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Concentrations of pyridoxal 5′-phosphate (PLP) in serum and whole blood are routinely measured. The suitability of these markers in capturing vitamin B6 insufficiency is not well studied.

Background: We aimed to study the effect of long-term supplementation of B-vitamins on folate forms in serum and whole blood (WB) in elderly German subjects. Methods: 59 participants (mean age 67 years) were randomized to daily receive either vitamin D3 (1200 IU), folic acid (500 μg), vitamin B12 (500 μg), vitamin B6 (50 mg), and calcium carbonate (456 mg) or vitamin D3 plus calcium carbonate. Serum and WB folate forms were measured before and after 6 and 12 months. Results: B-vitamins supplementation for 6  months led to higher concentrations of 5-methyltetrahydrofolate (5-methylTHF) in serum (mean 49.1 vs. 19.6 nmol/L) and WB (1332 vs. 616 nmol/L). Also non-methyl-folate concentrations in serum and WB were higher after 6 months with B-vitamins supplementation. Unmetabolized folic acid (UFA) increased after supplementation. tHcy concentration was lowered after 1 year of B-vitamin supplementation (mean 13.1 vs. 9.6 μmol/L). A stronger reduction of tHcy after 1 year was found in participants who had baseline level  > 12.5 μmol/L (mean 17.0 vs. 11.9 μmol/L) compared to those with baseline tHcy lower than this limit (mean 9.1 vs. 7.4 μmol/L). In contrast, the increases in serum and WB 5-methylTHF were comparable between the two group Conclusions: One year B-vitamins supplementation increased the levels of 5-methylTHF and non-methylfolate in serum and WB, normalized tHcy, but caused an increase in the number of cases with detectable UFA in serum. Lowering of tHcy was predicted by baseline tHcy, but not by baseline serum or WB 5-methylTHF. [ABSTRACT FROM AUTHOR]

Background: Methylmalonic acid (MMA) is a functional marker of vitamin B12 status and a valuable diagnostic tool. The gas chromatography mass spectrometry (GCMS) MMA assay has been used for decades in clinical studies. Methods: In this study, we compared a newly developed liquid chromatography tandem mass spectrometry assay for MMA (ClinMass® Complete Kit) with the GCMS method and further measured other relevant vitamin B12 markers (homocysteine, total B12 and holotranscobalamin) to validate the new liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method. For this purpose, 138 samples that were sent to our routine laboratory total B12 assay were used. Results: The GCMS and LC-MS/MS assays showed a strong correlation (r=0.92, p<0.001) particularly at low holoTC levels (deficiency is more probable). Forty six cases had MMA>300 nmol/L with both methods. Only five subjects showed MMA GCMS>300 nmol/L, but MMA LC-MS/MS≤300 nmol/L. However, the LC-MS/MS method showed a slightly better correlation with other B12 markers (holoTC, total B12). In addition, the LC-MS/MS method offers several advantages over the GCMS method, such as saving time and costs, precision, flexibility and popularity in modern labs. Conclusions: The new LC-MS/MS assay for MMA showed an excellent correlation to the GCMS method. The two methods showed similar agreements with other vitamin B12 markers. [ABSTRACT FROM AUTHOR]

Background: Disturbed DNA methylation is causally related to chronic diseases like cancer and atherosclerosis. B vitamins are cofactors required for methyl group synthesis and may therefore affect DNA methylation. Vitamin D has epigenetic effects. We tested if B and D vitamin supplementation has an effect on genomic long interspersed nuclear element-1 (LINE-1) methylation and the metabolites S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Methods: Fifty subjects (median age 68.0 years) were supplemented with a daily oral dose of B vitamins (500 µg folic acid, 500 µg vitamin B12 and 50 mg vitamin B6), 1200 IU vitamin D and 456 mg calcium. Fasting blood samples were collected before and after 1 year of supplementation. LINE-1 methylation was determined in genomic DNA from blood cells as a surrogate for whole genome methylation. In addition, SAM, SAH and total homocysteine (tHcy) were measured in plasma samples. Results: Plasma homocysteine decreased significantly after supplementation (12.8 vs. 9.1 µmol/L; p<0.05), whereas SAM, SAH, the SAM/SAH ratio and LINE-1 methylation did not change significantly. LINE-1 methylation was not significantly correlated with SAH, homocysteine or B vitamins. Conclusions: Long-term vitamin B supplementation had no effect on LINE-1 methylation in blood cells nor on plasma levels of SAM and SAH. Vitamin B and D supplementation seems to have no effect on DNA methylation, especially in cases where no severe deficiency exists. [ABSTRACT FROM AUTHOR]

