Current Drug Metabolism - Volume 6, Issue 1, 2005

When Vincent du Vigneaud isolated homocysteine in 1932, it was his intention to identify the origin of sulfur within the insulin molecule, discovered by Banting and Best in Toronto (1921). Cystine and methionine were likely candidates. However, du Vigneaud was to later recall that all of the work that led ultimately to the discovery of the transsulfuration and transmethylation pathways would not have taken place had he known that methionine was in fact not part of insulin. Around 1960 all reactions and metabolites participating in the metabolism of methionine and homocysteine were identified and known, but clinicians only began to realize the relevance when homocystine was first identified in the urine of children with inherited enzyme deficiencies in 1962. A few cases of extremely rare inborn genetic defects with the common feature of hyperhomocysteinemia enabled McCully in 1968 / 69 to establish the hypothesis that homocysteine might have a role to play in the etiology of vascular pathologies. The full metabolism as it is known today was published in “Science” (1964) and after another decade and with the development of more sensitive diagnostic technology and new markers and its introduction for routine use, homocysteine research began to explode and continues to be a very active field of research today. In fact it has become almost impossible to keep up with the number of daily published works. Beyond its potential role as a major risk factor in atherothrombotic disease, homocysteine has increasingly been found to participate directly and indirectly in a large number of basic functions of cell physiology. Consequently research has expanded to new fields of interest and these promise to yield new insights and understanding in molecular pathology with important clinical implications. In this special issue of Current Drug Metabolism the reader will find a number of selected reviews that are very closely linked to each other - by the amino acid homocysteine. My intention as guest editor is to cover some of the new fields of research that homocysteine has led the way to. The authors are dedicated researchers and much respected investigators in their own particular and very demanding fields. I am very grateful to them for their valuable contributions to this special issue. Their reviews provide current insight into homocysteine-mediated interactions with DNA-methylation, ADMA, betaine, cobalamin metabolism, renal function and oxygen radical formation. As such, they add to our understanding of the etiology and progression of diseases and serve to encourage further research.

Hyperhomocyst(e)inemia is associated with an increased risk for atherosclerotic disease and venous thromboembolism. The impact of elevated plasma homocysteine levels seems to be clinically relevant, since the total cardiovascular risk of hyperhomocyst(e)inemia is comparable to the risk associated with hyperlipidemia or smoking. There is substantial evidence for impairment of endothelial function in human and animal models of atherosclerosis, occurring even before development of overt plaques. Interestingly endothelial dysfunction appears to be a sensitive indicator of the process of atherosclerotic lesion development and predicts future vascular events. NO is the most potent endogenous vasodilator known. It is released by the endothelium, and reduced NO bioavailability is responsible for impaired endothelium-dependent vasorelaxation in hyperhomocyst(e)inemia and other metabolic disorders associated with vascular disease. Substances leading to impaired endothelial function as a consequence of reduced NO generation are endogenous NO synthase inhibitors such as ADMA. Indeed there is accumulating evidence from animal and human studies that ADMA, endothelial function and homocyst(e)ine might be closely interrelated. Specifically elevations of ADMA associated with impaired endothelium-dependent relaxation were found in chronic hyperhomocyst(e)inemia, as well as after acute elevation of plasma homocyst(e)ine following oral methionine intake. The postulated mechanisms for ADMA accumulation are increased methylation of arginine residues within proteins, as well as reduced metabolism of ADMA by the enzyme DDAH, but they still need to be confirmed to be operative in vivo. Hyperhomocyst(e)inemia, as well as subsequent endothelial dysfunction can be successfully treated by application of folate and B vitamins. Since ADMA seems to play a central role in homocyst(e)ine-induced endothelial dysfunction, another way of preventing vascular disease in patients with elevated homocyst(e)ine concentrations could be supplementation with L-arginine to reverse the detrimental effects of ADMA.

High plasma concentrations of homocysteine may increase risk of cardiovascular disease. Folic acid lowers plasma homocysteine by 25% maximally, because 5-methyltetrahydrofolate is a methyl donor in the remethylation of homocysteine to methionine. Betaine (trimethylglycine) is also a methyl donor in homocysteine remethylation, but effects on homocysteine have been less thoroughly investigated. Betaine in high doses (6 g / d and higher) is used as homocysteine-lowering therapy for people with hyperhomocysteinemia due to inborn errors in the homocysteine metabolism. Betaine intake from foods is estimated at 0.5-2 g / d. Betaine can also be synthesized endogenously from its precursor choline. Studies in healthy volunteers with plasma homocysteine concentrations in the normal range show that betaine supplementation lowers plasma fasting homocysteine dose-dependently to up to 20% for a dose of 6 g / d of betaine. Moreover, betaine acutely reduces the increase in homocysteine after methionine loading by up to 50%, whereas folic acid has no effect. Betaine doses in the range of dietary intake also lower homocysteine. This implies that betaine can be an important food component that attenuates homocysteine rises after meals. If homocysteine plays a causal role in the development of cardiovascular disease, a diet rich in betaine or choline might benefit cardiovascular health through its homocysteine-lowering effects. However betaine and choline may adversely affect serum lipid concentrations, which can of course increase risk of cardiovascular disease. However, whether the potential beneficial health effects of betaine and choline outweigh the possible adverse effects on serum lipids is as yet unclear.

