Plasma methionine metabolic profile is associated with longevity in mammals.

Methionine metabolism arises as a key target to elucidate the molecular adaptations underlying animal longevity due to the negative association between longevity and methionine content. The present study follows a comparative approach to analyse plasma methionine metabolic profile using a LC-MS/MS platform from 11 mammalian species with a longevity ranging from 3.5 to 120 years. Our findings demonstrate the existence of a species-specific plasma profile for methionine metabolism associated with longevity characterised by: i) reduced methionine, cystathionine and choline; ii) increased non-polar amino acids; iii) reduced succinate and malate; and iv) increased carnitine. Our results support the existence of plasma longevity features that might respond to an optimised energetic metabolism and intracellular structures found in long-lived species.

One-Carbon Metabolism in Nepalese Infant-Mother Pairs and Child Cognition at 5 Years Old.

BACKGROUND: One-carbon metabolism (OCM) refers to the transfer of methyl groups central to DNA methylation and histone modification. Insufficient access to methyl donors and B-vitamin cofactors affects epigenetic maintenance and stability, and when occurring in early life may impact future health and neurodevelopment. OBJECTIVE: The objective of this study was to examine the relative associations between one-carbon metabolites in Nepalese mother-infant pairs and child cognition measured at 5 y of age. METHODS: This is a cross-sectional study from Bhaktapur, Nepal, in a population at high risk of subclinical B-vitamin deficiencies and cumulative infection burden. Venous blood samples from 500 mother-infant pairs were collected when the infants were 2 to 12 mo old, and metabolite concentrations measured by microbiological assays and GC-tandem MS. We re-enrolled 321 of these children at 5 y and assessed cognition by the Ages and Stages Questionnaire, 3rd edition, and subtests from the Developmental Neuropsychological Assessment, 2nd edition (NEPSY-II). The associations of the independent metabolites or unobserved metabolic phenotypes (identified by latent class analysis) with the cognitive outcomes were estimated by seemingly unrelated regression. We explored direct and indirect relations between the OCM pathway and the cognitive outcomes using path analysis. RESULTS: Infant cystathionine concentration was inversely associated with 4 cognitive outcomes (standardized ßs ranging from -0.22 to -0.11, P values from <0.001 to 0.034). Infants with a metabolic phenotype indicating impaired OCM and low vitamin B-12 status had poorer cognitive outcomes compared with infants with normal OCM activity and adequate vitamin B-12 status (standardized ßs ranging from -0.80 to -0.40, P < 0.001 and 0.05). In the path analysis, we found several OCM biomarkers were associated with affect recognition through infant plasma cystathionine. CONCLUSIONS: Elevated plasma cystathionine during infancy reflects a metabolic phenotype of impaired OCM and low vitamin B-12 status and is associated with poorer cognitive function when the children are 5 y old.

Maternal Plasma Pyridoxal 5'-Phosphate Concentration Is Inversely Associated with Plasma Cystathionine Concentration across All Trimesters in Healthy Pregnant Women.

BACKGROUND: Vitamin B-6 (B-6), in the form of pyridoxal 5'phosphate (PLP), is critical for one-carbon metabolism reactions and cellular function. Plasma PLP concentration decreases throughout pregnancy, but the functional consequences of this have not been studied. Plasma cystathionine is a sensitive indicator of suboptimal B-6 status in healthy adults. OBJECTIVES: The aim of this study was to determine the relation between plasma PLP and cystathionine concentrations, and to assess longitudinal changes in plasma concentrations of metabolites of one-carbon metabolism, including total homocysteine (tHcy), cysteine, methionine, glycine, serine, and glutathione, over the course of pregnancy. DESIGN: This was a prospective cohort study of 186 healthy Brazilian pregnant women (20-40 y). Plasma PLP and metabolite concentrations were quantified in fasting maternal blood samples collected between 5-13, 20-26, and 30-36 weeks of gestation. Linear mixed regression models were used to determine the association of 1) first-trimester PLP tertiles, and 2) the variation of PLP concentration throughout pregnancy, with related metabolite concentrations across weeks of gestation. RESULTS: Median (IQR) PLP concentration decreased from 36.2 (29.2-44.5) to 21.0 (15.9-26.0) to 16.8 (12.9-21.4) nmol/L in the first, second, and third trimester, respectively, whereas cystathionine concentration increased from 63.2 (49.7-78.9) to 122 (98.0-167) to 143 (114-193) nmol/L, respectively (both P < 0.001). The variation of PLP throughout pregnancy was inversely associated with cystathionine concentration across weeks of gestation, after adjusting for confounding factors; ß (95% CI) = -0.387 (-0.752, -0.219), P = 0.04. This association significantly differed by trimester and was strongest in the third trimester. Plasma concentrations of glycine, serine, methionine, cysteine, and tHcy decreased, and that of glutathione increased, between the first and second trimesters (all P < 0.05). CONCLUSIONS: The variation of PLP concentration predicted cystathionine concentration throughout pregnancy. Increases in plasma cystathionine across trimesters may reflect maternal intracellular B-6 deficiency.