Background: Vitamin D and vitamin B deficiency are common in elderly subjects and are important risk factors for osteoporosis and age-related diseases. Supplementation with these vitamins is a promising preventative strategy. The objective of this study was to evaluate the effects of vitamins D3 and B supplementation on bone turnover and metabolism in elderly people. Methods: Healthy subjects (n=93; >54 years) were randomly assigned to receive either daily vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg), and calcium carbonate (456 mg) (group A) or only vitamin D3 plus calcium carbonate (group B) in a double blind trial. We measured at baseline and after 6 and 12 months of supplementation vitamins, metabolites, and bone turnover markers. Results: At baseline mean plasma 25-hydroxy vitamin D [25(OH)D] was low (40 or 30 nmol/L) and parathormone was high (63.7 or 77.9 pg/mL). 25(OH)D and parathormone correlated inversely. S-Adenosyl homocysteine and S-adenosyl methionine correlated with bone alkaline phosphatase, sclerostin, and parathormone. One year vitamin D3 or D3 and B supplementation increased plasma 25(OH)D by median 87.6% (group A) and 133.3% (group B). Parathormone was lowered by median 28.3% (A) and 41.2% (B), bone alkaline phosphatase decreased by 2.8% (A) and 16.2% (B), osteocalin by 37.5% (A) and 49.4% (B), and tartrate-resistant-acid-phosphatase 5b by 6.1% (A) and 36.0% (B). Median total homocysteine (tHcy) was high at baseline (group A: 12.6, group B: 12.3 µmol/L) and decreased by B vitamins (group A) to 8.9 µmol/L (29.4%). tHcy lowering had no additional effect on bone turnover. Conclusions: One year vitamin D3 supplementation with or without B vitamins decreased the bone turnover significantly. Vitamin D3 lowered parathormone. The additional application of B vitamins did not further improve bone turnover. The marked tHcy lowering by B vitamins may modulate the osteoporotic risk. [ABSTRACT FROM AUTHOR]

Background: Vitamin B12 deficiency and bone fractures are common in vegetarians. However, a direct relationship between vitamin B12 status and bone metabolism in vegetarians has not been tested sufficiently. Methods: Our study included 96 vegetarians (23 German vegans, and 54 German and 19 Indian lacto-, lacto-ovo-vegetarians) and 89 omnivores (Germans and Asian-Indian immigrants in Oman). Blood concentrations of total vitamin B12, holotranscobalamin (holoTC), 25OH-vitamin D (25(OH)D), total homocysteine (tHcy), methylmalonic acid (MMA), and the bone turnover markers (BTMs) bone alkaline phosphatase (BAP), osteocalcin (OC), pro-collagen type I N-terminal peptide (PINP) and C-terminal telopeptides of collagen I (CTx) were measured. Results: Vegetarians from both population groups exhibited significantly higher concentrations of tHcy, MMA, folate, and BAP, but lower concentrations of holoTC and cobalamin compared with omnivores from the same population. Additionally, German vegetarians had higher circulating activities of BAP as well as higher CTx, OC, and PINP compared with their omnivorous controls. HoloTC and MMA were correlated with OC, CTx and BAP. Subjects with low vitamin B12 status (holoTC ≤35 pmol/L and MMA >271 nmol/L) had significantly lower serum concentrations of 25(OH)D, but higher tHcy and the BTMs P1NP, BAP, OC, and CTx, compared with subjects with normal vitamin B12 status. Multiple regression analysis showed that the association between BTMs and markers of vitamin B12 status was independent from the association with 25(OH)D. Approximately 12%–14% of the variation in the concentration of BTMs was explained by a regression model including holoTC, MMA and 25(OH)D. The strictness of the diet was not related to the magnitude of change in BTMs. Conclusions: Low vitamin B12 status is related to increased bone turnover in vegetarians which is independent from vitamin D status. Clin Chem Lab Med 2009;47:1381–7. [ABSTRACT FROM AUTHOR]

