Volume 45, Issue 12

Background : Elevated plasma total homocysteine is a risk factor for cardiovascular disease (CVD), but the randomized trials of dietary supplementation with B-vitamins to lower homocysteine have not yet provided clear evidence of benefit on vascular risk. Methods : Cumulative meta-analysis of all randomized trials assessing the effects of lowering homocysteine levels with B-vitamins on risk of CVD. Results : An individual patient data meta-analysis of all randomized trials of the effects on vascular risk of lowering homocysteine with B-vitamins will maximize the power to assess the epidemiologically predicted differences in risk. Among the 12 randomized homocysteine-lowering trials for prevention of CVD, involving more than 1000 participants, data should be available on approximately 52,000 participants (32,000 with prior CVD in unfortified populations; and 14,000 with prior CVD and 6000 with renal disease in fortified populations). To minimize bias, the design and primary analyses to be carried out have been pre-specified. The analyses will include assessment of effects on major vascular events (MVE), stroke, major coronary events (MCE), in addition to venous thrombosis, cancer and fractures. Additional analyses will assess effects on vascular outcomes in sub-groups defined by population, prior disease, per 3 μmol/L difference in homocysteine levels achieved by treatment, pre-treatment vitamin status, duration, age, sex and vascular events excluding revascularizations and, separately, excluding vascular events occurring during the first year of treatment. Conclusions : A cumulative meta-analysis of the homocysteine-lowering trials should ensure that reliable evidence emerges about the effects of lowering homocysteine on risk of vascular and non-vascular outcomes. Clin Chem Lab Med 2007;45:1575–81.

Background : The pathogenesis of stroke is very different from that of myocardial infarction; most strokes are embolic, and as elevated plasma total homocysteine (tHcy) increases thrombosis, it increases the risk of cardioembolic strokes, particularly in atrial fibrillation, as well as the risk of deep vein thrombosis leading to paradoxical embolism, and elevated tHcy also increases strokes from cortical vein thrombosis. Purpose and methods : The main study results of the Vitamin Intervention for Stroke Prevention (VISP) trial showed no benefit of vitamin therapy for homocysteine lowering. However, there were a number of problems with the trial including: 1. We did not use a placebo control; we used a multiple vitamin containing low doses of folate and B6, and the recommended daily intake of B12; 2. Patients with low serum B12 levels in both arms of the trial received injections of B12; and 3. Folate fortification of grain products in North America coincided with the initiation of the trial. We therefore conducted a hypothesis-driven analysis of a subgroup of VISP from which patients who could not have responded to the study treatment were excluded, i.e., those with low or very high serum B12, and those with renal failure. Results : In the remaining 2155 patients, there was a significant reduction of stroke, coronary events and death (p=0.049). When the participants were stratified according to the median entry level of B12 (313 pmol/L), it became apparent that serum B12 status was the key determinant of response, those with high serum B12 at entry who received high-dose vitamin had a 33% reduction of events compared to those with low B12 who received low-dose vitamins; the other two groups were intermediate (p=0.02 for survival free of stroke, coronary and death in the Kaplan-Meier analysis). Discussion and conclusions : In this efficacy analysis, we found a significant reduction of vascular risk with vitamin therapy. Similarly, the Heart Outcomes Prevention Evaluation 2 trial showed a 25% significant reduction of stroke with vitamin therapy (p=0.03). The authors thought this result was a chance finding, because they did not think that stroke and myocardial infarction would be biologically different; however, stroke and myocardial infarction have very different pathogenesis, so that conclusion is unwarranted. It seems likely that even if vitamin therapy does not significantly reduce coronary events, it will reduce the risk of stroke. Clin Chem Lab Med 2007;45:1582–5.

In Italy, the mortality rate of hemodialysis patients is approximately 14% per year. Cardiovascular disease is the most important cause of morbidity and mortality in hemodialysis patients. High plasma homocysteine levels are commonly detected in these patients, but hyperhomocysteinemia and cardiovascular mortality are not always strictly correlated. The Dialysis Outcomes and Practice Pattern Study (DOPPS) showed a direct association between regular use of water-soluble vitamins and lower cardiovascular mortality. We recently performed a long-term prospective trial to study the effects of folic acid therapy on cardiovascular events in hemodialysis patients. We observed not only a lower rate of combined cardiovascular events in patients treated with folate, but also a direct correlation between hyperhomocysteinemia and cardiovascular morbidity. On the contrary, the distribution of deaths was similar in treated and untreated patients, because, almost certainly, sudden death is not always due to atherosclerotic events, and non-cardiovascular deaths, such as cachexia, septicemia and malignancy were characterized by low levels of homocysteine, which may be, in addition, a nutritional index similar to albumin and protein catabolic rate. As it is known that diabetic hemodialysis patients have a higher mortality rate, but lower homocysteine levels as compared to non-diabetic patients, we performed an equal allocation of diabetic patients in treated and untreated groups. We observed a similar homocysteine reduction rate in diabetic patients as compared to non-diabetic patients, and a trend towards a lower rate of composite cardiovascular events in treated diabetic patients as compared to untreated diabetic patients. To summarize, the strong relationship between homocysteine and nutritional, inflammatory markers may hide its association with cardiovascular disease. Homocysteine-lowering vitamin B therapy may lower cardiovascular events in dialysis patients. It is mandatory to perform large prospective trials to confirm our results. Clin Chem Lab Med 2007;45:1586–9.

