Heike Schorr

Functional vitamin B12 deficiency and determination of holotranscobalamin in populations at risk.

Abstract Background: The prevalence of a sub-clinical functional vitamin B12 deficiency in the general population is higher than previously expected. Total serum vitamin B12 may not reliably indicate vitamin B12 status. To get more specificity and sensitivity in diagnosing vitamin B12 deficiency, the concept of measuring holotranscobalamin II (holoTC), a sub-fraction of vitamin B12, has aroused great interest. HoloTC as a biologically active vitamin B12 fraction promotes a specific uptake of its vitamin B12 by all cells. In this study we investigated the diagnostic value of storage (holoTC) of vitamin B12 and functional markers (methylmalonic acid (MMA)) of vitamin B12 metabolism in populations who are at risk of vitamin B12 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 B12 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 B12) 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 B12 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 marker for vitamin B12 deficiency followed by MMA. Vitamin B12 deficiency causes folate trapping. A higher folate level is required to keep Hcy normal. The relationship between MMA and holoTC seemed dependent on renal function. In renal patients with a glomerular filtration rate below 36 ml/min, a significantly lower mean level of MMA was detected within the highest tertile of holoTC concentration, compared to the lowest tertile. Thus, in renal patients, a higher serum concentration of circulating holoTC is required to deliver sufficient amounts of holoTC into the cells. Conclusion: Our data support the concept that the measurement of holoTC and MMA provides a better index of cobalamin status than the measurement of total vitamin B12. HoloTC is the most sensitive marker, followed by MMA. The use of holoTC and MMA enables us to differentiate between storage depletion and functional vitamin B12 deficiency. Renal patients have a higher requirement of circulating holoTC. In renal dysfunction, holoTC cannot be used as a marker of vitamin B12 status.

The usefulness of holotranscobalamin in predicting vitamin B12 status in different clinical settings

Serum concentrations of homocysteine (Hcy) and methylmalonic acid (MMA) become increased in B12-deficient subjects and are therefore, considered specific markers of B12 deficiency. Serum level of holotranscobalamin (holoTC) becomes decreased before the development of the metabolic dysfunction. We investigated the usefulness of holoTC in diagnosing B12 deficiency in some clinical settings. We measured serum concentrations of holoTC, MMA, Hcy and total B12 in omnivores, vegetarians, elderly people and haemodialysis patients. Our results indicated that the incidence of holoTC <35 pmol/L was highest in the vegans (76%). Low holoTC and elevated MMA were detected in 64% of the vegans and 43% of the lacto- and lacto-ovovegetarians. An elevated MMA and a low holoTC were found in subjects with total serum B12 as high as 300 pmol/L. The distribution of holoTC in elderly people was similar to that in younger adults (median holoTC 55 pmol/L in both groups). A low holoTC and an elevated MMA were found in 16% of the elderly group. An elevated MMA and a normal holoTC were found in 20% of the elderly group who had a relatively high median serum concentration of creatinine (106.1 micromol/L). Serum concentrations of holoTC in dialysis patients were considerably higher than all other groups (median 100 pmol/L). This was also associated with severely increased serum levels of MMA (median 987 nmol/L). From these results it can be concluded that serum concentration of holoTC is a much better predictor of B12 status than total B12. This was particularly evident in case of dietary B12 deficiency. Serum concentrations of holoTC as well as MMA can be affected by renal dysfunction. Elevated MMA and normal holoTC in patients with renal insufficiency may not exclude vitamin B12 deficiency. HoloTC seems not to be a promising marker in predicting B12 status in renal patients.

