Water soluble selenometabolome of Cardamine violifolia.

Low molecular weight selenium containing metabolites in the leaves of the selenium hyperaccumulator Cardamine violifolia (261 mg total Se per kg d.w.) were targeted in this study. One dimensional cation exchange chromatography coupled to ICP-MS was used for purification and fractionation purposes prior to LC-Unispray-QTOF-MS analysis. The search for selenium species in full scan spectra was assisted with an automated mass defect based filtering approach. Besides selenocystathionine, selenohomocystine and its polyselenide derivative, a total number of 35 water soluble selenium metabolites other than selenolanthionine were encountered, including 30 previously unreported compounds. High occurrence of selenium containing hexoses was observed, together with the first assignment of N-glycoside derivatives of selenolanthionine. Quantification of the most abundant selenium species, selenolanthionine, was carried out with an ion pairing LC - post column isotope dilution ICP-MS setup, which revealed that this selenoamino acid accounted for 30% of the total selenium content of the leaf (78 mg (as Se) per kg d.w.).

A large-scale analysis of targeted metabolomics data from heterogeneous biological samples provides insights into metabolite dynamics.

INTRODUCTION: We previously developed a tandem mass spectrometry-based label-free targeted metabolomics analysis framework coupled to two distinct chromatographic methods, reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), with dynamic multiple reaction monitoring (dMRM) for simultaneous detection of over 200 metabolites to study core metabolic pathways. OBJECTIVES: We aim to analyze a large-scale heterogeneous data compendium generated from our LC-MS/MS platform with both RPLC and HILIC methods to systematically assess measurement quality in biological replicate groups and to investigate metabolite abundance changes and patterns across different biological conditions. METHODS: Our metabolomics framework was applied in a wide range of experimental systems including cancer cell lines, tumors, extracellular media, primary cells, immune cells, organoids, organs (e.g. pancreata), tissues, and sera from human and mice. We also developed computational and statistical analysis pipelines, which include hierarchical clustering, replicate-group CV analysis, correlation analysis, and case-control paired analysis. RESULTS: We generated a compendium of 42 heterogeneous deidentified datasets with 635 samples using both RPLC and HILIC methods. There exist metabolite signatures that correspond to various phenotypes of the heterogeneous datasets, involved in several metabolic pathways. The RPLC method shows overall better reproducibility than the HILIC method for most metabolites including polar amino acids. Correlation analysis reveals high confidence metabolites irrespective of experimental systems such as methionine, phenylalanine, and taurine. We also identify homocystine, reduced glutathione, and phosphoenolpyruvic acid as highly dynamic metabolites across all case-control paired samples. CONCLUSIONS: Our study is expected to serve as a resource and a reference point for a systematic analysis of label-free LC-MS/MS targeted metabolomics data in both RPLC and HILIC methods with dMRM.

Mapping of selenium metabolic pathway in yeast by liquid chromatography-Orbitrap mass spectrometry.

A high-resolution mass spectrometric detection method is described for the identification of key metabolites in the selenium pathway in selenium enriched yeast. Iodoacetic acid (IAA) was used as the derivatizing reagent to stabilize the selenols. Oxidized forms of selenocysteine (Se-Cys), selenohomocystine (Se-HCys), selenoglutathione (Se-GSH), seleno-γ-glutamyl-cysteine (Se-Glu-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (Se-DOP-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (Se-DOP-HCys), selenomethionine (SeMet), seleno-S-adenosyl-homocysteine (Se-AdoHcy), the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (GSH-Se-DOP-Cys), and the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (GSH-Se-DOP-HCys) were found in the selenium enriched yeast certified reference material (SELM-1). Selenols were also derivatized with a mercury tag, p-hydroxymercurybenzoate (PHMB). The selenol-PHMB complexes showed the overlapped isotopic patterns of selenium and mercury, which provided supporting information for the identification of selenols. Both methods showed good agreement (<4 ppm difference) between the theoretical masses of the target compounds and the measured masses in the yeast matrix. The method using IAA as the derivatizing reagent was used to study the response of Saccharomyces cerevisiae to three forms of selenium, Se-Met, Na(2)SeO(3) (Se(IV)), and Na(2)SeO(4)·10H(2)O (Se(VI)) (concentration of Se: 100 mg/L). The production of selenocompounds observed over a 6 h period was high in the Se-Met treated group compared to the groups treated with Se(IV) and Se(VI).

Experimental hyperhomocysteinaemia: differences in tissue metabolites between homocystine and methionine feeding in a rat model.