Plasma homocysteine concentration exhibits a strong relationship with (indices of) renal function. Hyperhomocysteinemia has been implicated in the high vascular event rate in patients with chronic renal failure. The precise pathophysiological explanation for the occurrence of hyperhomocysteinemia in renal failure is not yet elucidated. A defective intrinsic renal metabolism of homocysteine seems unlikely. There are several indications that whole body homocysteine metabolism is altered in renal insufficiency. Stable isotope studies in dialysis patients have shown a decreased homocysteine clearance by transsulfuration and decreased homocysteine remethylation and methionine transmethylation. Several, but not all, prospective studies have linked hyperhomocysteinemia to adverse cardiovascular outcomes in renal failure patients. Treatment of hyperhomocysteinemia in renal insufficiency is based on folic acidcontaining regimens, but so far, none of the regimens has been shown to successfully normalize plasma homocysteine concentration. Intervention studies have not yet demonstrated beneficial vascular effects of homocysteine-lowering treatment in dialysis patients.

Elevated plasma levels of homocysteine are associated with an increased generation of reactive oxygen species in aortas of hyperhomocysteinemic animals and in endothelial cells. This may contribute to endothelial dysfunction observed in hyperhomocysteinemia, and promote atherosclerotic vascular disease. Homocysteine seems to promote the formation of reactive oxygen species primarily by a biochemical mechanism involving endothelial nitric oxide synthase, as increased endothelial lipid peroxidation and oxidation of the redox-sensitve dye 2',7'-dichlorofluoresceine could only be observed after incubation of endothelial cells with L-, but not with D-homocysteine, and could be prevented by inhibition of endothelial nitric oxide synthase. An increased oxidation rate of aminothiols in plasma, as observed in patients with hyperhomocysteinemia, further contributes to increased generation of reactive oxygen species. These effects are amplified by a homocysteine-specific inhibition of cellular antioxidant enzymes, like superoxide dismutase and the cellular isoform of gluthatione peroxidase. All mechanisms together result in increased levels of superoxide anion and peroxyl radicals in the vasculature that react with nitric oxide to form peroxnitrites. This abolishes nitric oxide's bioactivity and contributes to endothelial dysfunction. In addition, increased vascular oxidant stress in hyperhomocysteinemia has been shown to activate proinflammatory signaling pathways in endothelial cells, like the transcription factor NF-κB. This leads to increased endothelial expression of chemokines and adhesion molecules that promote the recruitment, adhesion and transmigration of circulating leukocytes to the vessel wall. All these mechamisms may contribute to the increased risk for cardiovascular diseases associated with hyperhomocysteinemia.

Advances in molecular biology greatly contributed, in the past decades, to a deeper understanding of the role of gene function in disease development. Environmental as well as nutritional factors are now well acknowledged to interact with the individual genetic background for the development of several diseases, including cancer, cardiovascular disease, and neurodegenerative diseases. The precise mechanisms of such gene-nutrient interactions, however, are not fully elucidated yet. Many micronutrients and vitamins are crucial in regulating mechanisms of DNA metabolism. Indeed, folate has been most extensively investigated for its unique function as mediator for the transfer of one-carbon moieties for nucleotide synthesis / repair and biological methylation. Cell culture, animal, and human studies, clearly demonstrated that folate deficiency induces disruption of DNA synthesis / repair pathways as well as DNA methylation anomalies. Remarkably, a gene-nutrient interaction between folate status and a polymorphism in methylenetetrahydrofolate reductase gene has been reported to modulate genomic DNA methylation. This observation suggests that the interaction between a nutritional status and a mutant genotype may modulate gene expression through DNA methylation, especially when such polymorphism affects a key enzyme in one-carbon metabolism and limits the methyl supply. DNA methylation, both genome-wide and gene-specific, is of particular interest for the study of aging, cancer, and other pathologic conditions, because it affects gene expression without permanent alterations in the DNA sequence such as mutations or allele deletions. Understanding the patterns of DNA methylation through the interaction with nutrients is a critical issue, not only to provide pathophysiological explanations of a disease state, but also to identify individuals at-risk to conduct targeted diet-based interventions.

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 μmol / 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.