Hyperhomocysteinemia (HHcy) is related to central nervous system diseases. Epidemiological studies show a positive, dose-dependent relationship between plasma total homocysteine (tHcy) concentration and neurodegenerative disease risk. tHcy is a marker of B-vitamin (folate, B 12 , B 6 ) status. Hypomethylation, caused by low B-vitamin status and HHcy, is linked to key pathomechanisms of dementia; B-vitamin supplementation could potentially reduce neurological damage. In retrospective studies, the association between tHcy and cognition is impressive; there is also evidence that tHcy-lowering treatment could be effective in primary and secondary stroke prevention. Increased tHcy and low serum folate occur in patients with Parkinson's disease, especially those receiving L-dopa. There is also an association between HHcy and multiple sclerosis, and between B-vitamin status and depression. Studies also confirm a causal role for tHcy in epilepsy, and certain anti-epileptics enhance HHcy. B-vitamin status should be optimized by ensuring sufficient intake in patients with neuropsychiatric diseases. HHcy occurs commonly in the elderly and can contribute to age-related neurodegeneration. Treatment with folic acid, B 12 and B 6 lowers tHcy. For secondary and primary prevention from several neuropsychiatric disorders, it seems prudent to actively identify deficient subjects and ensure sufficient vitamin intake. Clin Chem Lab Med 2007;45:1590–606.

In recent years, L-Dopa treatment has been indicated as an acquired cause of hyperhomocysteinemia (HHcy). The mechanism underlying L-Dopa-related HHcy is the O-methylation of the drug catalyzed by the enzyme catechol-O-methyltransferase (COMT). Folate and cobalamin status also influences the effects of L-Dopa on plasma homocysteine (Hcy) levels. Although clinical correlations of HHcy in Parkinson's disease still remain uncertain, management of elevated plasma Hcy levels has been advocated, due to multiple cytotoxic effects of Hcy on neurons. This review summarizes data available in the literature concerning the two main therapeutic approaches to L-Dopa-related HHcy (use of COMT inhibitors or B vitamins diet supplementation). Clin Chem Lab Med 2007;45:1607–13.

Folate and vitamin B 12 are essential cofactors for the methionine/homocysteine cycle in the brain. These vitamins mediate the remethylation of homocysteine (Hcy), which affects the production of the universal methyl donor, S-adenosylmethionine (SAM), in the brain among other organs. Additionally, increased plasma concentrations of total Hcy (tHcy) are associated with cerebrovascular disease and can compromise the blood-brain barrier. tHcy concentrations in the brain and cerebrospinal fluid become increased in several psychiatric and neurological disorders. Disturbances in the transmethylation pathway indicated by abnormal SAM, S-adenosylhomocysteine or their ratio have been reported in many neurodegenerative diseases, such as dementia, depression or Parkinson's disease. Cobalamin is essential for neuronal generation and its deficiency can cause degeneration of the nervous system. Available data emphasize that deficiency of folate and vitamin B 12 can lead to elevated concentrations of tHcy and disturbed methylation potential in the brain. Therefore, acquired or inherited disorders in these metabolic pathways are associated with brain abnormalities and severe neurological symptoms that are mostly irreversible, even after providing the missing cofactors. This review discusses the relationship between brain and blood levels of key vitamins and metabolites related to one carbon metabolism. Clin Chem Lab Med 2007;45:1614–20.

Hyperhomocysteinemia (HHCY) has been suggested as a new risk factor for osteoporosis. Recent epidemiological, clinical and experimental studies provide a growing body of data, which is reviewed in this article. Epidemiological and (randomized) clinical trials suggest that HHCY increases fracture risk, but has minor effects on bone mineral density. Measurement of biochemical bone turnover markers indicates a shift of bone metabolism towards bone resorption. Animal studies confirm these observations showing a reduced bone quality and stimulation of bone resorption in hyperhomocysteinemic animals. Homocysteine (HCY) has been found to accumulate in bone by collagen binding. Cell culture studies demonstrate that high HCY levels stimulate osteoclasts but not osteoblasts, indicating again a shift of bone metabolism towards bone resorption. Regarding B-vitamins, only a few in vivo studies with equivocal results have been published. However, two large cell culture studies confirm the results obtained with exogenous HCY administration. In addition, HHCY seems to have adverse affects on extracellular bone matrix by disturbing collagen crosslinking. In conclusion, existing data suggest that HHCY (and possibly B-vitamin deficiencies) adversely affects bone quality by a stimulation of bone resorption and disturbance of collagen crosslinking. Clin Chem Lab Med 2007;45:1621–32.