Elevated plasma cystathionine is associated with increased risk of mortality among patients with suspected or established coronary heart disease.

BACKGROUND: Elevated circulating cystathionine levels are related to atherosclerotic cardiovascular disease, a leading cause of death globally. OBJECTIVE: We investigated whether plasma cystathionine was associated with mortality in patients with suspected or established coronary heart disease (CHD). METHODS: Data from 2 independent cohorts of patients with suspected stable angina pectoris (SAP) (3033 patients; median 10.7 y follow-up; 648 deaths) or acute myocardial infarction (AMI) (3670 patients; median 7.0 y follow-up; 758 deaths) were included. Hazard ratios with 95% CIs per SD increment of log-transformed cystathionine were calculated using Cox regression modeling. Endpoint data was obtained from a national health registry. RESULTS: Among patients with SAP, there was a positive association between plasma cystathionine and death (age- and sex-adjusted HRs [95% CI] per SD: 1.23 [1.14, 1.32], 1.29 [1.16, 1.44], and 1.17 [1.05, 1.29] for total, cardiovascular, and noncardiovascular mortality, respectively). Corresponding risk estimates were 1.28 (1.19, 1.37) for all-cause, 1.33 (1.22, 1.45) for cardiovascular, and 1.19 (1.06, 1.34) for noncardiovascular death among AMI patients. In both cohorts, estimates were slightly attenuated after multivariate adjustments for established CHD risk factors. Subgroup analyses showed that the relation between cystathionine and all-cause mortality in SAP patients was stronger among nonsmokers and those with lower plasma concentration of pyridoxal-5'-phosphate (P-interaction ≤ 0.01 for both). CONCLUSIONS: Elevated plasma cystathionine is associated with both cardiovascular and noncardiovascular mortality among patients with suspected or established CHD. The joint risk associations of high plasma cystathionine with lifestyle factors and impaired vitamin B-6 status on mortality need further investigation. This trial was registered at clinicaltrials.gov as NCT00354081 and NCT00266487.

Plasma Cystathionine and Risk of Incident Stroke in Patients With Suspected Stable Angina Pectoris.

Background Cystathionine is an intermediate product in the transsulfuration pathway and formed during the B6-dependent conversion of methionine to cysteine. Elevated plasma cystathionine has been related to atherosclerosis, which is a major etiological factor for ischemic stroke. However, the role of cystathionine in stroke development is unknown. Therefore, we prospectively assessed the association of circulating levels of cystathionine with risk of total and ischemic stroke. Methods and Results Two-thousand thirty-six patients (64% men; median age, 62 years) undergoing coronary angiography for suspected stable angina pectoris were included. Stroke cases were identified by linkage to the CVDNOR (Cardiovascular Disease in Norway) project. Hazard ratios with confidence intervals (95% confidence interval) were estimated by using Cox-regression analyses. During 7.3 years of median follow-up, 124 (6.1%) incident strokes were ascertained, which comprised 100 cases of ischemic stroke. There was a positive association of plasma cystathionine with risk of total stroke and ischemic stroke. Comparing the fourth versus the first cystathionine quartiles, age- and sex-adjusted hazard ratios (95% confidence interval) were 2.11 (1.19-3.75) and 2.56 (1.31-4.99) for total and ischemic stroke, respectively. Additional adjustment for major stroke risk factors only slightly attenuated the associations, which tended to be stronger in patients without previous or existing atrial fibrillation at baseline (hazard ratio [95% confidence interval], 2.43 [1.27-4.65] and 2.88 [1.39-5.98] for total and ischemic stroke, respectively). Conclusions In patients with suspected stable angina pectoris, plasma cystathionine was independently related to increased risk of total stroke and, in particular, ischemic stroke. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 00354081.

