Volume 41, Issue 11

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 B12, and vitamin B6 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 μmol/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 homocysteinemediated oxidative stress. Especially when acting as direct or indirect antagonists of cofactors and enzyme activities, numerous agents, drugs, diseases, and lifestyle factors have an impact on homocysteine metabolism. Folic acid deficiency is considered the most common cause of hyperhomocysteinemia. An adequate intake of at least 400 μg of folate per day is difficult to maintain even with a balanced diet, and highrisk groups often find it impossible to meet these folate requirements. Based on the available evidence, there is an increasing call for the diagnosis and treatment of elevated homocysteine levels in high-risk individuals in general and patients with manifest vascular disease in particular. Subjects of both populations should first have a baseline homocysteine assay. Except where manifestations are already present, intervention, if any, should be guided by the severity of hyperhomocysteinemia. Consistent with other working parties and consensus groups, we recommend a target plasma homocysteine level of <10 μmol/l. Based on various calculation models, reduction of elevated plasma homocysteine concentrations may theoretically prevent up to 25% of cardiovascular events. Supplementation is inexpensive, potentially effective, and devoid of adverse effects and, therefore, has an exceptionally favorable benefit/risk ratio. The results of ongoing randomized controlled intervention trials must be available before screening for, and treatment of, hyperhomocysteinemia can be recommended for the apparently healthy general population.

Classical homocystinuria is associated with arterial vascular diseases and venous thrombosis. In the last decade, many studies, including some prospective studies, have been published indicating that moderate hyperhomocysteinaemia is also a risk factor for venous thrombosis. The 677C>T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is an important cause of mild hyperhomocysteinaemia. Recent meta-analyses show an elevated risk of venous thrombosis for subjects with the TT-genotype. Based on the concept of ‘Mendelian randomisation’, this observation supports the hypothesis that hyperhomocysteinaemia is a causal risk factor for venous thrombosis. The results of one homocysteine-lowering trial regarding venous thrombosis are awaited at the end of 2003. In this paper the current evidence for hyperhomocysteinaemia as a risk factor for venous thrombosis is being discussed.

Several studies, some population-based, have plasma homocysteine levels linked to blood pressure, especially systolic pressure. In one large and carefully conducted epidemiological study, each 5 μmol/l increase in plasma homocysteine was associated with an increase in systolic and diastolic blood pressure of 0.7/0.5 mmHg in men and 1.2/0.7 mmHg in women, which was independent of renal function and B vitamin status. In addition, observations that homocysteine-lowering therapies with folic acid-based treatments have been followed by decreases in blood pressure raise the possibility that the link between homocysteine and blood pressure is causal, which is important since homocysteine levels can easily be lowered by folic acid-based regimens. Mechanisms that could explain the relationship between homocysteine and blood pressure include homocysteine-induced arteriolar constriction, renal dysfunction and increased sodium reabsorption, and increased arterial stiffness. However, there is only circumstantial evidence that these mechanisms are operative in humans. In addition, confounding by subtle renal dysfunction or by unmeasured dietary and lifestyle factors cannot be excluded as an explanation for the association between homocysteine and blood pressure. At present, therefore, the hypothesis that homocysteine increases blood pressure must be considered unproven. Ongoing large intervention studies with homocysteine-lowering vitamins may show whether blood pressure is indeed lowered by these vitamins, whether the blood pressure decrease, if any, is explained by the decrease in homocysteine levels, and whether a vitamin treatment-associated decrease in cardiovascular morbidity, if any, is explained by the decrease in blood pressure.

Hyperhomocysteinemia, a new cardiovascular risk factor, occurs in 85–100% of patients with end-stage renal disease. The exact mechanism by which renal function is linked to plasma homocysteine has not been definitively established. There is reasonably good clinical evidence that hyperhomocysteinemia in itself does not cause renal insufficiency. Two, not mutually exclusive, hypotheses are that in renal failure: i) homocysteine disposal is impaired in the kidneys themselves and ii) extra-renal homocysteine metabolism is defective, possibly due to uremic toxins. Several methods have been applied to investigate kidney and whole-body sulfur amino acid metabolism in healthy subjects and in patients with different degrees of renal failure. Arteriovenous extraction studies have not found a significant homocysteine disposal in the human kidney. Methods to study whole-body homocysteine metabolism have included measurement of plasma metabolites, calculation of plasma homocysteine elimination after oral loading and the use of stable isotope techniques with methionine tracers. The results implicate a decreased homocysteine clearance instead of an increased production as the cause of hyperhomocysteinemia in renal failure, but the exact site of the impaired clearance remains controversial.