AIM: Hyperhomocysteinaemia, diagnosed by serum levels, is regarded as an independent risk indicator for cardiovascular events and is associated with various diseases. The pathomechanisms seem to be partly due to concentrations of homocysteine metabolites and their effect on the cellular transmethylation processes. METHODS: We compared two common models for experimental hyperhomocysteinaemia - high methionine diet and homocystine-enriched diet - regarding their effects on tissue concentrations of homocysteine metabolites. RESULTS: Both diets induced hyperhomocysteinaemia without affecting renal function or vitamine status. However, the tissue contents of homocysteine and its precursors S-adenosyl-homocysteine (SAH) and S-adenosyl-methionine exhibited major differences between both models. Transmethylation potential was elevated 1.7-fold in liver of rats fed the methionine diet, whereas it was unaltered after homocystine-enriched diet. Kidneys of rats fed the methionine diet did not show any alterations in tissue content of homocysteine and its precursors, whereas in the homocystine group homocysteine and the transmethylation inhibitor SAH were elevated from 23.1 +/- 10.4 to 78.0 +/- 26.0 nmol g(-1) and from 106 +/- 4 to 170 +/- 22 nmol g(-1) respectively. Homocysteine tissue content was elevated in the homocystine, but not in the methionine group. CONCLUSIONS: Alterations to homocysteine metabolism are distinct in both models. These findings may explain divergent results, which have been published for these models of hyperhomocysteinaemia and which have resulted in controversial discussions in the past.

Oxidative DNA damage and level of thiols as related to polymorphisms of MTHFR, MTR, MTHFD1 in Alzheimer's and Parkinson's diseases.

Neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are accompanied by increased levels of 8-oxo-2'-deoxyguanosine (8-oxo2dG) and alterations in levels of homocysteine (Hcy), methionine (Met), and cysteine (Cys). Hcy may undergo remethylation due to involvement of MTHFR, MTR and MTHFD1 proteins. Present studies are aimed at determination of 8-oxo2dG, Hcy, Met, and Cys in AD and PD patients as well as in control groups, using HPLC/EC/UV, as well as estimation, by restriction analysis, frequency of following gene polymorphisms: MTHFR (C677T, A1298C, G1793A), MTHFD1 (G1958A), and MTR (A2756G). In AD there were significant differences of the levels of only Cys (GG, MTHFR, G1793A) and Met/Hcy (AA, MTHFD1, G1958A) whereas in PD there were more significant differences of the levels of thiols: Hcy [MTHFR: CT (C677T) and GG (G1793A); MTR, AG (A2756G)], Met [MTR, AA (A2756G)], Cys [MTR, AG (A2756G)], and Met/Hcy [MTHFR: CC, CT (C677T) and AA (A1298C), and GG (G1793A); MTHFD1 AA(G1958A); MTR AA(A2756G)]. Significant differences in the levels of Cys/Hcy, MTHFD1 GA (G1958) were varied between AD and PD groups. The results indicate that of the enzymes studied only polymorphisms of folate-dependent enzyme MTHFD1 have pointed to significant differences in intensity of turnover of circulating thiols between AD and PD patients.

Dynamic regulation of MTHFR mRNA expression and C677T genotype modulate mortality in coronary artery disease patients after revascularization.