Plasma cystathionine and risk of acute myocardial infarction among patients with coronary heart disease: Results from two independent cohorts.

BACKGROUND: Cystathionine is a thio-ether and a metabolite formed from homocysteine during transsulfuration. Elevated plasma cystathionine levels are reported in patients with cardiovascular disease; however prospective relationships with acute myocardial infarction (AMI) are unknown. We investigated associations between plasma cystathionine and AMI among patients with suspected and/or verified coronary heart disease (CHD). METHODS: Subjects from two independent cohort studies, the Western Norway Coronary Angiography Cohort (WECAC) (3033 patients with stable angina pectoris; 263 events within 4.8 years of median follow-up) and the Norwegian Vitamin Trial (NORVIT) (3670 patients with AMI; 683 events within 3.2 years of median follow-up) were included. RESULTS: In both cohorts, plasma cystathionine was associated with several traditional CHD risk factors (P < 0.001). Comparing the cystathionine quartile 4 to 1, age and gender adjusted hazard ratios (95% confidence intervals) for AMI were 2.08 (1.43-3.03) and 1.41 (1.12-1.76) in WECAC and NORVIT, respectively. Additional adjustment for traditional risk factors slightly attenuated the risk estimates, which were generally stronger in both cohorts among non-smokers, patients with higher age, and lower BMI or PLP status (P-interaction ≤ 0.04). Risk associations also tended to be stronger in patients not treated with B-vitamins. Additionally, in a subset of 80 WECAC patients, plasma cystathionine associated strongly negatively with glutathione, an important antioxidant and positively with lanthionine, a marker of H2S production (P < 0.001). CONCLUSIONS: Plasma cystathionine is associated with increased risk of AMI among patients with either suspected or verified coronary heart disease, and is possibly related to altered redox homeostasis.

Effects of high ambient temperature on plasma metabolomic profiles in chicks.

Exposure to high ambient temperature is detrimental to the poultry industry. To understand the influence from a metabolic perspective, we investigated the effects of exposure to high ambient temperature on plasma low-molecular-weight metabolite levels in chicks using gas chromatography/mass spectrometry-based non-targeted metabolomic analysis. Heat exposure for 4 days suppressed growth and food intake. Of the 92 metabolites identified, the levels of 29 decreased, whereas the levels of nine increased. We performed an enrichment analysis on the identified metabolites and found 35 candidates for metabolic processes affected by heat exposure. Among them, the sulfur amino acid metabolic pathway was clearly detected and the levels of the following related metabolites were decreased: cystathionine, cysteine, cystine, homocysteine and hypotaurine. Changes in the kynurenine pathway in tryptophan metabolism, which is linked to the immune system and oxidative stress, were also observed: kynurenine and quinolinic acid levels increased, whereas nicotinamide levels decreased. These results suggest the possible involvement of various metabolic processes in heat-exposed chicks. Some of these metabolites would be important to understand the mechanism of biological responses to high ambient temperature in chicks.

DNA damage in homocystinuria: 8-oxo-, 8-dihydro-2'-deoxyguanosine levels in cystathionine-ß-synthase deficient patients and the in vitro protective effect of N-acetyl­L­cysteine

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine ß-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS­deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2'- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 µM and 200 µM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria.

Metabolomic Evaluation of the Consequences of Plasma Cystathionine Elevation in Adults with Stable Angina Pectoris.