Measurement of total homocysteine (tHcy) in healthy and diseased children has documented the utility of this marker in pediatric research and diagnostics. This article focuses on novel data obtained in infants, children and adolescents, with emphasis on cobalamin status in infants. In children, determinants of plasma tHcy are similar to those established in adults, and include age, gender, nutrition, B-vitamin status, and some drugs interfering with B-vitamin function. In infants (age <1 year), tHcy is moderately elevated and related to serum cobalamin, whereas in older children and throughout childhood, plasma tHcy is low (about 60% of adult levels), and folate status becomes a strong tHcy determinant. As in adults, hyperhomocysteinemia in childhood is a risk factor for stroke, and folate-responsive hyperhomocysteinemia has been detected in children with renal failure. tHcy seems to be a sensitive indicator of folate deficiency in children on a poor diet, in HIV-infected children, and in children treated with anti-folate drugs. In children at increased risk of cobalamin deficiency, which includes children born to vegetarian mothers or children in developing countries on a poor diet, tHcy and methylmalonic acid are responsive indicators of a deficiency state. In newborns and infants born to mothers with an adequate nutrition, there are consistent observations of low cobalamin, elevated tHcy and methylmalonic acid, and reduction of both metabolites by cobalamin supplementation. These data have raised the question whether cobalamin deficiency may be widespread and undetected in babies born to non-vegetarian women on a Westernized diet.

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 B 12 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 12 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 B 12 metabolism, this effect did not result in overt homocysteine elevation.

Over the last few years we have reproduced all of the key morphological and biochemical features of human subacute combined degeneration in the central nervous system and peripheral nervous system of rats made cobalamin-deficient by means of total gastrectomy or a chronic cobalamin-deficient diet. We have also recently clarified the pathogenesis of experimental subacute combined degeneration induced in the rat by cobalamin deprivation. The results of our studies strongly support the notion that cobalamin plays a pivotal role in regulating the balance of the network of cytokines and growth factors in the central nervous system of the rat. We have demonstrated that cobalamin tightly regulates the central nervous system synthesis and/or the cerebrospinal fluid level of two cytokines, tumor necrosis factor-αand interleukin-6, and a growth factor, epidermal growth factor. Of these neuroactive agents, one, tumor necrosis factor-α, is neurotoxic, whereas the others are neurotrophic. Therefore, it becomes clear that cobalamin-deficient central neuropathy is caused not by the withdrawal of the vitamin, but reflects a locally increased production of neurotoxic agents, combined with the locally decreased production of neurotrophic agents.

Hyperhomocysteinemia is an established risk factor for atherosclerosis, thrombosis and other vascular diseases. Homocysteine auto-oxidation is considered to be crucially involved in the pathogenesis of these diseases. However, the question remains to be elucidated whether vitamin deficiency and homocysteine accumulation are causal for disease development or rather comprise a secondary phenomenon. Most diseases accompanied by hyperhomocysteinemia are also associated with ongoing activation of the immune system. In vitro experiments show homocysteine to accumulate in stimulated peripheral blood mononuclear cells. In patients with coronary heart disease, with rheumatoid arthritis and in patients with dementia, an association between cellular immune activation and homocysteine metabolism is found. Homocysteine concentrations not only correlate inversely with folate concentrations, they also show a positive relationship with concentrations of immune activation markers like neopterin. Moreover, in patients with various kinds of dementia, increased concentrations of serum peroxides, homocysteine and neopterin correlate with each other. Studies support a role of immune system activation in the development of hyperhomocysteinemia. Stimulation and proliferation of immune cells may lead to the production of reactive oxygen species that may oxidize antioxidants and oxidation-sensitive B-vitamins. An enhanced demand for antioxidants as well as folate and vitamin B 12 may develop, together with hyperhomocysteinemia, despite sufficient dietary intake.

The endothelium exerts fundamental control over vascular tone, and injury to the endothelium followed by dysfunction is an early key event preceding manifestation of vessel pathology. Both elevated plasma homocysteine and low folate status have been identified as major and independent risk factors for atherosclerosis and have stirred an enormous and still increasing interest. The damaging effects of hyperhomocysteinemia on endothelial function are, at least in part, reversible through folate supplementation. Because of the inverse relationship between plasma folate and homocysteine levels, however, it is difficult to discriminate between their respective effects. Endothelial dysfunction refers mainly to reduced bioavailability of nitric oxide (NO), which is involved in homocysteinemediated vascular damage. Accumulating evidence further suggests that radical oxygen species are fundamentally involved in hyperhomocysteinemia. NO production is determined by cofactors such as tetrahydrobiopterin, which is oxidized and depleted in conditions of oxidant stress by peroxynitrite. Deficiency of tetrahydrofolate contributes to uncoupling, turning the NO synthase into a superoxide radical-producing enzyme. It appears that progression of vascular disease is likely to determine the multiple interactions between homocysteine, NO, oxygen radicals and folate. Folate has only recently been found to exert direct anti-oxidative effects and contribute to restoration of impaired NO metabolism. Understanding of the complex interactions between homocysteine, radicals, NO and folate offers promising perspectives in the individual treatment of vascular disease. Thus, preventive and therapeutic strategies may require a more distinct approach and better discrimination of target groups for greatest possible efficacy.