Chronic heart failure (CHF) is a major public health problem causing considerable morbidity and mortality. Recently, plasma homocysteine (HCY) has been suggested to be significantly increased in CHF patients. This article reviews the relation between hyperhomocysteinemia (HHCY) and CHF. Clinical data indicate that HHCY is associated with an increased incidence, as well as severity, of CHF. In addition, HCY correlates with brain natriuretic peptide (BNP), a modern biochemical marker of CHF, which is used for diagnosis, treatment guidance and risk assessment. Animal studies showed that experimental HHCY induces systolic and diastolic dysfunction, as well as an increased BNP expression. Moreover, hyperhomocysteinemic animals exhibit an adverse cardiac remodeling characterized by accumulation of interstitial and perivascular collagen. In vitro superfusion experiments with increasing concentrations of HCY in the superfusion medium stimulated myocardial BNP release independent from myocardial wall stress. Thus, clinical and experimental data underline a correlation between HHCY and BNP supporting the role of HHCY as a causal factor for CHF. The mechanisms leading from an elevated HCY level to reduced pump function and adverse cardiac remodeling are a matter of speculation. Existing data indicate that direct effects of HCY on the myocardium, as well as nitric oxide independent vascular effects, are involved. Preliminary data from small intervention trials have initiated the speculation that HCY lowering therapy by micronutrients may improve clinical as well as laboratory markers of CHF. In conclusion, HHCY might be a potential etiological factor in CHF. Future studies need to explore the pathomechanisms of HHCY in CHF. Moreover, larger intervention trials are needed to clarify whether modification of plasma HCY by B-vitamin supplementation improves the clinical outcome in CHF patients. Clin Chem Lab Med 2007;45:1633–44.

Experimental and observational studies support a role of plasma homocysteine levels (tHcy) in coronary artery disease (CAD). In the GENICA (Genetic and Environmental factors In Coronary Atherosclerosis) study, we found that high tHcy predicted cardiovascular mortality in hypertensive, but not in normotensive, patients independently of CAD and history of myocardial infarction. Moreover, despite not being associated with the coronary atherosclerotic burden, tHcy was inversely associated with left ventricular (LV) ejection fraction. This inverse relationship between LV systolic function and tHcy, which has been independently confirmed, might explain the association of tHcy with the risk of incident heart failure documented in the Framingham Heart Study. Thus, additional mechanistic investigation taking into consideration the effects of tHcy on LV function is necessary to further explore the potential therapeutic usefulness of tHcy lowering treatment in CAD. Clin Chem Lab Med 2007;45:1645–51.

Hyperhomocysteinemia (HHcy) is a significant and independent risk factor for cardiovascular disease (CVD) and the underlying mechanism is unclear. We and others have reported that homocysteine (Hcy) is inversely correlated with plasma high-density lipoprotein cholesterol (HDL-C) and apolipoprotein AI (apoA-I) in patients with coronary heart disease (CHD). We confirmed this negative correlation in mice with targeted deletions of the genes for apolipoprotein E (apoE) and cystathionine β-synthase (CBS). Severe HHcy (plasma Hcy 210 μmol/L) accelerates spontaneous arthrosclerosis in the CBS −/− /apoE −/− mice, reduces the concentration of circulating HDL, apoA-I, and large HDL particles, inhibits HDL function, and enhances HDL-C clearance. We have demonstrated further that Hcy (0.5–2 mmol/L) reduces apoA-I protein synthesis and secretion, but not RNA transcription in mouse primary hepatocytes. A different mechanism was proposed based on studies using the HepG2 cells showing that Hcy (5–10 mmol/L) inhibits apoA-I transcription via peroxisome proliferator-activated receptor-α (PPARα)-inhibition-dependent and -independent mechanisms. These studies suggest that Hcy-induced HDL-C and apoA-I inhibition represent a novel mechanism by which Hcy induces atherosclerotic CVD. Clin Chem Lab Med 2007;45:1652–9.

Hyperhomocysteinemia (HHcy) has been established as a potent independent risk factor for cardiovascular disease (CVD) and the underlying mechanism is largely unknown. We were the first to propose that hypomethylation is the key biochemical mechanism by which homocysteine (Hcy) inhibits endothelial cell (EC) growth. We reported that clinically relevant concentrations of Hcy (10–50 μmol/L) exerts highly selective inhibitory effects on cyclin A transcription and EC growth through a hypomethylation related mechanism, which blocks cell cycle progression and endothelium regeneration. Recently, we demonstrated that Hcy reduces DNA methyltransferase 1 (DNMT1) activity and demethylates cyclin A promoter leading to cyclin A chromatin remodeling. We found that adenovirus-transduced DNMT1 gene expression reverses the inhibitory effect of Hcy on cyclin A expression and EC growth inhibition. We hypothesize that DNA hypomethylation is a key biochemical mechanism responsible for Hcy-induced cyclin A suppression and growth inhibition in EC and contributes to CVD. Clin Chem Lab Med 2007;45:1660–6.