INTRODUCTION: A large body of evidence links plasma homocysteine (Hcy) concentrations and cardiovascular disease. A common MTHFR polymorphism (C677T) leads to a variant with reduced activity and associated with increased Hcy levels. Coronary surgery precipitates a significant and sustained increase in the blood concentrations of Hcy and elevated levels of plasma Hcy have been associated to saphenous vein (SV) graft disease after CABG. However, the effects of MTHFR genotypes in the incidence of cardiovascular events after CABG have not been investigated prospectively. Here, we investigate whether MTHFR gene variants are associated with an increased cardiovascular risk in individuals submitted to CABG. We also propose a molecular mechanism to explain our findings. METHODS: We performed MTHFR C677T genotypes in 558 patients with two or three vessel-disease and normal left ventricular function prospectively followed in the MASS II Trial, a randomized study to compare treatments for multivessel CAD and preserved left ventricle function. Follow-up time was 5 years. Survival curves were calculated with the Kaplan-Meier method, and evaluated with the log-rank statistic. We assessed the relationship between baseline variables and the composite end-point of death, myocardial infarction and refractory angina using a Cox proportional hazards survival model. Finally, using an ex-vivo organ culture we have reproduced the arterialization of SV implants by culturing human SV either under venous hemodynamic condition (flow: 5 mL/min; no pressure) or arterial hemodynamic condition (flow: 50 mL/min; pressure: 80 mm Hg) for 1 day. MTHFR gene expression was quantified by real time RT-PCR in 15 SV from different individuals in both experimental conditions. RESULTS: There were no significant differences among individuals within each genotype group for baseline clinical characteristics. A statistically significant association between the TT genotype, associated with increased serum levels of Hcy, and cardiovascular mortality after 5 years was verified (p=0.007) in individuals submitted to CABG surgery. In addition, MTHFR TT genotype was still significantly associated with a 4.4 fold increased risk in cardiovascular outcomes (p=0.01) even after adjustment of a Cox multivariate model for age, sex, hypertension, diabetes, LDL, HDL, triglycerides, and number of diseased vessels in this population. Finally, a significant reduction in MTHFR gene expression was demonstrated in human SV when submitted to an arterial hemodynamic condition (p=0.02). CONCLUSIONS: There is a dynamic regulation of MTHFR gene expression during the arterialization process of human saphenous vein grafts resulting in lower levels of gene expression when in an arterial hemodynamic condition. In addition, the C677T MTHFR functional variant is associated with a worse outcome in individuals submitted to CABG. Taken together, these data suggest an important role of Hcy metabolism in individuals after CABG.

The organotellurium compound ammonium trichloro(dioxoethylene-o,o')tellurate reacts with homocysteine to form homocystine and decreases homocysteine levels in hyperhomocysteinemic mice.

Ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) is an organotellurium compound with pleiotropic functions that has been associated with antitumoral, immunomodulatory and antineurodegenerative activities. Tellurium compounds with a +4 oxidation state, such as AS101, react uniquely with thiols, forming disulfide molecules. In light of this, we tested whether AS101 can react with the amino acid homocysteine both in vitro and in vivo. AS101 conferred protection against homocysteine-induced apoptosis of HL-60 cells. The protective mechanism of AS101 against homocysteine toxicity was directly mediated by its chemical reactivity, whereby AS101 reacted with homocysteine to form homocystine, the less toxic disulfide form of homocysteine. Moreover, AS101 was shown here to reduce the levels of total homocysteine in an in vivo model of hyperhomocysteinemia. As a result, AS101 also prevented sperm cells from undergoing homocysteine-induced DNA fragmentation. Taken together, our results suggest that the organotellurium compound AS101 may be of clinical value in reducing total circulatory homocysteine levels.

Coronary calcification, homocysteine, C-reactive protein and the metabolic syndrome in Type 2 diabetes: the Prospective Evaluation of Diabetic Ischaemic Heart Disease by Coronary Tomography (PREDICT) Study.

AIMS: The PREDICT Study aims to determine: (i) the association between cardiovascular risk factors and coronary artery calcification score (CACS) obtained by electron beam tomography and (ii) the predictive value of CACS for coronary heart disease (CHD) events in Type 2 diabetes. METHODS: Having previously reported relationships between CACS and conventional risk factors, we have now studied the novel risk factors, plasma high-sensitivity C-reactive protein (CRP) and homocysteine, insulin resistance, serum apoprotein A1 and B concentrations, the serum triglyceride/high-density lipoprotein cholesterol ratio and metabolic syndrome (International Diabetes Federation definition) in 573 subjects of the PREDICT Type 2 diabetes cohort. RESULTS: In univariate analyses, the only significant positive novel correlate of CACS was homocysteine (P = 0.0004). CRP was increased in those with detectable calcification, but decreased with increasing calcification score (P = 0.006). In a multivariate model that included all significant univariate correlates, CACS was independently associated with age (P < 0.0001), waist-hip ratio (P < 0.02), male gender (P < 0.05) and duration of diabetes (P < 0.05), but the association with homocysteine was no longer significant. The negative association between CACS and CRP remained in multivariate analysis, and was independent of statin use. CONCLUSIONS: Age was the major factor influencing CACS in Type 2 diabetes, with weaker contributions from waist hip-ratio and duration of diabetes. Other novel cardiovascular risk factors appear to have little positive effect.

Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs.

Methionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis, i.e. methylation of homocysteine. A search of the Schizosaccharomyces pombe genomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, and thus the gene was identified as a methionine synthase and designated met26. The met26 mutant was found to exhibit a remarkable growth defect in the absence of adenine even in medium supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeast Saccharomyces cerevisiae, which has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes from Sac. cerevisiae constituting the cystathionine pathway (CYS4 and CYS3) into Sch. pombe Deltamet26 cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in Deltamet26 cells was higher than in other methionine auxotrophs and that introduction of the Sac. cerevisiae cystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in purine biosynthesis in the met26 mutant.