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. An increasing body of evidence has implicated oxidative stress as being contributory to homocysteine's deleterious effects on the vasculature. Elevated levels of homocysteine may lead to increased generation of superoxide by a biochemical mechanism involving nitric oxide synthase, and, to a lesser extent, by an increase in the chemical oxidation of homocysteine and other aminothiols in the circulation. The resultant increase in superoxide levels is further amplified by homocysteinedependent alterations in the function of cellular antioxidant enzymes such as cellular glutathione peroxidase or extracellular superoxide dismutase. One direct clinical consequence of elevated vascular superoxide levels is the inactivation of the vasorelaxant messenger nitric oxide, leading to endothelial dysfunction. Scavenging of superoxide anion by either superoxide dismutase or 4,5-dihydroxybenzene 1,3-disulfonate (Tiron) reverses endothelial dysfunction in hyperhomocysteinemic animal models and in isolated aortic rings incubated with homocysteine. Similarly, homocysteine-induced endothelial dysfunction is also reversed by increasing the concentration of the endogenous antioxidant glutathione or overexpressing cellular glutathione peroxidase in animal models of mild hyperhomocysteinemia. Taken together, these findings strongly suggest that the adverse vascular effects of homocysteine are at least partly mediated by oxidative inactivation of nitric oxide.

Indirect pathways, involving homocysteine (Hcy)-thiolactone and S-nitroso -Hcy, allow incorporation of Hcy into protein. Hcy-thiolactone, synthesized by methionyl-tRNA synthetase in all organisms investigated, including human, modifies proteins post-translationally by forming adducts in which Hcy is linked by amide bonds to e-amino group of protein lysine residues. SNitroso -Hcy, synthesized in human vascular endothelial cells, is incorporated translationally into peptide bonds in protein at positions normally occupied by methionine. Hcy- N -hemoglobin and Hcy- N -albumin constitute a major pool of Hcy in human blood. Hcy-thiolactone is present in human plasma. Modification with Hcy-thiolactone leads to protein damage. Hcy-thiolactone is detoxified by Hcy-thiolactonase/paraoxonase present in a subset of high-density lipoprotein particles in humans.

There is abundant evidence that the endothelium plays a crucial role in the maintenance of vascular tone and structure. One of the major endothelium-derived vasoactive mediators is nitric oxide (NO), which has been characterized as an “endogenous anti-atherosclerotic molecule”. Synthesis of NO can be selectively inhibited by guanidino-substituted analogs of L-arginine, which act as competitive inhibitors at the active site of the enzyme. One such analog is asymmetric dimethylarginine (ADMA), a compound that has been found in human plasma and urine and exerts the activity of an endogenous inhibitor of NO synthase. In contrast to ADMA, its regioisomer symmetric dimethylarginine (SDMA) does not inhibit NO synthase. The methyl groups contained within the dimethylarginine molecules are derived from S-adenosylmethionine, an intermediate in the homocysteine/methionine pathway. There is experimental evidence that homocysteine may affect endothelium-dependent vascular function by increasing the formation of ADMA. Both ADMA and SDMA are eliminated from the body by renal excretion. In addition, the metabolism of ADMA, but not SDMA, occurs via hydrolytic degradation to citrulline and dimethylamine by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Data from experimental studies suggest that ADMA inhibits vascular NO elaboration at concentrations found in pathophysiological conditions ( i.e. , 3–15 μM). ADMA likely acts as an autocrine regulator of endothelial NO synthase activity. When rabbits are placed on a diet enriched with 1% cholesterol, ADMA levels are increased within 4 weeks of dietary intervention as compared to control animals. Elevated plasma concentrations of ADMA are also present in hypercholesterolemic and hypertensive patients, in patients with chronic heart failure, and in other patient groups at high risk of developing cardiovascular disease. Elevation of ADMA induces dysfunction of the endothelium, which becomes clinically evident by impaired endothelium-dependent vasodilation, hyperaggregability of platelets, and enhanced monocyte adhesion. Recent prospective studies suggest that endothelial dysfunction indicates an increased risk of future cardiovascular events. In line with these observations, we and others found evidence that ADMA is a novel cardiovascular risk factor.

