Elastin modulation and modification by homocysteine: a key factor in the pathogenesis of Pseudoexfoliation syndrome?

BACKGROUND: Pseudoexfoliation syndrome (PXF) is an idiopathic, elastogenesis-associated systemic disease characterised by amyloid-like material aggregates in the eye. Elevated plasma and aqueous humour (aqH) homocysteine (Hcy) is reportedly associated with PXF. This study is aimed to probe Hcy-mediated alterations in elastin expression. METHODOLOGY: Lens level of Hcy (total Hcy (tHcy)), mRNA expression of Eln, CBS and MTR in lens capsule, protein expression of elastin in aqH were estimated by enzyme immunoassay, quantitative PCR and western blot, respectively in PXF, PXF with glaucoma (PXF-G) cases, in comparison with cataract-alone disease controls. Human lens epithelial cells (hLECs) were exposed to Hcy and homocysteine thiolactone (HCTL) to evaluate elastin expression in vitro. Furthermore, elastin recombinant protein was incubated with Hcy and HCTL to assess secondary and tertiary structural modifications based on circular dichroism spectroscopy, spectrophotometric and SEM studies. RESULTS: The lens tHcy was significantly high in PXF (p=0.02) and PXF-G (p=0.009). Eln expression was elevated in PXF and PXF-G (p=0.0007). Elastin level in aqH was elevated in PXF (p=0.01) and PXF-G (p=0.002). Hcy (200 µM) and HCTL (1 µM) promoted elastin expression at mRNA level by 36-fold (p=0.02) and 10-fold (p=0.05), respectively, and at protein level by nearly two-fold in cultured hLECs. Secondary structure changes in elastin protein caused by Hcy were evident from 34.11% drop in α-helix and 6.17% gain in ß-sheet. Fluorescence, spectral assays and SEM analyses showed aggregation and amyloid formation of elastin with homocysteinylation. CONCLUSION: The study reveals that lens accumulation of Hcy associated with hyperhomocysteinaemia is characteristic of PXF that augments elastin expression. Hcy causes structural changes promoting elastin aggregation, thereby contributing to defective elastin in PXF and PXF-G.

Aldosterone modulates blood homocysteine and cholesterol in coronary artery disease patients - a possible impact on atherothrombosis?

Aldosterone plays a key role in maintaining the homeostasis of the whole organism. Under some circumstances, aldosterone can contribute to the progression of cardiovascular diseases, including coronary artery disease. This study demonstrates that aldosterone associates negatively with some lipidogram parameters and positively with the concentration of homocysteine. These associations are characteristic for coronary artery disease and are not present in control subjects. The findings also indicate that in vitro aldosterone stimulates homocysteine production by rat adrenal glands, which may explain the associations observed with coronary artery disease. Moreover, we have found that aldosterone significantly modulates in vitro platelet reactivity to arachidonate and collagen - aldosterone increases the pro-aggregatory action of collagen, but decreases the pro-aggregatory potential of arachidonate. Therefore, the findings of these in vitro and ex vivo experiments indicate the existence of new pathways by which aldosterone modulates lipid- homocysteine- and platelet-dependent atherogenesis.

Hyperhomocysteinemia and cardiovascular disease in animal model.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and is associated with primary causes of mortality and morbidity throughout the world. Several studies have been carried out to evaluate the effects of a diet inducing cystathionine-ß-synthase, methyltetrafolate, folic acid, and vitamin B supplemented with methionine on the homocysteine metabolism and in lowering the plasma total homocysteine levels. A large number of molecular and biomedical studies in numerous animals, such as mice, rabbits, and pigs, have sought to elevate the plasma total homocysteine levels and to identify a disease model for human hyperhomocysteinemia. However, a specific animal model is not suitable for hyperhomocysteinemia in terms of all aspects of cardiovascular disease. In this review article, the experimental progress of animal models with plasma total homocysteine levels is examined to identify a feasible animal model of hyperhomocysteinemia for different aspects.

Contribution of guanine nucleotide exchange factor Vav2 to NLRP3 inflammasome activation in mouse podocytes during hyperhomocysteinemia.