Background: An elevated circulating cystathionine concentration, which arises in part from insufficiencies of vitamin B-6, B-12, or folate, has been shown to be associated with cardiovascular disease (CVD) risk. Hydrogen sulfide (H2S) is a gasotransmitter involved in vasodilation, neuromodulation, and inflammation. Most endogenously produced H2S is formed by pyridoxal phosphate (PLP)-dependent enzymes by noncanonical reactions of the transsulfuration pathway that yield H2S concurrently form lanthionine and homolanthionine. Thus, plasma lanthionine and homolanthionine concentrations can provide relative information about H2S production in vivo.Objective: To determine the metabolic consequences of an elevated plasma cystathionine concentration in adults with stable angina pectoris (SAP), we conducted both targeted and untargeted metabolomic analyses.Methods: We conducted NMR and LC-mass spectrometry (MS) metabolomic analyses on a subset of 80 plasma samples from the Western Norway Coronary Angiography Cohort and selected, based on plasma cystathionine concentrations, a group with high cystathionine concentrations [1.32 ± 0.60 µmol/L (mean ± SD); n = 40] and a group with low cystathionine concentrations [0.137 ± 0.011 µmol/L (mean ± SD); n = 40]. Targeted and untargeted metabolomic analyses were performed and assessed with the use of Student's t tests corrected for multiple testing. Overall differences between the cystathionine groups were assessed by untargeted NMR and LC-MS metabolomic methods and evaluated by partial least squares discriminant analysis (PLS-DA) with significant discriminating metabolites identified with 99% confidence.Results: Subjects with high cystathionine concentrations had 75% higher plasma lanthionine concentrations (0.12 ± 0.044 µmol/L) than subjects with low cystathionine concentrations [0.032 ± 0.013 µmol/L (P < 0.001)]. Although plasma homolanthionine concentrations were notably higher than lanthionine concentrations, they were not different between the groups (P = 0.47). PLS-DA results showed that a high plasma cystathionine concentration in SAP was associated with higher glucose, branched-chain amino acids, and phenylalanine concentrations, lower kidney function, and lower glutathione and plasma PLP concentrations due to greater catabolism. The high-cystathionine group had a greater proportion of subjects in the postprandial state.Conclusion: These data suggest that metabolic perturbations consistent with higher CVD risk exist in SAP patients with elevated plasma cystathionine concentrations.

Comfortably Numb and Back: Plasma Metabolomics Reveals Biochemical Adaptations in the Hibernating 13-Lined Ground Squirrel.

Hibernation is an evolutionary adaptation that affords some mammals the ability to exploit the cold to achieve extreme metabolic depression (torpor) while avoiding ischemia/reperfusion or hemorrhagic shock injuries. Hibernators cycle periodically out of torpor, restoring high metabolic activity. If understood at the molecular level, the adaptations underlying torpor-arousal cycles may be leveraged for translational applications in critical fields such as intensive care medicine. Here, we monitored 266 metabolites to investigate the metabolic adaptations to hibernation in plasma from 13-lined ground squirrels (57 animals, 9 time points). Results indicate that the periodic arousals foster the removal of potentially toxic oxidative stress-related metabolites, which accumulate in plasma during torpor while replenishing reservoirs of circulating catabolic substrates (free fatty acids and amino acids). Specifically, we identified metabolic fluctuations of basic amino acids lysine and arginine, one-carbon metabolism intermediates, and sulfur-containing metabolites methionine, cysteine, and cystathionine. Conversely, reperfusion injury markers such as succinate/fumarate remained relatively stable across cycles. Considering the cycles of these metabolites with the hibernator's cycling metabolic activity together with their well-established role as substrates for the production of hydrogen sulfide (H2S), we hypothesize that these metabolic fluctuations function as a biological clock regulating torpor to arousal transitions and resistance to reperfusion during arousal.

Methionine and Choline Supply during the Periparturient Period Alter Plasma Amino Acid and One-Carbon Metabolism Profiles to Various Extents: Potential Role in Hepatic Metabolism and Antioxidant Status.