One-carbon metabolism is under the influence of folate, vitamin B12 and genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR 677 C→T and 1298 A→C), of methionine synthase (MTR 2756 C→G), methionine synthase reductase (MTRR 66 A→G) and transcobalamin (TCN 776 C→G). The pathogenesis of neural tube defect (NTD) may be related to this metabolism. The influence of the MTHFR 677 C→T polymorphism reported in The Netherlands and Ireland can be questioned in southern Italy, France and Great Britain. MTRR, combined with a low level of vitamin B12, increases the risk of NTD and of having a child with NTD in Canada, while TCN 776 GG and MTRR 66 GG mutated genotypes associated with the MTHFR 677 CC wild-type are predictors of NTD cases in Sicily. Down syndrome (DS) is due to a failure of normal chromosomal segregation during meiosis, possibly related to one-carbon metabolism. MTHFR 677 C→T and MTRR 66 A→G polymorphisms are associated with a greater risk of having a child with DS in North America, Ireland and The Netherlands. In contrast, MTHFR 677 C→T has no influence on DS risk in France and Sicily, while homocysteine and MTR 2756 AG/GG genotypes are predictors of DS risk in Sicily. In conclusion, NTD and DS are influenced by the same genetic determinants of onecarbon metabolism. The distinct data produced in different geographical areas may be explained by differences in the nutritional environment and genetic characteristics of the populations.

Background: The prevalence of a sub-clinical functional vitamin B 12 deficiency in the general population is higher than previously expected. Total serum vitamin B 12 may not reliably indicate vitamin B 12 status. To get more specificity and sensitivity in diagnosing vitamin B 12 deficiency, the concept of measuring holotranscobalamin II (holoTC), a sub-fraction of vitamin B 12 , has aroused great interest. HoloTC as a biologically active vitamin B 12 fraction promotes a specific uptake of its vitamin B 12 by all cells. In this study we investigated the diagnostic value of storage (holoTC) of vitamin B 12 and functional markers (methylmalonic acid (MMA)) of vitamin B 12 metabolism in populations who are at risk of vitamin B 12 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 12 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 12 ) 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 12 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 B 12 deficiency followed by MMA. Vitamin B 12 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 B 12 . 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 B 12 deficiency. Renal patients have a higher requirement of circulating holoTC. In renal dysfunction, holoTC cannot be used as a marker of vitamin B 12 status.

We report on the performance of a new test, holotranscobalamin, as compared to well established markers of vitamin B12 deficiency (plasma cobalamins, methylmalonic acid, and homocysteine). Holotranscobalamin was analyzed in 143 samples by a competitive radiobinding assay (Axis-Shield). Employing a cut-off value of 50 pmol/l, holotranscobalamin showed a sensitivity of 1.00 and a specificity of 0.89 as regards discriminating between individuals with test results indicating vitamin B12 deficiency (methylmalonic acid >0.70 μmol/l and plasma cobalamins <200 pmol/l, n = 35) and individuals with test results inside the reference intervals (methylmalonic acid <0.29 μmol/l and plasma cobalamins ≥200 pmol/l, n = 35). In a group (n = 37) with low plasma cobalamins (<200 pmol/l) and normal methylmalonic acid (<0.29 μmol/l), 27 individuals had low holotranscobalamin, and in nine of these individuals plasma homocysteine supported the deficiency state (homocysteine >15 μmol/l). Holotranscobalamin was low in 12 individuals with increased methylmalonic acid (>0.40 μmol/l) and normal plasma cobalamins ≥200 pmol/l) (n = 36), and plasma homocysteine supported the deficiency state in four of these individuals. We conclude that holotranscobalamin is likely to be a sensitive marker of vitamin B12 deficiency that also has a reasonable specificity. Large-scale clinical studies are warranted in order to clarify the usefulness of holotranscobalamin in the clinical setting.

Although hyperhomocysteinemia is an established risk factor for venous thromboembolism there is no consensus for routine determination of circulating homocysteine in the UK, either at the beginning or end of oral anticoagulation therapy. The purpose of this study was to evaluate the prevalence of hyperhomocysteinemia and its relationship to folate and vitamin B12 status in subjects with venous thromboembolism 4 weeks after discontinuation of warfarin therapy. In 78 consecutively recruited patients, plasma homocysteine was significantly higher (p < 0.001) and red cell folate significantly lower (p = 0.03) than in controls. Plasma vitamin B12 was similar in both groups. Strikingly, 38.5% of patients had hyperhomocysteinemia (>15 μmol/l). Retrospective analysis revealed a significant positive association between plasma total homocysteine and duration of warfarin therapy (p < 0.001) but a negative, though non-significant (p = 0.06), trend with warfarin dose. The results do not suggest any direct interaction between warfarin and plasma homocysteine but raise the possibility of reduced intake of a common food source of folate and vitamin K. One possibility is the shortage of green-leafy vegetables since patients are often advised to limit their intake of this major source of vitamin K. On the basis of this study we suggest that homocysteine screening should be carried out at the time that patients begin warfarin therapy.

