly lower in the VD compared to the MD group. The primary parameters did not differ between the groups and BMI remained stable in the two groups. CONCLUSION: Four weeks of a controlled VD affected the number of neutrophils, monocytes and platelets but not the number or function of lymphocytes. The relation with branched-chain amino acids and GM-CSF suggests a mode of action via the mTOR signaling pathway. REGISTERED AT: http://www.drks.de (German Clinical Trial register) at DRKS00011963.

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.

Catalytic effect of riboflavin on electron transfer from NADH to aquacobalamin.

Reduction of cobalamin by non-dedicated cellular reductases has been reported in earlier work, however, the sources of reducing power and the mechanisms are unknown. This study reports results of kinetic and mechanistic investigation of the reaction between aquacobalamin, H2OCbl, and reduced ß-nicotinamide adenine dinucleotide, NADH. This interaction leads to the formation of one-electron reduced cobalamin, cob(II)alamin, and proceeds via water substitution on aquacobalamin by NADH and further decomposition of NADH-Co(III) complex to cob(II)alamin and NADH·+. Riboflavin catalyzes the reduction of aquacobalamin by NADH both in free form and with aquacobalamin bound to the cobalamin processing enzyme CblC. The rate-determining step of this catalytic reaction is the interaction between riboflavin and NADH to produce a charge transfer complex that reacts with aquacobalamin. Aquacobalamin quenches the fluorescence of NADH and riboflavin predominantly via a static mechanism.

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.

Proteome Profiling of Primary Pancreatic Ductal Adenocarcinomas Undergoing Additive Chemoradiation Link ALDH1A1 to Early Local Recurrence and Chemoradiation Resistance.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with frequent post-surgical local recurrence. The combination of adjuvant chemotherapy with radiotherapy is under consideration to achieve a prolonged progression-free survival (PFS). To date, few studies have determined the proteome profiles associated with response to adjuvant chemoradiation. We herein analyzed the proteomes of primary PDAC tumors subjected to additive chemoradiation after surgical resection and achieving short PFS (median 6 months) versus prolonged PFS (median 28 months). Proteomic analysis revealed the overexpression of Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) and Monoamine Oxidase A (MAOA) in the short PFS cohort, which were corroborated by immunohistochemistry. In vitro, specific inhibition of ALDH1A1 by A37 in combination with gemcitabine, radiation, and chemoradiation lowered cell viability and augmented cell death in MiaPaCa-2 and Panc 05.04 cells. ALDH1A1 silencing in both cell lines dampened cell proliferation, cell metabolism, and colony formation. In MiaPaCa-2 cells, ALDH1A1 silencing sensitized cells towards treatment with gemcitabine, radiation or chemoradiation. In Panc 05.04, increased cell death was observed upon gemcitabine treatment only. These findings are in line with previous studies that have suggested a role of ALDH1A1 chemoradiation resistance, e.g., in esophageal cancer. In summary, we present one of the first proteome studies to investigate the responsiveness of PDAC to chemoradiation and provide further evidence for a role of ALDH1A1 in therapy resistance.