The objective of this study was to profile plasma amino acids (AA) and derivatives of their metabolism during the periparturient period in response to supplemental rumen-protected methionine (MET) or rumen-protected choline (CHOL). Forty cows were fed from -21 through 30 days around parturition in a 2 × 2 factorial design a diet containing MET or CHOL. MET supply led to greater circulating methionine and proportion of methionine in the essential AA pool, total AA, and total sulfur-containing compounds. Lysine in total AA also was greater in these cows, indicating a better overall AA profile. Sulfur-containing compounds (cystathionine, cystine, homocystine, and taurine) were greater in MET-fed cows, indicating an enriched sulfur-containing compound pool due to enhanced transsulfuration activity. Circulating essential AA and total AA concentrations were greater in cows supplied MET due to greater lysine, arginine, tryptophan, threonine, proline, asparagine, alanine, and citrulline. In contrast, CHOL supply had no effect on essential AA or total AA, and only tryptophan and cystine were greater. Plasma 3-methylhistidine concentration was lower in response to CHOL supply, suggesting less tissue protein mobilization in these cows. Overall, the data revealed that enhanced periparturient supply of MET has positive effects on plasma AA profiles and overall antioxidant status.

The association of single nucleotide polymorphisms of the maternal cystathionine-ß-synthase gene with early-onset preeclampsia.

OBJECTIVES: Preeclampsia (PE) is a pregnancy complication, characterized by hypertension and proteinuria. The transsulfuration pathway may be involved in its pathophysiology, since homocysteine, cystathionine and cysteine are increased in PE. Cystathionine-ß-synthase (CBS) is a key-enzyme in the pathway, converting homocysteine into cysteine via cystathionine. Another product of CBS is hydrogen sulfide (H2S), a vasodilatory, proangiogenic and cytoprotective gas that is thought to play a role in placental and vascular function during pregnancy. Since single nucleotide polymorphisms (SNPs) can affect CBS expression and/or function, we studied tag-SNPs in the CBS gene in PE patients. STUDY DESIGN: Controls (n=75), early-onset (n=45), and late-onset PE (n=52) cases were genotyped for six tag-SNPs in the CBS gene; rs12329764, rs2851391, rs234713, rs234706, rs1789953, and rs11203172. Plasma homocysteine, cysteine and cystathionine were determined during pregnancy. MAIN OUTCOME MEASURES: Early-onset PE, late-onset PE. RESULTS: Women with the minor allele of rs11203172 have a reduced risk for early-onset PE. Compared to women without the minor allele, normotensive pregnant women with the minor allele of rs11203172 and rs234713 have lower cysteine levels. Women with the minor allele of rs1789953 have increased levels of cysteine and cystathionine, compared to women without. CONCLUSION: The CBS tag-SNP rs11203172 is associated with a decreased risk for early-onset PE. Decreased cysteine concentrations in normotensive pregnant women carrying the minor allele of rs11203172, may be due to increased cysteine conversion to H2S by CBS. Higher H2S levels may positively affect placentation and vascular function during pregnancy and decrease their risk for PE.

Reduced folate and serum vitamin metabolites in patients with rectal carcinoma: an open-label feasibility study of pemetrexed with folic acid and vitamin B12 supplementation.

The objectives of this single-center, open-label, phase II study were to evaluate (a) the feasibility and safety of neoadjuvant administration of pemetrexed with oral folic acid and vitamin B12 (FA/B12) in newly diagnosed patients with resectable rectal cancer and (b) intracellular and systemic vitamin metabolism. Patients were treated with three cycles of pemetrexed (500 mg/m, every 3 weeks) and FA/B12 before surgery. The reduced folates tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate were evaluated from biopsies in tumor tissue and in adjacent mucosa. Serum levels of homocysteine, cystathionine, and methylmalonic acid were also measured. All 37 patients received three cycles of pemetrexed; 89.2% completed their planned dosage within a 9-week feasibility time frame. Neither dose reductions nor study drug-related serious adverse events were reported. Reduced folate levels were significantly higher in tumor tissue compared with adjacent mucosa at baseline. After FA/B12 administration, tissue levels of reduced folates increased significantly and remained high during treatment in both tumor and mucosa until surgery. Serum levels of cystathionine increased significantly compared with baseline after FA/B12 administration, but then decreased, fluctuating cyclically during pemetrexed therapy. Homocysteine and methylmalonic acid levels decreased significantly after FA/B12 administration, and remained below baseline levels during the study. These results indicate that administration of three neoadjuvant cycles of single-agent pemetrexed, every 3 weeks, with FA/B12 in patients with resectable rectal cancer is feasible and tolerable. Tissue and serum vitamin metabolism results demonstrate the influence of pemetrexed and FA/B12 on vitamin metabolism and warrant further study.