Hyperhomocysteinemia induces a tissue specific accumulation of homocysteine in bone by collagen binding and adversely affects bone

BACKGROUND Recently, hyperhomocysteinemia (HHCY) has been suggested to have adverse effects on bone. This study investigated if an experimental HHCY in rats induces an accumulation of homocysteine (HCY) in bone tissue that is accompanied by bone loss and reduced bone strength. MATERIAL AND METHODS HHCY was induced in healthy rats by either a methionine (Meth)- or a homocystine (Homo)-enriched diet and compared with controls. Homocystine is the product of two disulfide linked HCY molecules. Tissue and plasma concentrations of HCY, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were measured. Bones were assessed by biomechanical testing, histomorphometry, microCT and the measurement of biochemical bone turnover markers in plasma. RESULTS Meth and Homo animals developed a significant HHCY that was accompanied by a tissue specific accumulation of HCY (1300 to 2000% vs. controls). 65% of HCY in bone was bound to collagen of the extracellular matrix. The SAH / SAM-ratio in bone and plasma of Meth and Homo animals exhibited a tissue specific increase indicating a reduced methylation capacity. Accumulation of HCY in bone was characterized by a distinct reduction of cancellous bone (proximal femur: -25 to -35%; distal femur -56 to -58%, proximal tibia: -28 to -43%). Accordingly, bone strength was significantly reduced (-9 to -12%). CONCLUSION A tissue specific accumulation of HCY in bone may be a promising mechanism explaining adverse effects of HHCY on bone. A reduced methylation capacity of bone cells might be another relevant pathomechanism.

The impact of vegetarianism on some haematological parameters

Abstract: Objective : Subjects adopting a vegetarian diet are liable to vitamin B12 and iron deficiencies. Co‐existing vitamin B12 and iron deficiencies may give an equivocal haematological picture, which may, in turn, delay making an early diagnosis. The current work was undertaken to investigate some haematological parameters in relation to vitamin B12 and iron status in vegetarians. Subjects and methods : Twenty‐nine vegans, 64 lacto‐ and lacto‐ovo‐vegetarians, in addition to 20 occasional meat eaters, were enrolled for this study. The total group included 49 males and 64 females aged [mean (SD) = 46(15) yr]. Complete blood count, methylmalonic acid (MMA), homocysteine (HCY), ferritin, and transferrin concentrations and percentage transferrin saturation were assayed, using conventional methods. Results : Vegans displayed the highest MMA and HCY levels (median MMA = 708 nmol L −1 ; HCY = 12.8 µmol L −1 ). A lower lymphocyte count and a higher mean corpuscular volume (MCV) were found in vegans compared with lacto‐ or lacto‐ovo‐vegetarians (median = 1.51 × 10 9 vs. 1.83 × 10 9  L −1 ; 92 vs. 89 fL, respectively). Vitamin B12‐deficient subjects in the higher range of transferrin saturation percentage had higher MCV than vitamin B12‐deficient subjects in the lower transferrin saturation range (mean MCV = 92 vs. 89 fL). A lower platelet count was found in the highest quartile of MMA (mean = 211 × 10 9  L −1 ) and in the highest quartile of HCY (mean = 215 × 10 9  L −1 ), compared with the other quartiles. Lower lymphocyte and platelet counts and higher MCV were found in subjects with elevated MMA and HCY, compared to those with normal metabolites. Factors that explained the variations in MCV were red blood cell count, ferritin, transferrin saturation, and methylmalonic acid levels. Conclusion : vitamin B12 and iron status were compromised by a vegetarian diet. Variations in mean corpuscular volume were determined by iron and vitamin B12 status. Lower lymphocyte and platelet count were accompanied by metabolic evidence that indicated vitamin B12 deficiency.