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) have been demonstrated to mediate the activation of NOD-like receptor protein 3 (NLRP3) inflammasomes in podocytes in response to elevated levels of homocysteine (Hcys). However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The present study tested whether the guanine nucleotide exchange factor Vav2 mediates Rac1-mediated NOX activation in response to elevated Hcys leading to NLRP3 inflammasome activation in podocytes and consequent glomerular injury. In a mouse model of hyperhomocysteinemia (hHcys), we found that mice with hHcys (on the FF diet) or oncoVav2 (a constitutively active form of Vav2) transfection in the kidney exhibited increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1 and elevated IL-1ß levels in glomeruli, indicating the formation and activation of the NLRP3 inflammasome. This glomerular NLRP3 inflammasome activation was accompanied by podocyte dysfunction and glomerular injury, even sclerosis. Local transfection of Vav2 shRNA plasmids significantly attenuated hHcys-induced NLRP3 inflammasome activation, podocyte injury, and glomerular sclerosis. In cultured podocytes, Hcys treatment and oncoVav2 transfection were also found to increase NLRP3 inflammasome formation and activation, which were all inhibited by Vav2 shRNA. Furthermore, Vav2 shRNA prevented Hcys-induced podocyte damage as shown by restoring Hcys-impaired VEGF secretion and podocin production. This inhibitory action of Vav2 shRNA on Hcys-induced podocyte injury was associated with reduction of Rac1 activity and ROS production. These results suggest that elevated Hcys levels activate Vav2 and thereby increase NOX activity leading to ROS production, which triggers NLRP3 inflammasome activation, podocyte dysfunction and glomerular injury.

Persulfide Formation Mediates Cysteine and Homocysteine Biosynthesis in Methanosarcina acetivorans.

The mechanisms of sulfur uptake and trafficking in methanogens inhabiting sulfidic environments are highly distinctive. In aerobes, sulfur transfers between proteins occur via persulfide relay, but direct evidence for persulfides in methanogens has been lacking. Here, we use mass spectrometry to analyze tryptic peptides of the Methanosarcina acetivorans SepCysS and MA1821 proteins purified anaerobically from methanogen cells. These enzymes insert sulfide into phosphoseryl(Sep)-tRNACys and aspartate semialdehyde, respectively, to form Cys-tRNACys and homocysteine. A high frequency of persulfidation at conserved cysteines of each protein was identified, while the substantial presence of persulfides in peptides from other cellular proteins suggests that this modification plays a general physiological role in the organism. Purified native SepCysS containing persulfide at conserved Cys260 generates Cys-tRNACys in anaerobic single-turnover reactions without exogenously added sulfur, directly linking active-site persulfide formation in vivo with catalytic activity.

Deep analyses of the associations of a series of biomarkers with insulin resistance, metabolic syndrome, and diabetes risk in nondiabetic middle-aged and elderly individuals: results from a Chinese community-based study.

OBJECTIVE: The current study was designed to perform deep analyses of the associations of biomarkers, including high-sensitivity C-reactive protein (hs-CRP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and homocysteine (Hcy), with insulin resistance (IR), metabolic syndrome (MetS), and diabetes risk and evaluate the abilities of biomarkers to identify IR, MetS, and diabetes risk in Chinese community-dwelling middle-aged and elderly residents. PARTICIPANTS AND METHODS: A total of 396 participants older than 45 years underwent physical examinations and laboratory analyses following standardized protocol. RESULTS: Serum hs-CRP concentrations were able to identify MetS, Chinese diabetes risk score (CDRS) ≥4, high-density lipoprotein-cholesterol (HDL-c) <0.9/1.0 mmol/L, and HDL-c <1.0/1.3 mmol/L (P<0.05 for all). Serum NT-proBNP concentrations were able to identify homeostasis model assessment of IR >1.5, CDRS ≥4, overweight, and blood pressure (BP) ≥140/90 mmHg (P<0.05 for all). Serum Hcy concentrations were able to identify CDRS ≥4, general obesity, overweight, and BP ≥140/90 mmHg (P<0.05 for all). Serum hs-CRP concentrations were independently associated with MetS as well as HDL-c <1.0/1.3 mmol/L and HDL-c <0.9/1.0 mmol/L (P<0.05 for all). Serum NT-proBNP concentrations were independently associated with BP ≥140/90 mmHg (P<0.05). Serum Hcy concentrations were independently associated with CDRS ≥4 (P<0.05). CONCLUSION: Serum HDL-c levels were the major determinant of the associations between serum hs-CRP levels and MetS and the key link between inflammation and MetS. There was no other association of these biomarkers with IR, MetS, and diabetes risk after full adjustment.

MTHFD1 regulates nuclear de novo thymidylate biosynthesis and genome stability.