Accumulation of homolanthionine and activation of a novel pathway for isoleucine biosynthesis in Corynebacterium glutamicum McbR deletion strains.

In the present work, the metabolic consequences of the deletion of the methionine and cysteine biosynthesis repressor protein (McbR) in Corynebacterium glutamicum, which releases almost all enzymes of methionine biosynthesis and sulfate assimilation from transcriptional regulation (D. A. Rey, A. Pühler, and J. Kalinowski, J. Biotechnol. 103:51-65, 2003), were studied. C. glutamicum ATCC 13032 DeltamcbR showed no overproduction of methionine. Metabolome analysis revealed drastic accumulation of a single metabolite, which was not present in the wild type. It was identified by isotopic labeling studies and gas chromatography/mass spectrometry as L-homolanthionine {S-[(3S)-3-amino-3-carboxypropyl]-L-homocysteine}. The accumulation of homolanthionine to an intracellular concentration of 130 mM in the DeltamcbR strain was accompanied by an elevated intracellular homocysteine level. It was shown that cystathionine-gamma-synthase (MetB) produced homolanthionine as a side reaction. MetB showed higher substrate affinity for cysteine (Km = 260 microM) than for homocysteine (Km = 540 microM). The cell is able to cleave homolanthionine at low rates via cystathionine-beta-lyase (MetC). This cleavage opens a novel threonine-independent pathway for isoleucine biosynthesis via 2-oxobutanoate formed by MetC. In fact, the deletion mutant exhibited an increased intracellular isoleucine level. Metabolic flux analysis of C. glutamicum DeltamcbR revealed that only 24% of the O-acetylhomoserine at the entry of the methionine pathway is utilized for methionine biosynthesis; the dominating fraction is either stored as homolanthionine or redirected towards the formation of isoleucine. Deletion of metB completely prevents homolanthionine accumulation, which is regarded as an important step in the development of C. glutamicum strains for biotechnological methionine production.

2 minute non-invasive screening for cardio-vascular diseases: relative limitation of C-Reactive Protein compared with more sensitive L-Homocystine as cardio-vascular risk factors; safe and effective treatment using the selective drug uptake enhancement method.

Contrary to the present practice of measurement of cardio-vascular risk factors or inflammatory risk factors such as C-Reactive Protein (CRP) from a blood sample from the vein of one arm, by using the Bi-Digital O-Ring Test Resonance Phenomena between 2 identical substances, one can non-invasively detect the approximate location on the body of abnormally increased risk factors in just 2 minutes, by detecting the resonance with L-Homocystine, even when blood CRP failed to detect any abnormality. This is performed by projecting a 0.5 to approximately 5mW red spectral laser beam with 560-670nm wavelength, to at least 6 standard parts of the body, when one of the control risk markers placed next to the laser beam also exists in the part of the body tested. It is generally believed that CRP is increased in the presence of acute myocardial infarct, chronic rheumatoid arthritis, ulcerative colitis, metabolic abnormalities such as often detected in diabetes, inflammation and underlying infection of the cardio-vascular system, and in some cancers. However, in our study, when the clinical significance of CRP and L-Homocystine was compared, we found that CRP often was not increased when there was extensive infection of Mycobacterium Tuberculosis as well as asymptomatic infection by Cytomegalovirus, Herpes Simplex Virus Type I, Human Herpes Virus Type 6, Borrelia Burgdorferi, or Chlamydia Trachomatis in the heart (and other parts of the body), particularly when there was liver cell dysfunction such as an increase in ALT. In contrast, L-Homocystine was often increased in the presence of localized infections of the heart and other parts of the body. For screening of Cardio-Vascular diseases by this method, 0.5mg of L-Homocystine as a control marker was found to be the most sensitive and reliable, compared with most effective amount of CRP, 0.5ng, for detecting early Cardio-Vascular problems due to various localized infections. About 0.5ng of cardiac Troponin T and cardiac Troponin I were also useful for detecting early stages of heart disease but they are not as sensitive as L-Homocystine. Once the pathogenic factors were identified, the effective medication was given, and the Selective Drug Uptake Enhancement Method (originally discovered by the first author in 1990) was applied after the effective drug was administered, to selectively deliver the medication to the pathological area, while reducing drug uptake to the normal parts of the body. As a result, the therapeutic effect was markedly accelerated.