Background: p66 Shc is a stress response protein and partially regulated by epigenetic modifications. Mice lacking p66 Shc have reduced atherosclerosis, increased resistance to oxidative stress and a prolonged life time. The aim of the present study was to compare promoter methylation of the p66 Shc gene between healthy controls and patients with end-stage renal disease (ESRD). There are two reasons for studying patients with ESRD. First, patients with ESRD have a disturbed homocysteine metabolism, and second an increased risk of morbidity and mortality from cardiovascular disease is a constant finding in these patients. Methods: In our study, we measured fasting levels of homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 8-isoprostane in 22 patients and in 26 healthy, age- and sex-matched controls. The methylation of the p66 Shc promoter and Line-1, as surrogate marker of whole genome methylation was quantified in peripheral blood mononuclear cells. Results: In comparison to the control group, homocysteine, SAM, SAH, 8-isoprostane and whole genome methylation were significantly elevated in ESRD patients, while the p66 Shc promoter methylation was significantly reduced. A significant correlation was found between SAH and p66 Shc promoter methylation in the patient group. This observation underlines the role of SAH as a potent inhibitor of methyltransferases. Using backward regression analysis, we demonstrated that 8-isoprostane has a significant influence on p66 Shc promoter methylation. In the control group and in patients with ESRD, increasing 8-isoprostane levels were linked to an elevated promoter methylation. Conclusions: Under physiological conditions, based on the results of the control group, the p66 Shc expression is more silenced through epigenetic modifications. The atherosclerotic risk is dramatically increased in ESRD patients; therefore, our experimental results of methylation are in accordance with the clinical situation. Clin Chem Lab Med 2007;45:1764–70. [ABSTRACT FROM AUTHOR]