Homocysteine Increases during Endurance Exercise

Abstract Hyperhomocysteinemia is a risk factor for cardiovascular and other diseases. Recently many endogenous and exogenous modulators of homocysteine (Hcy) have become known, e.g., B-vitamins. However, little is known about the effect of exercise on Hcy. The purpose of this study was to investigate the effect of three different types of acute endurance exercise on serum Hcy. We measured Hcy in 100 recreational athletes (87 males, 13 females) who participated in a marathon race (n = 46), a 100 km run (100 km; n = 12) or a 120 km mountain bike race (n = 42). Blood samples were taken before, 15 min and 3 h after the race. In athletes with pre-race Hcy >12 μmol/l we also determined folate and vitamin B12. Marathon running induced a Hcy increase of 64%, while mountain biking and 100 km running had no significant effect on Hcy. Pre-race Hcy (25th–75th percentile) overall; marathon race; 100 km; mountain bike race was 9.7 (7.1–11.5) μmol/l; 9.8 (7.4–11.1) μmol/l; 10.2 (6.6–13.2) μmol/l; 9.1 (6.9–13.5) μmol/l, respectively. At 15 min and 3 h post-race, Hcy was 11.9 (8.4–16.4) μmol/l; 16.1 (12.7–20.4) μmol/l; 9.5 (7.8–15.9) μmol/l; 8.8 (7.1–11.2) μmol/l, respectively, and 11.5 (8.9–15.7) μmol/l; 14.9 (11.5–20.0) μmol/l; 10.0 (8.1–11.8) μmol/l; 9.4 (7.4–12.1) μmol/l, respectively. The change in Hcy correlated negatively with the running time. Twenty-three athletes had pre-race Hcy levels >12 μmol/l, which were associated with relatively low folate (14.3 (11.6–18.9) nmol/l) and vitamin B12 levels (231 (183–261) pmol/l). Endurance exercise may induce a considerable Hcy increase, which varies between different disciplines and is most probably determined by the duration

Genetic defects as important factors for moderate hyperhomocysteinemia

Abstract The genes for the enzymes methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), methionine synthase reductase (MSR) and cytathionine-Β-synthase (CBS) play an important role in homocysteine metabolism. Rare mutations in these genes cause severe hyperhomocysteinemia and clinical symptoms. Growing interest has focused on common mutations with moderate effects on homocysteine levels. We studied 280 subjects of different age groups for the following mutations: MTHFR677C→T and 1298A→C, MS2756A→G, MSR66A→G and the 68 bp insertion in the CBS gene. The median value for homocysteine increased significantly with age (median homocysteine levels: 7.5, 12.4 and 16.5 μmol/l in the age groups 20–43, 65–75 and 85–96 years, respectively). The genotypes of the MTHFR677C→T mutation were associated with differences in plasma homocysteine levels, but without reaching significance. Individuals homozygous for the MTHFR677C→T mutation had a 2.3 μmol/l higher median homocysteine level compared to individuals with the wild-type allele. This effect was pronounced in combination with low folate levels and abolished with higher folate in plasma. For the other three mutations no association with homocysteine values could be determined. The analysis of homocysteine metabolite cystathionine by backward regression analysis revealed a significant correlation of the MS2756A→G mutation with cystathionine level. This increase could indicate a disturbed remethylation. In summary, larger and homogeneous study populations are necessary to quantify the small effects of common mutations on homocysteine levels. This may also be the reason that no effects of genetic interactions between two genotypes were observed.

Homocysteine, cystathionine, methylmalonic acid and B-vitamins in patients with renal disease.