Objectives : We systematically reviewed published studies looking at the relationship between total serum homocysteine (tHcy) and subclinical markers of atherosclerosis, such as carotid intimal-medial thickness (C-IMT), coronary artery calcium (CAC) and ankle-brachial index (ABI) in asymptomatic individuals. We analyzed these studies to examine this relationship as well as to guide future avenues of investigation by identifying studies that will help in the inclusion of tHcy levels in current guidelines on atherosclerotic disease management. Background : tHcy has been known to be associated with clinically evident atherosclerotic disease. However, tHcy is not incorporated in current guidelines for diagnosis of subclinical disease in high-risk asymptomatic individuals. Methods : We searched online databases (e.g., PUBMED, MEDLINE) for published articles assessing the relationship between tHcy and C-IMT, CAC and ABI. We limited the studies to asymptomatic populations, and excluded any study including symptomatic patients or individuals with a history of coronary, peripheral or cerebrovascular disease. Results : A systemic review of 19 articles revealed a significant association between elevated levels of tHcy and subclinical markers of atherosclerosis in asymptomatic individuals in most studies. Mean tHcy levels were consistently found to be higher in men compared to women. A total of 12 studies showed a significant association between tHcy and other risk markers of atherosclerosis, even after adjusting for age, sex and conventional risk factors. There were seven studies in unique populations that showed no significant relationship. We also observed that studies lowering baseline tHcy levels did not lead to an improvement in C-IMT, CAC or ABI scores. This might indicate that tHcy has a stronger role as a marker of atherosclerotic disease than as a risk factor for the same. Conclusions : Based on our review, we conclude that there is a significant association between the subclinical atherosclerotic process and tHcy, and it shows potential as a cheap marker for risk stratification of asymptomatic patients. However, future studies further elucidating this association and elaborating the exact role of tHcy in the atherosclerotic disease process are required. The results of these studies suggest the incorporation of plasma tHcy levels in future risk reduction protocols for identification of individuals at higher risk of atherosclerotic events, and thus to categorize them for more aggressive treatment with established preventive and therapeutic measures. Clin Chem Lab Med 2007;45:1667–77.

Protein homocysteinylation is proposed as one of the mechanisms of homocysteine toxicity. It occurs through various means, such as the post-biosynthetic acylation of free amino groups (protein-N-homocysteinylation, mediated by homocysteine thiolactone) and the formation of a covalent -S-S- bond found primarily with cysteine residues (protein-S-homocysteinylation). Both protein modifications are a cause of protein functional derangements. Hemodialysis patients in the majority of cases are hyperhomocysteinemic, if not malnourished. Protein-N-homocysteinylation and protein-S-homocysteinylation are significantly increased in hemodialysis patients compared to controls. Oral folate treatment normalizes protein-N-homocysteinylation levels, while protein-S-homocysteinylation is significantly reduced. Albumin binding experiments after in vitro homocysteinylation show that homocysteinylated albumin is significantly altered at the diazepam, but not at the warfarin and salicilic acid binding sites. Clin Chem Lab Med 2007;45:1678–82.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is formed by methylation of arginine residues in proteins and released after proteolysis. In this reaction, S-adenosylmethionine is methyldonor and S-adenosylhomocysteine the demethylated product. ADMA and homocysteine are thus biochemically linked. Both plasma homocysteine and ADMA concentrations are increased in patients with renal dysfunction, probably as a result of an impairment in their metabolic, but not urinary, clearance. Hyperhomocysteinemia has been associated with an increased risk of cardiovascular disease in end-stage renal disease, especially in patients without malnutrition and inflammation. Also, plasma ADMA levels have been associated with cardiovascular disease in renal failure patients. Both homocysteine and ADMA are thought to mediate their adverse vascular effects by impairing endothelial, nitric oxide-dependent function resulting in decreased vasodilatation, increased smooth muscle cell proliferation, platelet dysfunction and increased monocyte adhesion. At the same time, it has been shown that the correlation between plasma ADMA and homocysteine is weak and that, in renal patients, the association of plasma ADMA carotid intima-media thickness, cardiovascular events and overall mortality is independent of homocysteine. This indicates that the negative vascular effects of ADMA and homocysteine have a different etiology. Treatment with folic acid substantially lowers homocysteine, but not ADMA concentration. So far, homocysteine-lowering therapy has not been very successful in decreasing cardiovascular disease. In patients with renal failure, ADMA reduction may be an interesting new goal in the prevention of cardiovascular disease. Clin Chem Lab Med 2007;45:1683–7.