A Vitamin B-6 Antagonist from Flaxseed Perturbs Amino Acid Metabolism in Moderately Vitamin B-6-Deficient Male Rats.

BACKGROUND: Pyridoxal 5'-phosphate (PLP) plays a crucial role as a cofactor in amino acid metabolism. There is a prevalence of moderate vitamin B-6 deficiency in the population that may be exacerbated through the ingestion of 1-amino d-proline (1ADP), a vitamin B-6 antagonist found in flaxseed. OBJECTIVE: Given prior evidence of the impact of synthetic 1ADP on indexes of pyridoxine metabolism, the current study was designed to investigate the effects of 1ADP derived from flaxseed on amino acid metabolism in moderately vitamin B-6-deficient rats. METHODS: Male weanling rats (n = 8/treatment) consumed a semipurified diet containing either 7 mg pyridoxine hydrochloride/kg diet [optimum vitamin B-6 (OB)] or 0.7 mg pyridoxine hydrochloride/kg diet [moderately vitamin B-6 deficient (MB)], each with 0 or 10 mg vitamin B-6 antagonist/kg diet, in either a synthetic form (1ADP) or as a flaxseed extract (FE), for 5 wk. At the end of the experiment, plasma vitamin B-6 and amino acid concentrations and the activities of hepatic PLP-dependent enzymes were analyzed. RESULTS: Compared with the MB control group, plasma PLP concentrations were 26% and 69% lower, respectively, in the MB+FE and MB+1ADP rats (P ≤ 0.001). In the MB+FE group, the plasma cystathionine concentration was 100% greater and the plasma α-aminobutyric acid and glutamic acid concentrations were 59% and 30% lower, respectively, than in the MB control group. Both synthetic 1ADP and FE significantly (P < 0.001) inhibited the in vitro hepatic activities of 2 PLP-dependent enzymes, cystathionine ß-synthase (up to 44%) and cystathionine γ-lyase (up to 60%), irrespective of vitamin B-6 concentrations. Because of vitamin B-6 antagonist exposure, observed perturbations in plasma biomarkers and hepatic enzyme activities were not evident or of lesser magnitude in rats consuming adequate vitamin B-6. CONCLUSION: The current data from a rat model provide evidence that a vitamin B-6 antagonist now prevalent in the human food supply may pose challenges to individuals of moderate vitamin B-6 status.

Folic acid causes higher prevalence of detectable unmetabolized folic acid in serum than B-complex: a randomized trial.

PURPOSE: Unmetabolized folic acid (UMFA) is common in serum of elderly individuals receiving folic acid (FA)-fortified foods or supplements. We studied the effect of supplementing FA or B-complex on serum concentrations of (6S)-5-methyltetrahydropteroylglutamate [(6S)-5-CH3-H4Pte] and UMFA in elderly people and explored factors associated with detectable UMFA post-supplementation. METHODS: This is a randomized single-blind non-controlled trial on 58 elderly people using daily 400 µg FA (n = 31) or 400 µg FA, 10 µg cyanocob(III)alamin and 8 mg pyridoxine (n = 27) for a median of 23 days. Main outcome includes changes in concentrations of serum (6S)-5-CH3-H4Pte and UMFA. RESULTS: Total homocysteine declined by a median of 1.6 (p = 0.074) in the FA and 1.3 µmol/L (p = 0.009) in the B-complex arms (p = 0.66 between the arms). Serum (6S)-5-CH3-H4Pte significantly (p < 0.001 vs. baseline) increased by a median of 9.2 and 6.5 nmol/L in the FA and B-complex groups, respectively (p = 0.152 between the groups). Compared to FA, B-complex reduced cystathionine and caused lower post-intervention serum UMFA, percentage of UMFA to (6S)-5-CH3-H4Pte and prevalence of UMFA ≥ 0.21 nmol/L. Higher serum cystathionine and whole-blood folate predicted higher post-intervention serum UMFA. CONCLUSIONS: FA caused higher UMFA as compared to B-complex. Pyridoxine appears to improve folate recycling. Data on serum UMFA should be interpreted in relation to other vitamins involved in folate metabolism. Serum UMFA is suggested to play a sensory role through which the cell recognizes FA available for metabolism via dihydrofolate reductase.

