A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid

Hempen, Christel; Wanschers, Harry; Veer, G. van der Sluijs

doi:10.1007/s00216-008-1953-8

Cited by 42 publications

(38 citation statements)

References 16 publications

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“…Methylmalonic acid was assayed by LC-MS with D 3-MMA as an internal standard (11). Formate in serum or plasma was measured by an enzyme assay, using formate dehydrogenase (10); urinary formate was measured by 1 H-NMR as follows: 60 l of buffer (1.5 M potassium phosphate, 2 mM NaN3, pH 7.4) was added to 540 l of urine, and this was syringe-filtered, through a 0.45-m filter, directly into NMR tubes.…”

Section: Methodsmentioning

confidence: 99%

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Lamarre

Molloy

Reinke

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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Lamarre SG, Molloy AM, Reinke SN, Sykes BD, Brosnan ME, Brosnan JT. Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration. 1 H-NMR metabolomic analysis of sera from vitamin B12-deficient rats. In addition to the expected increases in methylmalonate and homocysteine (Hcy), we observed an approximately sevenfold increase in formate levels, from 64 M in control rats to 402 M in vitamin B 12-deficient rats. Urinary formate was also elevated. This elevation of formate could be attributed to impaired one-carbon metabolism since formate is assimilated into the one-carbon pool by incorporation into 10-formyl-THF via the enzyme 10-formyl-THF synthase. Both plasma and urinary formate were also increased in folate-deficient rats. Hcy was elevated in both the vitamin B12-and folate-deficient rats. Although plasma Hcy was also elevated, plasma formate was unaffected in vitamin B6-deficient rats (impaired transsulfuration pathway). These results were in accord with a mathematical model of folate metabolism, which predicted that reduction in methionine synthase activity would cause increased formate levels, whereas reduced cystathionine ␤-synthase activity would not. Our data indicate that formate provides a novel window into cellular folate metabolism, that elevated formate can be a useful indicator of deranged one-carbon metabolism and can be used to discriminate between the hyperhomocysteinemia caused by defects in the remethylation and transsulfuration pathways. VITAMIN B 12 DEFICIENCY RESULTS IN A WIDE SPECTRUM of hematologic and neuropsychiatric disorders. The detection of vitamin B 12 deficiency has traditionally been based on low serum vitamin B 12 levels, with supportive clinical evidence of disease. It is now recognized that serum vitamin B 12 may be insufficient to detect a deficiency, especially in the case of elderly subjects without hematologic abnormalities. The measurement of metabolites such as homocysteine (Hcy) and methylmalonic acid (MMA) has been shown to be more sensitive in diagnosing vitamin B 12 deficiency (33, 34). In a search for novel biomarkers of vitamin B 12 deficiency, we fed rats a vitamin B 12 -deficient diet and subjected their sera to 1 H-NMR metabolomic analysis. Together with the expected increased concentrations of Hcy and MMA, we found an approximately sevenfold increase in both serum and urinary formate concentrations in the vitamin B 12 -deficient animals.We confirmed that elevated formate would also be found in folate-deficient rats, since impaired folate metabolism (via the methylfolate trap) is a well-recognized consequence of vitamin B 12 deficiency (12, 31, 32) and because formate is incorporated into 10-formyl-tetrahydrofolate (THF) via 10-formyl-THF synthase (37). Both folate and vitamin B 12 deficiencies are characterized by elevated plasma Hcy. A deficiency of pyridoxal (vitamin B 6 ) also causes elevated plasma Hcy, which becomes more pronounced after methionine loading (18). This is due to impaired removal of Hcy ...