[Homocysteine and hyperhomocysteinemia]. / Homocysteina i hiperhomocysteinemia.

This paper reviews the biochemical and physiological data underlying hyperhomocysteinemia. Elevation of plasma homocysteine arises from disrupted of its metabolism (remethylation to methionine and transsulfuration to cystathionine) and is a function of a complex interaction between multiple genetic and environmental factors. Hyperhomocysteinemia, a conditions that recent epidemiological studies have shown to be associated with an increased risk of vascular diseases, may be a equal importance to hypercholesterolemia, hypertension and smoking in etiology of atherosclerosis. Based on the role of folic acid, vitamin B12 and B6 in homocysteine metabolism supplementation with these vitamins in treatment of hyperhomocysteinemia both in cardiovascular diseases and neural tube defects is reviewed.

Homocyst(e)ine, atherosclerosis, and thrombosis.

Ample clinical and epidemiologic evidence exists to implicate homocyst(e)ine as a risk factor for atherosclerotic vascular disease and thrombosis. The precise mechanisms by which this occurs are uncertain but probably involve injury to endothelium, impairment of endothelial function, lipid peroxidation, oxidation of low-density lipoprotein, and creation of a prothrombotic environment in areas of endothelial injury. Plasma homocyst(e)ine concentration (PHC) can be effectively reduced with oral administration of folic acid. Whether vitamins B6 and B12 are also required in the absence of vitamin deficiency remains uncertain. Studies currently in progress may help to determine whether reduction of PHC will translate into a decrease in clinical vascular events.

Post- and prenatal diagnostic methods for the homocystinurias.

Diagnosis of the homozygous homocystinurias can be performed by investigations at the metabolite, enzyme and DNA level. The existence of variant forms due to the wide range of genetic variation may result in only small differences in various parameters between controls and affected subjects. 1. Sulphur amino acid concentrations in plasma, especially total homocysteine, are useful in first line diagnostic investigations. 2. Cystathionine-beta-synthase (CBS), methylenetetrahydrofolate reductase (MTHFR) and methylfolate homocysteine methyltransferase (MFMT) can be directly assayed in many tissues including fibroblasts (each) and blood cells (except CBS). Indirect whole cell assays which measure pathway activity dependent on a particular enzyme can provide useful diagnostic information. 3. Direct analysis of mutations is available for CBS, MTHFR and recently also for MFMT deficiencies. However the existence of a larger number of very rare, often private, mutations limits the usefulness of this approach in routine diagnosis. The above diagnostic approaches can generally be applied to prenatal diagnosis. Measurement of methylmalonic acid and other metabolites in amniotic fluid by stable isotope dilution / gas chromatography-mass spectrometry is well established for the methylmalonic acidurias. This method has also been applied to combined homocystinuria/methylmalonic aciduria supported by enzyme assays in cultured cells. Total homocysteine measurement in cell free amniotic fluid is also possible, performed so far in 14 cases with two affected fetuses. The indirect assay of methionine formation from [14C] labelled formate in intact cultured amniotic fluid cells has been for prenatal diagnosis of the remethylation defects.

Reduction-oxidation (Redox) and vascular tissue level of homocyst(e)ine in human coronary atherosclerotic lesions and role in extracellular matrix remodeling and vascular tone.