Despite substantial evidence indicating the association of hyperhomocysteinemia (hHcys) and end-stage renal disease (ESRD), the pathogenic role of increased plasma homocysteine (Hcys) levels in the progression of ESRD remains unclear. This review will briefly summarize recent findings regarding the role of hHcys in the development of glomerulosclerosis, the association of hHcys with reduced renal transsulfuration and Hcys-induced changes of redox signaling in the development of glomerulosclerosis in rat kidneys. Based on these results, it is concluded that hHcys is implicated in glomerular sclerosis in hypertension, elevated plasma Hcys in Dahl salt-sensitive (SS) hypertensive rats is due to downregulation of cystathionine β-synthase (CBS) expression and consequent abnormality of transsulfuration in the kidney compared with normotensive rats. Hcys-induced superoxide (O 2 · – ) production by activation of NADPH oxidase as a triggering mechanism contributes to the effects of Hcys on the homeostasis of extracellular matrix and consequent sclerosis in the glomeruli, and NADPH oxidase activation by Hcys is associated with enhanced Rac GTPase activity. Clin Chem Lab Med 2007;45:1688–93.

In mammalian liver, two intersecting pathways, remethylation and transsulfuration, compete for homocysteine that has been formed from methionine. Remethylation of homocysteine, employing either methyltetrahydrofolate or betaine as the methyl donor, forms a methionine cycle that functions to conserve methionine. In contrast, the transsulfuration sequence – cystathionine synthase and cystathionase – serves to irreversibly catabolize the homocysteine while synthesizing cysteine. The rate of homocysteine formation and its distribution between these two pathways are the sites for metabolic regulation and coordination. The mechanisms for regulation include both the tissue content and the kinetic properties of the component enzymes as well as the concentrations of their substrates and other metabolic effectors. Adenosylmethionine and adenosylhomocysteine are important regulatory metabolites and may use one or more mechanisms to affect the enzymes. Adenosylmethionine is a positive effector of its own synthesis, cystathionine synthase and glycine methyltransferase but impairs both homocysteine methylases. Thus, the concentration of adenosylmethionine may be self-regulatory in mammalian liver. By means of other enzymatic mechanisms, the hepatic concentration of adenosylhomocysteine, an index of homocysteine accumulation, is also self-regulated. These considerations pertain primarily to liver, which has the unique capacity to synthesize more adenosylmethionine in the presence of excess methionine. However, there are organ-specific patterns of methionine metabolism and its regulation. All tissues possess the methionine cycle with methyltetrahydrofolate as the methyl donor but only liver, kidney, pancreas, intestine and brain also contain the transsulfuration pathway. The limitation of adenosylmethionine concentrations may make adenosylhomocysteine a more significant metabolic regulator in extrahepatic tissues. However, estimates of regulatory changes based on determinations of the plasma concentrations of the two metabolites are of limited value and must be used with caution. In addition, the recent description of “cystathionine (CBS) domains” in proteins not involved with methionine metabolism raises the possibility that abnormal concentrations of the adenosyl metabolites may impact on other metabolic pathways. Clin Chem Lab Med 2007;45:1694–9.

There are now four genetic mouse models that induce hyperhomocyst(e)inemia by decreasing the activity of an enzyme involved in homocysteine metabolism: cystathionine β-synthase, methylenetetrahydrofolate reductase, methionine synthase and methionine synthase reductase. While each enzyme deficiency leads to murine hyperhomocyst(e)inemia, the accompanying metabolic profiles are significantly and often unexpectedly, different. Deficiencies in cystathionine β-synthase lead to elevated plasma methionine, while deficiencies of the remaining three enzymes lead to hypomethioninemia. The liver [S-adenosylmethionine]/[S-adenosylhomocysteine] ratio is decreased in mice lacking methylenetetrahydrofolate reductase or cystathionine β-synthase, but unexpectedly increased in mice with deficiencies in methionine synthase or methionine synthase reductase. Folate pool imbalances are observed in complete methylenetetrahydrofolate reductase deficiency, where methyltetra-hydrofolate is a minor component, and in methionine synthase reductase deficiency, where methyltetrahydrofolate is increased relative to wild-type mice. These differences illustrate the potential diversity among human patients with hyperhomocyst(e)inemia, and strengthen the argument that the pathologies associated with the dissimilar forms of the condition will require different treatments. Clin Chem Lab Med 2007;45:1700–3.

Accumulating evidence suggests that a metabolite of homocysteine (Hcy), the thioester Hcy-thiolactone, plays an important role in atherogenesis and thrombosis. Hcy-thiolactone levels are elevated in hyperhomocysteinemic humans and mice. The thioester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues, which impairs or alters the protein's function. Protein targets for the modification by Hcy-thiolactone in human blood include fibrinogen, low-density lipoprotein, and high-density lipoprotein. Protein N-homocysteinylation leads to pathophysiological responses, including increased susceptibility to thrombogenesis caused by N-Hcy-fibrinogen, and an autoimmune response elicited by N-Hcy-proteins. Chronic activation of these responses in hyperhomocysteinemia over many years could lead to vascular disease. This article reviews recent evidence supporting the hypothesis that Hcy-thiolactone contributes to pathophysiological effects of Hcy on the vascular system. Clin Chem Lab Med 2007;45:1704–16.