Abstract Moderate hyperhomocysteinemia is very frequent in renal patients. Aside from homocysteine (HCY) itself, the metabolites methylmalonic acid (MMA) and cystathionine (CYS) supply further information about disturbances in HCY metabolism. In two groups of renal patients, transplant and hemodialysis patients, we measured HCY, MMA and CYS and evaluated their diagnostic value for impaired HCY metabolism due to vitamin deficiency and renal insufficiency. We investigated serum samples from 63 transplant patients and 38 patients undergoing hemodialysis. HCY, MMA and CYS were assayed by gas chromatography-mass spectrometry, vitamin B6 by HPLC, B12 and folate by chemiluminescence immunoassay. The determination of HCY, MMA, and CYS in renal patients provides specific information about intracellular disturbances of HCY metabolism. The frequency of increased metabolite levels in renal patients was much higher than the frequency of lowered vitamin concentrations in serum. Furthermore, the metabolite levels in transplant patients were only moderately increased, whereas they were strongly increased in patients on hemodialysis (HCY 19.2 vs. 28.8 μmol/l, MMA 292 vs. 1025 nmol/l, CYS 733 vs. 2711 nmol/l). Our findings may support the use of MMA determination in the diagnosis of vitamin B12 deficiency in renal patients. Compared to vitamin B12 deficiency, renal dysfunction itself appears to cause only a modest elevation in serum MMA. Regression analysis revealed that the moderate elevation of HCY and CYS in transplant patients is mainly a consequence of impaired remethylation of HCY to methionine with activated transsulfuration, whereas the mildly elevated MMA level is attributable to renal dysfunction. In patients on hemodialysis, all three metabolites were markedly elevated, indicating a strongly disturbed HCY metabolism. Based on a backward regression, we discovered that the HCY metabolism was strongly disturbed by renal insufficiency and vitamin deficiency. The markedly elevated HCY level was mainly attributable to functional vitamin B12 deficiency indicated by high MMA, and the strong CYS elevation was due to renal dysfunction and inhibition of this pathway by low levels of vitamin B6. In conclusion, besides HCY, the determination of MMA and CYS levels supports an early diagnosis of B-vitamin deficiency in renal patients. MMA is a more sensitive indicator of intracellular vitamin B12 deficiency than vitamin B12 in serum.

Comparison of the influence of volume-oriented training and high-intensity interval training on serum homocysteine and its cofactors in young, healthy swimmers

Abstract Background: Since homocysteine (Hcy) is a risk factor for cardiovascular and other diseases, it is important to know how exercise can modify it. Previous studies have suggested that endurance training influences Hcy. However, little is known about the effect of training intensity on Hcy. Materials and Methods: We investigated Hcy, vitamin B12, vitamin B6, folate and methylmalonic acid (MMA) before and after 3 weeks of volume-oriented training (VOL) (30 km/week) and high-intensity interval training (HIT) (20 km/week) in 20 young swimmers (16±2 years). Afterward, the athletes completed 5 days of recovery training. Results: The training induced a Hcy increase in HIT and VOL (6.47±0.95 μmol/l vs. 7.44±1.17 μmol/l and 7.33± 1.92 μmol/l vs. 8.28±1.42 μmol/l, respectively) that persisted during the recovery period (8.02±1.69 μmol/and 8.00±1.81 μmol/l, respectively). Vitamin B12 was unchanged after the training (539±166 ng/l vs. 556±192 ng/l and 480±144 ng/l vs. 491±124 ng/l, respectively) but decreased during the recovery period (459±134 ng/l and 451±116 ng/l, respectively). Folate showed an increase during the training (9.07±2.01 μg/vs. 11.71±4.08 μg/l and 10.34±2.32 μg/l vs. 11.13± 4.64 μg/l, respectively), which was reversible by the end of the recovery training (8.57±1.98 μg/l and 9.60±2.38 μg/l, respectively). Vitamin B6 and MMA did not change. For none of the measured parameters were there significant differences between HIT and VOL. Conclusion: Three weeks of strenuous swimming caused a prolonged Hcy increase, which was accompanied by changes in vitamin B12 and folate. The magnitude of these effects was not influenced by the training intensity.

The vegetarian lifestyle and DNA methylation.

Abstract Vegetarians have a lower intake of vitamin B12 than omnivores do. Vitamin B12 deficiency (holotranscobalamin II <35pmol/L or methylmalonic acid >271nmol/L) was found in 58% of 71 vegetarians studied. Higher homocysteine levels (>12μmol/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.

The role of genetic factors in the development of hyperhomocysteinemia.

Abstract 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 B12 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 B12 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 B12 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, whereas the high activity COMT genotype (G/G) resulted in increased levels (35 μmol/l vs. 21 μmol/l for heterozygous and low activity genotypes). The MMA levels were inversely correlated to holoTC-II concentrations. In conclusion, the study on vegetarians and seniors documents interesting lifestyle-genotype interactions. Although the TC and COMT mutations influence cellular vitamin B12 metabolism, this effect did not result in overt homocysteine elevation.