Background: Many new therapies are being developed that are anti-atherosclerotic, but which do not change clinical parameters such as blood pressure or cholesterol. In order to measure the effects of these therapies it will be necessary to measure atherosclerosis. Elevated levels of plasma total homocysteine (tHcy) are associated with increased risk of stroke and myocardial infarction. Measurement of the effect of vitamin therapy on atherosclerosis has therefore been used as an example of this approach. Methods: 2-Dimensional measurement of carotid plaque cross-sectional area has been used to measure effects of vitamin therapy in patients whose plaque is progressing despite intensive treatment of traditional risk factors. In clinic patients, addition of vitamin therapy halted progression of atherosclerosis, in samples of 50 patients over 2.5 years. However, in patients randomized to high-dose vs . low-dose vitamins in the Vitamin Intervention for Stroke Prevention trial, no difference in plaque progression was seen between high-dose and low-dose vitamin therapy. New methods have been developed for the measurement of 3-dimensional plaque volume, and for the measurement of plaque surface roughness. Results: The accuracy and reliability of the measurement of plaque volume is 95%. This will permit measurement of effects of new anti-atherosclerotic therapies with much smaller sample sizes, in a much shorter time, than previously available methods such as intima-media thickness. Conclusion: Measurement of atherosclerotic plaque volume and roughness will greatly enhance the study of new anti-atherosclerotic therapies.

Elevated plasma homocysteine is associated with increased cardiovascular risk but it remains unproven that the effect is directly causal. Folate and homocysteine metabolism are closely linked such that administration of folic acid in doses ranging from 0.2–10 mg/day lowers plasma total homocysteine (tHcy) by up to 25%. Folic acid has been widely advocated as a therapy which may reduce cardiovascular risk, but the clinical benefit remains as yet unproven and the choice of dose remains unclear. The effect of folic acid on endothelial function has been investigated in patients with proven coronary heart disease (CHD) by measuring flow-mediated dilatation (FMD) in the brachial artery. Oral folic acid (5 mg/day) markedly enhances endothelial function (FMD) and lowers homocysteine. Studies of the acute effects of folic acid have shown that this improvement occurs within the first 2–4 hours following the first dose, at which times there was no significant reduction in plasma tHcy. Administration of 5-methyltetrahydrofolate directly into the brachial artery markedly enhances FMD, an effect that is blocked by monomethyl arginine (LNMMA), suggesting that the effects of folate are mediated by nitric oxide. This Review summarises studies which show that pharmacological doses of folate markedly enhance endothelial function in patients with CHD. The discordance with changes in plasma homocysteine suggests that these effects may occur by mechanisms distinct from homocysteine lowering.

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.

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 B 12 . 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 B 12 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

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 B 12 , vitamin B 6 , 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 B 12 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 B 6 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 B 12 and folate. The magnitude of these effects was not influenced by the training intensity.

A relatively new method for the detection of single nucleotide polymorphisms is the use of denaturing highperformance liquid chromatography (DHPLC). DHPLC was used to analyse the transcobalamin II 776C>G polymorphism in DNA from 159 healthy elderly. Furthermore, cobalamin, folate, homocysteine and holotranscobalamin II (holo-TC II) were measured. The allele frequency of the G -allele was 17% with n = 55 harbouring the CC genotype, n = 77 being heterozygous and n = 27 showing the GG genotype. Holo-TC II concentrations were significantly decreased in patients harbouring the GG genotype. There was no effect on cobalamin, methylmalonyl-CoA, folate or homocysteine concentrations. A new G>A variant at nucleotide position 810 in the TC II gene was detected by an altered peak pattern in the DHPLC and further elucidated by direct sequencing. The TC II G810A variant is a silent mutation without replacement of the corresponding amino acid (alanine) at position 270 in the TC II protein and was only found as a heterozygous genotype in a single patient. The new variant would have been undetected by other methods used for single nucleotide polymorphism detection, e.g., restriction fragment length polymorphism analysis. The results suggest that the common TC II 776C>G polymorphism has no major influence on vitamin B12 metabolism.