Although the beneficial effects of maternal folate supplementation in the periconceptional period have been shown to prevent neural tube defects, congenital heart defects and orofacial clefts, the exact protective mechanism of folates remains unknown. Folates affect DNA synthesis, amino acid metabolism and methylation of genes, proteins and lipids via S-adenosylmethionine-mediated one-carbon transfer reactions. Our laboratory has created several mouse knock out models of folate transport using gene targeting to inactivate folate receptor 1 ( Folr1 ), folate receptor 2 ( Folr2 ) and reduced folate carrier 1 ( Slc19a1 ) genes. Gene ablation of both Folr1 and Slc19a1 leads to lethality, but with maternal folate supplementation, nullizygous embryos for both genes present with neural tube defects (NTDs) and congenital heart defects (CHDs). Folr1 nullizygous mice also exhibit orofacial clefts when the dams are provided with low folate supplementation during pregnancy. Finally, women with NTD-affected pregnancies have been reported to have high autoantibody titers against the folate receptor, potentially inhibiting the transport of folate to the developing embryo. This may be an explanation for some of the folate-responsive NTDs and perhaps other congenital malformations. Herein, we propose how homocysteinylation of the folate receptor may contribute to generation of these autoantibodies against the folate receptor. Clin Chem Lab Med 2007;45:1717–27.

During the last years, a growing body of evidence has been accumulated on the role of hyperhomocysteinemia in the occurrence of coronary artery disease and other arterial occlusive diseases. The mechanism by which high circulating homocysteine concentrations are a risk factor for atherothrombosis is incompletely understood. The present review is aimed to evaluate the role of inflammation in influencing homocysteine (Hcy) and vitamin B 6 concentrations. Results of a large population-based study have suggested that inflammatory markers are the major determinants of Hcy and vitamin B 6 concentrations. This association, independent of the leading factor, may explain, at least in part, why subjects with high concentrations of Hcy and low concentrations of vitamin B 6 have a high risk of developing cardiovascular diseases. Clin Chem Lab Med 2007;45:1728–36.

Background : Derangements of one-carbon metabolism have been related to the development of chronic diseases. Metabolic profiling as part of epidemiological studies in this area should include intermediates involved in the transfer of one-carbon units, cofactors for the relevant enzymes and markers of inflammation, kidney function and smoking. Methods : We established five platforms that measured 6–16 analytes each. Platforms A (gas chromatography-mass spectrometry; GC-MS) and B (gas chromatography-tandem mass spectrometry; GC-MS/MS) involved methylchloroformate derivatization of primary amines, thiols and carboxylic acids. Platform C determined basic compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using an ether-linked phenyl reversed-phase column. Platforms D and E (LC-MS/MS) exploited the efficient ionization and high sensitivity obtained for a wide range of analytes, using a mobile phase containing a high concentration of acetic acid. The chromatographic run times ranged from 3 to 8 min. Results : The analyte concentrations ranged from 0.2 nmol/L to 400 μmol/L. Platforms A and B both measured methylmalonic acid, total homocysteine and related amino acids. Platform B also included sarcosine, cystathionine, tryptophan and kynurenine. Platform C was optimized for the measurement of choline and betaine, but also included arginine, asymmetric and symmetric dimethylarginine and creatinine. A diversity of low abundance compounds mainly occurring in the nanomolar range were measured on platform D. These were vitamin B 2 and B 6 species, neopterin, cotinine and tryptophan metabolites. Platform E measured folates and folate catabolites. Conclusions : Approximately 40 analytes related to one-carbon metabolism were determined in less than 1 mL of plasma/serum using five complementary analytical platforms. As a method control, several metabolites were measured on two or more platforms. Logistics and data handling were carried out by specially designed software. This strategy allows profiling of one-carbon metabolism in large-scale epidemiological studies. Clin Chem Lab Med 2007;45:1737–45.