Pyridoxine supplementation does not alter in vivo kinetics of one-carbon metabolism but modifies patterns of one-carbon and tryptophan metabolites in vitamin B-6-insufficient oral contraceptive users.

BACKGROUND: Low chronic vitamin B-6 status can occur in a subset of women who use oral contraceptives (OCs) with uncertain metabolic consequences. An insufficiency of cellular pyridoxal 5'-phosphate (PLP), which is the coenzyme form of vitamin B-6, may impair many metabolic processes including one-carbon and tryptophan metabolism. OBJECTIVE: We investigated the effects of vitamin B-6 supplementation on the in vivo kinetics of one-carbon metabolism and the concentration of one-carbon and tryptophan metabolites in vitamin B-6-deficient OC users. DESIGN: A primed, constant infusion of [(13)C5]methionine, [3-(13)C]serine, and [(2)H3]leucine was performed on 10 OC users (20-40 y old; plasma PLP concentrations <30 nmol/L) before and after 28 d of supplementation with 10 mg pyridoxine hydrochloric acid/d. In vivo fluxes of total homocysteine remethylation, the remethylation of homocysteine from serine, and rates of homocysteine and cystathionine production were assessed. Targeted metabolite profiling was performed, and data were analyzed by using orthogonal partial least-squares-discriminant analysis and paired t tests adjusted for multiple testing. RESULTS: Pyridoxine supplementation increased the mean ± SD plasma PLP concentration from 25.8 ± 3.6 to 143 ± 58 nmol/L (P < 0.001) and decreased the leucine concentration from 103 ± 17 to 90 ± 20 nmol/L (P = 0.007) and glycine concentration from 317 ± 63 to 267 ± 58 nmol/L (P = 0.03). Supplementation did not affect in vivo rates of homocysteine remethylation or the appearance of homocysteine and cystathionine. A multivariate analysis showed a clear overall effect on metabolite profiles resulting from supplementation. Leucine, glycine, choline, cysteine, glutathione, trimethylamine N-oxide, and the ratios glycine:serine, 3-hydroxykynurenine:kynurenine, 3-hydroxykynurenine:3-hydroxyanthranilic acid, and 3-hydroxykynurenine:anthranilic acid were significant discriminating variables. CONCLUSIONS: Consistent with previous vitamin B-6-restriction studies, fluxes of one-carbon metabolic processes exhibited little or no change after supplementation in low-vitamin B-6 subjects. In contrast, changes in the metabolic profiles after supplementation indicated perturbations in metabolism, suggesting functional vitamin B-6 deficiency. This study was registered at clinicaltrials.gov as NCT01128244.

Oral exposure to the anti-pyridoxine compound 1-amino D-proline further perturbs homocysteine metabolism through the transsulfuration pathway in moderately vitamin B6 deficient rats.