Vegetarians have a lower intake of vitamin B12 than omnivores do. Vitamin B12 deficiency (holotranscobalamin II <35 pmol/L or methylmalonic acid >271 nmol/L) was found in 58% of 71 vegetarians studied. Higher homocysteine levels (>12 μmol/L) found in 45% indicate disturbed remethylation of homocysteine to methionine. The methylation of DNA is strongly linked to homocysteine metabolism. Since DNA methylation is an important epigenetic factor in the regulation of gene expression, alteration of the methylation pattern has been associated with aging, cancer, atherosclerosis and other diseases. Three observations indicate that DNA methylation could be diminished by a vegetarian lifestyle. The vegetarian diet has a low content of methionine, remethylation of homocysteine is reduced by vitamin B12 deficiency and elevated homocysteine levels can induce the generation of S-adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases. In our study we observed a significant correlation between SAH and whole-genome methylation (r=−0.36, p<0.01). This observation underlines the role of SAH as a potent inhibitor of methyltransferases. The methylation status was not correlated with homocysteine or S-adenosylemethionine (SAM). These results indicate that the degree of methylation does not depend on the supply of methyl groups and that the reverse generation of SAH has no influence. In addition to whole-genome methylation, the specific promoter methylation of the p66Shc gene was studied. However, the latter did not correlate with SAH, SAM or homocysteine. Obviously, the promoter methylation of the p66Shc gene is controlled in a specific way, without following the general regulating influence of SAH. In conclusion, an inhibitory effect of SAH on whole-genome methylation was found, but from our data no interaction between vegetarian lifestyle and DNA methylation could be determined. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia is a risk factor in obstetrical complications such as pre-eclampsia, `hemolysis, elevated liver enzymes, low platelet' (HELLP)-syndrome and placental insufficiency. The aim of our study was to investigate the alterations of homocysteine catabolism in these patients in relation to serum B-vitamins and renal function. Maternal fasting serum from pre- eclampsia (n=24), HELLP (n=20) and placental insufficiency (n=25) patients at the time of diagnosis and pregnant controls (n=34) was analyzed for homocysteine and its metabolites cystathionine and methyl- malonic acid, the vitamins B6, B12 and folate, renal and additional parameters. Cystathionine, a parameter of homocysteine catabolism, was significantly increased in pre-eclampsia and HELLP compared with controls and placental insufficiency patients (mean concentrations: 343, 324, 248, 227 nmol/l; p=0.001). Homocysteine, folic acid, vitamin B6 and methyl- malonic acid, however, did not differ significantly between groups. The main determinants of cystathionine are cystatin C and vitamin B6, whereas the main determinants of homocysteine are folate and uric acid. The strongest dependency of cystathionine on vitamin B6 was observed in pre-eclampsia and HELLP patients. The results suggest that the vitamin B6- dependent trans-sulfuration pathway is activated in pre-eclampsia and HELLP syndrome, probably by oxidative stress. Therefore, the demand for vitamin B6 is increased in these patients. Furthermore, renal dysfunction and low vitamin B6 levels contribute to the increase of cystathionine in pre-eclampsia and HELLP patients. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the diagnostic utility of complexed prostate-specific antigen (cPSA) with total PSA (tPSA) in screening for prostate cancer. Serum concentrations of tPSA and cPSA were measured in 4479 adult men during the prostate cancer screening program in the Saarland region (Germany). The percentage of men with c/tPSA ratio above the cut-off value of 0.75 increased with increasing tPSA intervals: tPSA 0-0.9 μg/l, 4.4%; 1.0-1.9 μg/l, 24.3%; 2.0-2.9 μg/l, 43.9%; 3.0-3.9 μg/l, 50.4%; and 4.0-20 μg/l, 60.2%. The commonly accepted tPSA cutoff value of 3.9 μg/l matched to the 93rd percentile the overall population (corresponding cPSA value, 2.9 μg/l). A total of 202 men out of 313 with increased cPSA had increased c/tPSA ratio (cut-off ≥0.75) vs. 186 out of 312 men with increased tPSA. Thus, an additional 16 men at high risk for prostate cancer were selected only if cPSA was utilised as a first line parameter. Our data show that, compared to tPSA, cPSA measurement will always detect more high-risk patients, independent of the cut-off levels utilised for cPSA, tPSA and c/tPSA ratio. cPSA is more effective than tPSA in selecting subjects with an elevated c/tPSA ratio who are at high risk of prostate cancer. Thus, cPSA might be seen as the superior first-line parameter in screening for prostate cancer. Using lower cut-off values for tPSA or cPSA than the commonly accepted values seems reasonable for screening purposes. [ABSTRACT FROM AUTHOR]

Presents a correction to the article "DACH-LIGA Homocystein (German, Austrian and Swiss Homocysteine Society): Consensus Paper on the Rational Clinical Use of Homocysteine, Folic Acid and B-Vitamins in Cardiovascular and Thrombotic Diseases: Guidelines and Recommendations," by Olaf Stanger, Wolfgang Herrmann, Klaus Pietrzik, Brian Fowler, Jürgen Geisel, Jutta Dierkes and Martin Weger.

Coronary heart disease often occurs in the absence of traditional risk factors. Consequently, epidemiological studies exploring novel risk factors are necessary to improve the prediction of coronary heart disease. This study evaluated five promising markers of cardiovascular risk: homocysteine, C-reactive protein, fibrinogen, lipoprotein(a) (Lp(a)), free apolipoprotein(a) (apo(a)) and Lp(a) phenotypes. The study included 135 patients with angiographically confirmed atherosclerosis. The control group consisted of 93 sex- and age-matched individuals. The Mann-Whitney U-test was used for group comparison. New risk factors were evaluated by binary logistic regression. The odds ratios were calculated continuously for homocysteine in dependence on C-reactive protein. Low density lipoprotein (LDL)-cholesterol was nearly identical in controls and patients. Homocysteine, C-reactive protein, fibrinogen, high density lipoprotein (HDL)-cholesterol and Lp(a) discriminated highly significantly between both groups. The continuously calculated odds ratio for homocysteine demonstrated a distinct influence of C-reactive protein. In the group with high C-reactive protein levels, homocysteine levels above 9.6 µmol/l resulted in a markedly elevated risk (odds ratio 12), in the group with C-reactive protein levels below 5 mg/dl, a comparable risk increase was observed at a homocysteine level of 16.6 µmol/l. This data strongly suggests that plasma homocysteine helps identify individuals at risk, especially among those with elevated C-reactive protein levels. [ABSTRACT FROM AUTHOR]