Background : Cobalamin-saturated transcobalamin, also called holotranscobalamin (holoTC), constitutes only between 6% and 20% of total plasma B 12 . Serum concentration of holoTC is a new marker in laboratory diagnosis of cobalamin deficiency. We tested the utility of holoTC in assessing vitamin B 12 status. Methods : We measured concentrations of holoTC and methylmalonic acid (MMA) in 1018 serum samples that were referred to our laboratory for total cobalamin testing. Results : Concentrations of MMA were lower in females compared to males and this difference was no more significant after adjusting for serum creatinine. Moreover, age was associated with higher concentrations of serum MMA, higher holoTC and slightly higher concentrations of total cobalamin. Higher concentrations of serum creatinine were associated with higher concentrations of MMA and holoTC. However, no association between serum creatinine and total cobalamin was observed. Only subjects with normal serum creatinine showed a negative correlation between serum holoTC and MMA (r= –0.36, p<0.001). In subjects with MMA ≥300 nmol/L and holoTC ≤35 pmol/L, concentrations of total cobalamin were well within the normal range (median; 25th/75th percentiles=212; 171/272 pmol/L). Receiver operating characteristic (ROC) curve analysis displayed a higher sensitivity and specificity for holoTC compared with vitamin B 12 for detecting concentrations of MMA ≥300 nmol/L in individuals with normal renal function. Conclusions : Compared to total cobalamin, we observed a better performance of holoTC assay in detecting elevated concentrations of MMA in subjects with normal renal function. The majority of subjects with combined low holoTC and elevated MMA had normal concentrations of total cobalamin. HoloTC can be used as a first line parameter in detecting cobalamin deficiency. Clin Chem Lab Med 2007;45:1746–50.

Background : Evolutionary haptocorrin is the youngest of the cobalamin-binding proteins. It evolved by duplication of the intrinsic factor gene and has been identified in most mammals examined. Its ability to bind both cobalamin and analogues is well established, but apart from that, our knowledge concerning its function and its distribution in adult and foetal life is limited. In this study, we present data on the tissue expression of haptocorrin and on the relation between analogues on haptocorrin and vitamin B 12 status in humans. Methods : Polyclonal antibodies towards haptocorrin were used to study the localisation in foetal and adult tissues by immunohistochemistry. Positive immunoreactions were primarily observed in exocrine glands, the gastrointestinal tract and the respiratory system. ELISA was used for measurement of holo- and total haptocorrin in blood samples from individuals diagnosed with vitamin B 12 deficiency, based on measurement of methylmalonic acid (μmol/L) as evident (>0.75, n=61), suspected (0.29–0.75, n=155) or not present (<0.29, n=170). Cobalamins and holotranscobalamin were measured in the same individuals. Results : Holohaptocorrin was considerably higher than holohaptocorrin-cobalamins (cobalamins minus holotranscobalamin). The median (25th–75th percentile, pmol/L) for holohaptocorrin analogues (holohaptocorrin minus holohaptocorrin-cobalamins) was higher in deficient [200 (130–240)] compared to the non-deficient [140 (80–200)] individuals (analysis of variance and Tukey's multiple comparison test, p<0.01). Conclusions : Our results indicate that haptocorrin is widely distributed also in foetal tissues and suggest analogues to accumulate on haptocorrin in vitamin B 12 -deficient individuals, a result that warrants further studies employing methods directly measuring cobalamins and analogues attached to haptocorrin. Clin Chem Lab Med 2007;45:1751–9.

Background : Impairments in folate-mediated one-carbon metabolism are associated with pathologies and developmental anomalies, including cardiovascular disease, cancer, neurological disorders and neural tube defects. The mechanisms that detail the role of folate and one-carbon metabolism in these disorders remain to be established. Folate deficiency impairs folate-dependent thymidylate biosynthesis resulting in depleted dTTP levels, increased rates of uracil incorporation into DNA and genomic instability. Folate-dependent enzymes involved in the de novo thymidylate pathway include cytoplasmic serine hydroxymethyltransferase (cSHMT), thymidylate synthase (TS) and dihydrofolate reductase (DHFR). Previously, we demonstrated that cSHMT-derived folate activated one-carbon units are preferentially incorporated into thymidylate, and we provided evidence that this was achieved through modification with small ubiquitin-like modifier (SUMO) enabling SUMO-dependent nuclear localization of cSHMT during S-phase. Methods and results : Here, we provide evidence that TS and DHFR are also substrates for UBC9-catalyzed SUMOylation in vitro by SUMO-1. Conclusions : The SUMOylation of cSHMT, TS and DHFR provides a mechanism by which all three enzymes in the thymidylate synthesis pathway are directed and compartmentalized in the nucleus. Clin Chem Lab Med 2007;45:1760–3.

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.