Hyperhomocyst(e)inemia in patients with coronary and peripheral arterial occlusion has been demonstrated by others. Redox-state of homocyst(e)ine causes dysfunction of endothelial cells and promote growth of vascular smooth muscle cells. The role of tissue, protein bound and unbound, oxidative mixed disulfides in the development of fibrous plaque in atherosclerotic lesion is not known. Redox-state around the fibroblasts and vascular smooth muscle cells modulates the expression of extracellular matrix (ECM) components (Tyagi et al. 1996, J Cell Biochem, 61: 139-151). To determine the role of tissue homocystine in fibrotic atherosclerotic plaque development, coronary arteries were isolated from ischemic explanted hearts (n = 10). Apparently normal vascular tissue was obtained from idiopathic cardiomyopathic explanted hearts (n = 10). Tissue extract were prepared from atherosclerotic lesions and from normal arteries devoid of adventitia. Interaction of homocystine with Ellman's reagent (5, 5'-dithio-bis-2-nitro benzoic acid) catalyzed by limiting amount of reducing agent (catalyst) generated change in optical density (OD) at 412 nm in dose dependent fashion. We have generated a standard curve between change at 412 nm and amount of homocystine. The change in OD at 412 nm with increasing amount (0-25 microg) of homocystine demonstrated linearity. The protein-bound oxidized disulfides were precipitated by trichloroacetic acid (TCA) and free-oxidative disulfides in the supernatant were collected. The pathophysiological amount of protein-bound disulfide in atherosclerotic tissue (1.0 +/- 0.2 microg/mg total protein) was 10 times that in normal tissue (0.1 +/- 0.01 microg/mg, p < 0.001). The amount of free oxidative disulfide in atherosclerotic tissue (1.5 +/- 0.3 microg/mg) was 15 times that in normal tissue (0.12 +/- 0.02 microg/mg, p < 0.001). To determine the role of homocystine in ECM expression, ECM collagenase activity in the presence and absence of homocystine was measured by zymography. The effect of homocysteine on collagenase activity was biphasic, increased at < [0.01 mM] and inhibited at > [0.1 mM]. To determine whether homocystine regulates vascular tone, isometric measurements were carried out using normal coronary rings. Results suggested that homocystine induced endothelial-modulated vasoconstriction in coronary vessels. Tissue oxidative disulfides and the homocystine may contribute to the development of fibrotic atherosclerotic lesions and vascular dysfunction.

[Homocystinuria--biochemical, clinical and genetic aspects]. / Homocistinurija--biohemijski, klinicki i genetski aspekti.

Homocystinuria is a rare autosomal recessive disease characterized by homocystinuria and multisystemic clinical disorders. The term denotes a biochemical abnormality of methionine metabolism caused both by transsulfuration pathway disorders and remethylation of homocysteine into methionine, and as such it can be a result of numerous specific and different genetic lesions. Homocystinuria is most commonly caused by deficiency of cystathionine beta-synthase (CBS) activity (EC 4.2.1.22). In this lesion, depending on specific characteristics of mutant enzyme molecules, in regard to existence of residual activity, responsive and nonresponsive homocystinuria can be differed regarding clinical response to high doses of pyridoxine. Although there are numerous different clinical abnormalities, changes on four organ systems are dominant. The most common symptoms of homocystinuria include lens dislocation, vascular disorders, skeletal abnormalities and mental retardation. Laboratory findings are the first diagnostic procedure, while determination of enzymatic activity is a direct parameter for making diagnosis. Prenatal diagnosis and early detection are extremely important for the course and prognosis of the disease as they enable application of currently available therapy as soon as possible. The presently available therapy can, only in such cases, prevent occurrence of serious clinical symptoms, prevent their advancement to some extent or improve reversible clinical manifestations.

Measurement of homocyst(e)ine in the prediction of arteriosclerosis.

Homocyst(e)ine [H(e)], the sum of homocysteine, homocystine, and the homocysteine-cysteine mixed disulfide, free and protein-bound, has been shown to be associated in retrospective case control studies, and in one prospective study, with vascular disease, including coronary artery disease (CAD), cerebrovascular disease, and peripheral vascular disease. Elevated levels of homocyst(e)ine severe enough to cause homocystinuria are seen in severe nutritional deficiencies of vitamin B12, folic acid and vitamin B6. Rare genetic disorders of vitamin B12 synthesis of 5'-10'-methylene tetrahydrofolate reductase, or the pyridoxal phosphate-dependent enzyme cystathionine beta-synthase may cause severe hyperhomocyst(e)inemia and homocystinuria. The clinical manifestation of these disorders are mental retardation, neurological disorders, and widespread thromboembolic phenomena. The measurement of H(e) is currently performed using high-pressure liquid chromatography with fluorescence detection. Other methods, especially mass spectroscopy, are also used. Internal standards using increasing concentrations of homocystine and acetylcysteine and several external standards are used to ensure accuracy of the assay. Milder elevations of H(e) have recently been associated with vascular disease, in both men and women. The strength of this association appears to be stronger for peripheral and cerebrovascular disease than for CAD. Nevertheless, several case control studies in Europe, Canada, and the United States have shown that H(e) levels are elevated in CAD patients compared with controls, and H(e) levels are independent of the conventional cardiovascular risk factors (age, gender, lipid and lipoprotein cholesterol levels, hypertension, or cigarette smoking). One prospective study, the Physicians' Health Study, has shown that H(e) levels are slightly but significantly higher in CAD cases vs controls in a population of US physicians.(ABSTRACT TRUNCATED AT 250 WORDS)