Moderate hyperhomocysteinemia has been identified as a new independent risk factor for cardiovascular and neurodegenerative diseases. This fact has produced interest in the study of genetic variants involved in homocysteine metabolism and its relationship to pathogenesis. Recently, more than 15 different genes were studied for their relationship to plasma homocysteine levels. We determined the influence of genetic variants in five genes (5,10-methylenetetrahydrofolate reductase (MTHFR) 677C→T, serine hydroxymethyltransferase (SHMT) 1420C→T, thymidylate synthase (TS) 2R→3R, catechol-O-methyltransferase (COMT) 1947G→A and transcobalamin (TC) 776C→G) on plasma homocysteine, folic acid and parameters of vitamin B[sub12] metabolism in 111 vegetarians (mean age: 46 ± 15 years) and 118 healthy seniors (mean age: 82 ± 6.5 years). Median homocysteine concentration in plasma was significantly influenced by the MTHFR genotypes in both populations. In the vegetarians the median homocysteine level was increased by 8 µmol/l in individuals homozygous for the mutation as compared to wild-type or heterozygous genotypes (20.4 µmol/l vs. 12.9 and 12.7 µmol/l, respectively). This unexpected increase was observed although the folate levels were in medium to elevated ranges. Our results suggest that vegetarians have a higher demand for folate to neutralize the genotype effect. Preclinical vitamin B[sub12] deficiency in vegetarians may be the cause for disturbed remethylation and folate trap. Plasma homocysteine was not significantly influenced by the SHMT, TS, COMT and TC mutations. In addition, for the TC mutation a trend toward cellular vitamin B[sub12 deficiency was observed. The methylmalonic acid (MMA) levels were slightly elevated and the holotranscobalamin-II (holoTC-II) levels decreased. In the vegetarian group a significant relationship between the COMT genotype and holoTC-II concentration in plasma was determined,... [ABSTRACT FROM AUTHOR]

Background: The prevalence of a sub-clinical functional vitamin B[sub12] deficiency in the general population is higher than previously expected. Total serum vitamin B[sub12] may not reliably indicate vitamin B[sub12] status. To get more specificity and sensitivity in diagnosing vitamin B[sub12] deficiency, the concept of measuring holotranscobalamin II (holoTC), a sub-fraction of vitamin B[sub12] , has aroused great interest. HoloTC as a biologically active vitamin B[sub12] fraction promotes a specific uptake of its vitamin B[sub12] by all cells. In this study we investigated the diagnostic value of storage (holoTC) of vitamin B[sub12] and functional markers (methylmalonic acid (MMA)) of vitamin B[sub12] metabolism in populations who are at risk of vitamin B[sub12] deficiency. Subjects and Methods: Our study included 93 omnivorous German controls, 111 German and Dutch vegetarian subjects, 122 Syrian apparently healthy subjects, 127 elderly Germans and finally 92 German pre-dialysis renal patients. Serum concentrations of homocysteine (Hcy) and MMA were measured by gas chromatographymass spectrometry, folate and vitamin B[sub12] by chemiluminescence immunoassay, and holoTC by utilizing a RIA test. Results: High Hcy (>12 µmol/l), high MMA (>271 nmol/l) resp. low holoTC (vitamin B[sub12]) in serum were detected in 15%, 8% resp. 13% (1%) of German controls, 36%, 60%, resp. 72% (30%) of vegetarians, 42%, 48% resp. 50% (6%) of Syrians, 75%, 42%, resp. 21% (7%) of elderly subjects and 75%, 67% resp. 4% (2%) of renal patients. The lowest median levels of holoTC were observed in vegetarians, followed by the Syrian subjects (23 and 35 pmol/l, respectively). Renal patients had significantly higher levels of holoTC compared to the German controls (74 vs. 54 pmol/l). In the vitamin B[sub12] range between 156 pmol/l (conventional cut-off level) and 241 pmol/l, both mean concentrations of holoTC and MMA were in the pathological range. HoloTC was the earliest... [ABSTRACT FROM AUTHOR]