Pyridoxal 5'-phosphate (PLP; a B6 vitamer) serves as an important cofactor in a myriad of metabolic reactions, including the transsulfuration (TS) pathway, which converts homocysteine (Hcy) to cysteine. While overt vitamin B6 deficiency is rare, moderate deficiency is common and may be exacerbated by anti-pyridoxine factors in the food supply. To this end, we developed a model of moderate B6 deficiency and a study was conducted to examine the in vivo effect of 1-amino D-proline (1ADP), an anti-pyridoxine factor found in flaxseed, on indices of Hcy metabolism through the TS pathway in moderately B6 deficient rats. Male weaning rats received a semi-purified diet containing either 7 mg/kg (control; CD) or 0.7 mg/kg (moderately deficient; MD) diet of pyridoxine·hydrochloride (PN∙HCl), each with 1 of 4 levels of 1ADP, viz. 0, 0.1, 1 and 10 mg/kg diet for 5 weeks. Perturbations in vitamin B6 biomarkers were more pronounced in the MD group. Plasma PLP was significantly reduced, while plasma Hcy (8-fold) and cystathionine (11-fold) were increased in rats consuming the highest amount of 1ADP in the MD group. The activities of hepatic cystathionine ß-synthase and cystathionine γ-lyase enzymes were significantly reduced in rats consuming the highest 1ADP compared to the lowest, for both levels of PN∙HCl. Dilation of hepatic central veins and sinusoids, mild steatosis and increased liver triglycerides were present in MD rats consuming the highest 1ADP level. The current data provide evidence that the consumption of an anti-pyridoxine factor linked to flaxseed may pose a risk for subjects who are moderate/severe vitamin B6 deficient.

Enzymatic diagnosis of homocystinuria by determination of cystathionine-ß-synthase activity in plasma using LC-MS/MS.

BACKGROUND: Cystathionine ß-synthase (CBS) is released into plasma from organs expressing this enzyme. Decreased plasma CBS activity has been demonstrated in CBS-deficient patients with 16 different genotypes. The aim of this study was to determine plasma CBS activity in patients carrying 11 additional genotypes using two LC-MS/MS methods. Patients and methods CBS activity was measured in EDTA or heparin plasma using either a previously described or a newly developed LC-MS/MS method optimized for analysis of the reaction product, 3,3-(2)H2-cystathionine, as its butyl ester derivative. We analyzed plasma samples from 26 CBS-deficient patients with known genotypes and 57 controls. RESULTS: We developed a new LC-MS/MS method for simple and sensitive determination of CBS activity. Plasma CBS activity was low (i.e., 0.001-0.036 of the multiples of median control values, MoM) in patients homozygous for the prevalent Hispanic mutation c.572C>T (p.T191M) but was highly elevated (2.95 MoM) in a single patient homozygous for the c.1330G>A (p.D444N) mutation. Patients with the remaining nine genotypes exhibited decreased activities (0.00-0.22 MoM), which did not overlap with the controls (0.29-2.10 MoM). CONCLUSIONS: The determination of CBS activity in plasma is a rapid and non-invasive procedure for detecting a subgroup of CBS-deficient patients with distinct genotypes.

Simultaneous determination of cystathionine, total homocysteine, and methionine in dried blood spots by liquid chromatography/tandem mass spectrometry and its utility for the management of patients with homocystinuria.

BACKGROUND: Disorders of homocysteine and B-vitamin metabolism represent a significant problem in clinical practice. Establishing the diagnosis requires specialized tests with demanding preanalytical requirements. To advance the detection of patients with these disorders, we developed a method for the simultaneous determination of cystathionine (Cysta), methionine (Met) and total homocysteine (tHcy) in dried blood spots (DBSs). METHODS: A punch from a DBS sample was mixed with a solution of isotopically labeled internal standards, and analytes were extracted using methanol/0.1% formic acid/0.5mol/L dithiothreitol. The extract was injected into an LC-MS/MS system operating in MRM mode. RESULTS: The analytical performance of the method employing DBS is adequate for its purpose and the type of sample. Compared with Cysta, tHcy and Met plasma levels, our method exhibited a negative bias between -3.8% and -42.2% due to the lower concentrations of these analytes in erythrocytes. The tHcy level and the Met/Cysta ratio in DBS enabled the clear detection of 12 patients with disorders of transsulfuration and with genetic and nutritional remethylation defects. CONCLUSIONS: The ease of collecting and transporting DBS samples may advance diagnostic procedures in patients with neuropsychiatric disorders and thromboembolism. Consequently, this approach may facilitate detection and simplify the monitoring of patients with homocystinuria.