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Section: Methodsmentioning

confidence: 99%

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Lamarre

Molloy

Reinke

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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“…42. Hcy, succinic acid and MMA were determined by a single UPLC-MS/MS procedure adapted from previously published articles, with an Acquity UPLC BEH C18 column (1.7 m, 2.1 ϫ 50 mm, Waters Corporation) (8,43,44).…”

Section: Pcdna3mentioning

confidence: 99%

Vitamin B12 deficiency reduces proliferation and promotes differentiation of neuroblastoma cells and up-regulates PP2A, proNGF, and TACE

Battaglia-Hsu

Akchiche

Noel

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Vitamin B12 (cobalamin, Cbl) is indispensable for proper brain development and functioning, suggesting that it has neurotrophic effects beside its well-known importance in metabolism. The molecular basis of these effects remains hypothetical, one of the reasons being that no efficient cell model has been made available for investigating the consequences of B12 cellular deficiency in neuronal cells. Here, we designed an approach by stable transfection of NIE115 neuroblastoma cells to impose the anchorage of a chimeric B12-binding protein, transcobalamin-oleosin (TO) to the intracellular membrane. This model produced an intracellular sequestration of B12 evidenced by decreased methyl-Cbl and Sadenosylmethionine and increased homocysteine and methylmalonic acid concentrations. B12 deficiency affected the proliferation of NIE115 cells through an overall increase in catalytic protein phosphatase 2A (PP2A), despite its demethylation. It promoted cellular differentiation by improving initial outgrowth of neurites and, at the molecular level, by augmenting the levels of proNGF and p75 NTR . The up-regulation of PP2A and pro-nerve growth factor (NGF) triggered changes in ERK1/2 and Akt, two signaling pathways that influence the balance between proliferation and neurite outgrowth. Compared with control cells, a 2-fold increase of p75 NTR -regulated intramembraneous proteolysis (RIP) was observed in proliferating TO cells (P < 0.0001) that was associated with an increased expression of two tumor necrosis factor (TNF)-␣ converting enzyme (TACE) secretase enzymes, Adam 10 and Adam 17. In conclusion, our data show that B12 cellular deficiency produces a slower proliferation and a speedier differentiation of neuroblastoma cells through interacting signaling pathways that are related with increased expression of PP2A, proNGF, and TACE.homocysteine ͉ neurotrophin V itamin B12 (B12, also named cobalamin, Cbl) deficiency has long been associated with pernicious anaemia (1) and neurological disorders that range from minor behavior changes to severe neurodegenerative disorders (2). Molecular mechanisms are still lacking to explain how the deficiency can bring about all of the symptoms observed. Indeed, only two enzymes are B12-dependent in mammalian cells: the mitochondrial enzyme L-methylmalonyl-CoA mutase (EC 5.4.99.2) and the cytoplasmic homocysteine methyltransferase, also referred as methionine synthase (EC 2.1.1.13). Inferences are thus based on the two direct consequences of B12 deficiency: the accumulation of methylmalonic acid (MMA) and homocysteine (Hcy). Until now, the consequences of B12 deficiency in the brain have been difficult to evidence because of the experimental limitations of the classical cell models. Indeed, the minute amount of vitamin B12 needed by cells can be provided in vitro by B12 from the FCS, which can bind the ''autocrine'' B12 carrier protein, transcobalamin (TC) (3, 4).To delineate the role of B12 in neurological disorders, we designed a neuronal cell model made deficient in B12 through the stable ...

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“…Water [15,16], buffers [9] and mixing HCY aqueous standards with pooled human plasma in various ratios [21] have all been utilised for preparation of calibration standards; however, ion suppression was problematic [18]. Li et al [17] used acetonitrile to precipitate plasma proteins in samples of pooled human plasma and then diluted the supernatant three-fold with water to prepare a 'blank' matrix reportedly free of endogenous HCY.…”

Section: Introductionmentioning

confidence: 99%

Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum: A new approach

Ghassabian

Rethwan

Griffiths

et al. 2014

Journal of Chromatography B

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A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid

Cited by 42 publications

References 16 publications

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Vitamin B12 deficiency reduces proliferation and promotes differentiation of neuroblastoma cells and up-regulates PP2A, proNGF, and TACE

Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum: A new approach

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