About half of all deaths are due to cardiovascular disease and its complications. The economic burden on society and the healthcare system from cardiovascular disability, complications, and treatments is huge and getting larger in the rapidly aging populations of developed countries. As conventional risk factors fail to account for part of the cases, homocysteine, a "new" risk factor, is being viewed with mounting interest. Homocysteine is a sulfur-containing intermediate product in the normal metabolism of methionine, an essential amino acid. Folic acid, vitamin B[sub12], and vitamin B[sub6] deficiencies and reduced enzyme activities inhibit the breakdown of homocysteine, thus increasing the intracellular homocysteine concentration. Numerous retrospective and prospective studies have consistently found an independent relationship between mild hyperhomocysteinemia and cardiovascular disease or all-cause mortality. Starting at a plasma homocysteine concentration of approximately 10 µmol/l, the risk increase follows a linear dose-response relationship with no specific threshold level. Hyperhomocysteinemia as an independent risk factor for cardiovascular disease is thought to be responsible for about 10% of total risk. Elevated plasma homocysteine levels (> 12 &micromol/l; moderate hyperhomocysteinemia) are considered cytotoxic and are found in 5 to 10% of the general population and in up to 40% of patients with vascular disease. Additional risk factors (smoking, arterial hypertension, diabetes, and hyperlipidemia) may additively or, by interacting with homocysteine, synergistically (and hence over-proportionally) increase overall risk. Hyperhomocysteinemia is associated with alterations in vascular morphology, loss of endothelial anti-thrombotic function, and induction of a procoagulant environment. Most known forms of damage or injury are due to homocysteine-mediated oxidative stress. Especially when acting as direct or indirect antagonists of cofactors... [ABSTRACT FROM AUTHOR]

Background: Methylenetetrahydrofolate reductase 677 (MTHFR 677) polymorphism may provoke hyperhomocysteinemia when folate status is low. The influence of MTHFR 677 mutation on homocysteine (HCY) levels in relation to vitamin B[sub12] and folate status was investigated in the current study. Subjects and methods: 113 vegetarians, 123 omnivorous Germans, and 117 omnivorous Syrians were recruited. MTHFR 677 genotype, HCY, methylmalonic acid (MMA), total serum vitamin B[sub12], serum folate, and vitamin B[sub6] were determined using conventional methods. Results: Omnivorous Germans displayed the lowest HCY levels compared with vegetarians and Syrians (median 8.0, 10.4, and 11.3 &micro'mol/l, respectively). The highest serum folate and the highest MMA levels were found in vegetarians (median folate = 30.0; MMA = 355 nmol/l). Among vegetarians and Syrians, TT subjects had higher HCY levels than other genotypes which were, however, no longer significant in the highest folate tertiles. When the data were pooled, the odds ratio (OR, 95% CI) for HCY > 12 µmol/l was 3.81 (1.55-9.34) in TT compared with CC subjects. The OR increased to 28.85 (4.63-179.62) in TT subjects who had folate in the lowest tertile, and to 21.84 (4.81-99.1) in TT subjects who had MMA in the highest MMA tertile. Conclusion: MTHFR 677 TT individuals are more liable to hyperhomocysteinemia under vitamin B[sub12] deficiency than the other two genotypes. In such a case, relative folate shortage may progressively increase HCY levels. TT individuals may have increased folate and vitamin B[sub12] requirements compared to the other CC and CT genotypes. [ABSTRACT FROM AUTHOR]