Background : Hyperhomocysteinemia (HHcy) is an independent risk factor of cardiovascular diseases. Extracellular signal-regulated kinase-1/2 (ERK-1/2) and the JAK/STAT pathway kinase, signal transducer and activator of transcription 3 (STAT3), are involved in matrix metalloproteinase-9 (MMP-9) induction and matrix remodeling. However, their role in homocysteine (Hcy)-mediated MMP-9 induction and matrix remodeling is unclear. Clinical and experimental evidence indicates that HHcy and activation of the renin-angiotensin system, mediated by angiotensin II type 1 (AT1) receptor, are involved in a variety of vascular pathologies. Despite this fact, the relationship between HHcy and activation of the renin-angiotensin system has not been comprehensively characterized. Therefore, we hypothesized that Hcy activates AT1 receptor that potentiates STAT3 via ERK-1/2 phosphorylation. STAT3 modulates target MMP-9 and collagen, resulting in vascular remodeling. Methods : Mouse aortic endothelial cells (MAEC) were treated with various doses of Hcy for different time periods. The levels of AT1 receptor, ERK-1/2, STAT3, MMP-9 and collagen type-1 were measured by immunoblot analyses. The activation of ERK-1/2 and STAT3 were determined by measuring ERK-1/2 phosphorylation and phosphoserine (727) STAT3. Results : Although Hcy dose-dependently induced AT1 receptor expression in the endothelial cells, a significant induction was observed at 100 μM at 48 h. We investigated Hcy-induced ERK-1/2 and STAT3 phosphorylation through AT1 receptor induction, and our results suggest that Hcy activated AT1 receptor which led to ERK-1/2 and STAT3 phosphorylation. In addition, findings of this study suggest that Hcy-mediated STAT3 activation regulated MMP-9 and collagen type-1. However, AT1 receptor blocker, valsartan, and the specific STAT3 inhibitor peptide attenuated MMP-9 and collagen type-1 induction. Conclusions : These findings demonstrate for the first time the contribution of AT1 receptor in HHcy-induced atherosclerotic diseases; Hcy-induced activation of AT1 receptor involves MMP-9 and collagen type-1 modulation using ERK-1/2 and STAT3 signaling cascades. Clin Chem Lab Med 2007;45:1771–6.

Background : γ-Aminobutyric acid (GABA) is a known inhibitory neurotransmitter in the mammalian central nervous system, and homocysteine (Hcy) behaves as an antagonist for GABA A receptor. Although the properties and functions of GABA A receptors are well studied in mouse neural tissue, its presence and significance in non-neural tissue remains obscure. The aim of the present study was to examine the expression of GABA A receptor and its subunits in non-neural tissue. Methods : The mice were analyzed. The presence of GABA A receptor and its subunits was evaluated using Western blot and reverse transcription polymerase chain reaction. Results : We report that GABA A receptor protein is abundant in the renal medulla, cortex, heart, left ventricle, aorta and pancreas. Low levels of GABA A receptor protein were detected in the atria of the heart, right ventricle, lung and stomach. The mRNA protein expression of GABA A receptor subunit shows that α1, β1, β3 and γ1 subunits are present only in brain. The mRNA protein expression levels of GABA A receptor α2, α6, β2 and γ3 subunits were highly expressed in brain compared to other tested tissue, while GABA A receptor γ2 subunit was expressed only in brain and kidney. Treatment of microvascular endothelial cells with Hcy decreased GABA A receptor protein level, which was restored to its baseline level in the presence of GABA A receptor agonist, muscimol. The distribution of GABA A and GABA B receptors in wild type mice was determined and tissue-specific expression patterns were found showing that several receptor subtypes were also expressed in the central nervous system. Conclusions : Hcy, a GABA A agonist, was found to decrease GABA A expression levels. These data enlarge knowledge on distribution of GABA receptors and give novel ideas of the effects of Hcy on different organs. Clin Chem Lab Med 2007;45:1777–84.

Background : Hyperhomocysteinemia is a new risk factor for osteoporosis. This study analyzed the effect of a homocysteine (HCY)-lowering treatment in osteoporotic individuals. Methods : Osteoporotic subjects (n=47, 55–82 years) were treated with either a combination of 2.5 mg folate, 0.5 mg vitamin B 12 and 25 mg vitamin B 6 or placebo. Bone mineral density (BMD) at lumbar spine and hip was measured at baseline and after 1 year. Urinary desoxypyridinoline cross-links (DPD) and plasma levels of tartrate resistant acid phosphatase (TRAP), C-terminal cross-links of collagen I (CTx), pro-collagen type I N-terminal peptide (PINP) and osteocalcin (OC) were measured after 0, 4, 8 and 12 months. Results : B-vitamin supplementation significantly reduced HCY (0 vs. 12 months: 13.6±4.8 vs. 8.9±2.4 μmol/L). Placebo treatment had no effect on HCY (0 vs. 12 months: 12.0±3.4 vs. 12.7±3.9μmol/L). BMD, TRAP, CTx, OC and PINP did not change throughout the study in both groups. Vitamin treatment decreased urinary DPD by –13% (p<0.01) after 8 and 12 months. In a sub-group analysis of hyperhomocysteinemic subjects (HCY>15 μmol/L, n=8), B-vitamin treatment tended to increase BMD at the lumbar spine, with a t-score from –2.7 to –1.7, and to decrease OC and PINP by approximately 50%. Conclusions : B-vitamin supplementation had no consistent effects on bone turnover or BMD. However, the situation may be different in patients with hyperhomocysteinemia. Clin Chem Lab Med 2007;45:1785–92.