Disruptions in folate-mediated one-carbon metabolism (FOCM) are associated with risk for several pathologies including developmental anomalies such as neural tube defects and congenital heart defects, diseases of aging including cognitive decline, neurodegeneration and epithelial cancers, and hematopoietic disorders including megaloblastic anemia. However, the causal pathways and mechanisms that underlie these pathologies remain unresolved. Because folate-dependent anabolic pathways are tightly interconnected and best described as a metabolic network, the identification of causal pathways and associated mechanisms of pathophysiology remains a major challenge in identifying the contribution of individual pathways to disease phenotypes. Investigations of genetic mouse models and human inborn errors of metabolism enable a more precise dissection of the pathways that constitute the FOCM network and enable elucidation of causal pathways associated with NTDs. In this overview, we summarize recent evidence that the enzyme MTHFD1 plays an essential role in FOCM in humans and in mice, and that it determines the partitioning of folate-activated one carbon units between the folate-dependent de novo thymidylate and homocysteine remethylation pathways through its regulated nuclear localization. We demonstrate that impairments in MTHFD1 activity compromise both homocysteine remethylation and de novo thymidylate biosynthesis, and provide evidence that MTHFD1-associated disruptions in de novo thymidylate biosynthesis lead to genome instability that may underlie folate-associated immunodeficiency and birth defects.

Autoantibodies against homocysteinylated protein in a mouse model of folate deficiency-induced neural tube defects.

BACKGROUND: Periconceptional supplementation with folic acid results in a significant reduction in the incidence of neural tube defects (NTDs). Nonetheless, NTDs remain a leading cause of perinatal morbidity and mortality worldwide, and the mechanism(s) by which folate exerts its protective effects are unknown. Homocysteine is an amino acid that accumulates under conditions of folate-deficiency, and is suggested as a risk factor for NTDs. One proposed mechanism of homocysteine toxicity is its accumulation into proteins in a process termed homocysteinylation. METHODS & RESULTS: Herein, we used a folate-deficient diet in pregnant mice to demonstrate that there is: (i) a significant inverse correlation between maternal serum folate levels and serum homocysteine; (ii) a significant positive correlation between serum homocysteine levels and titers of autoantibodies against homocysteinylated protein; and (iii) a significant increase in congenital malformations and NTDs in mice deficient in serum folate. Furthermore, in mice administered the folate-deplete diet before conception, supplementation with folic acid during the gestational period completely rescued the embryos from congenital defects, and resulted in homocysteinylated protein titers at term that are comparable to that of mice administered a folate-replete diet throughout both the pre- and postconception period. These results demonstrate that a low-folate diet that induces NTDs also increases protein homocysteinylation and the subsequent generation of autoantibodies against homocysteinylated proteins. CONCLUSION: These data support the hypotheses that homocysteinylation results in neo-self antigen formation under conditions of maternal folate deficiency, and that this process is reversible with folic acid supplementation.

Homocysteine is biosynthesized from aspartate semialdehyde and hydrogen sulfide in methanogenic archaea.

The biosynthetic route for homocysteine, intermediate in methionine biosynthesis, is unknown in some methanogenic archaea because homologues of the canonical required genes cannot be identified. Here we demonstrate that Methanocaldococcus jannaschii can biosynthesize homocysteine from aspartate semialdehyde and hydrogen sulfide. Additionally, we confirm the genes involved in this new pathway in Methanosarcina acetivorans. A possible series of reactions in which a thioaldehyde is formed and then reduced to a thiol are proposed. This represents a novel route for the biosynthesis of homocysteine and exemplifies unique aspects of sulfur chemistry occurring in prebiotic environments and in early life forms.

Rationally engineered variants of S-adenosylmethionine (SAM) synthase: reduced product inhibition and synthesis of artificial cofactor homologues.

S-Adenosylmethionine (SAM) synthase was engineered for biocatalytic production of SAM and long-chain analogues by rational re-design. Substitution of two conserved isoleucine residues extended the substrate spectrum of the enzyme to artificial S-alkylhomocysteines. The variants proved to be beneficial in preparative synthesis of SAM (and analogues) due to a much reduced product inhibition.

Role of homocysteinylation of ACE in endothelial dysfunction of arteries.

The direct impact of de novo synthesis of homocysteine (Hcy) and its reactive metabolites, Hcy-S-S-Hcy and Hcy thiolactone (HCTL), on vascular function has not been fully elucidated. We hypothesized that Hcy synthesized within endothelial cells affects activity of angiotensin-converting enzyme (ACE) by direct homocysteinylation of its amino- and/or sulfhydryl moieties. This covalent modification enhances ACE reactivity toward angiotensin II (ANG II)-NADPH oxidase-superoxide-dependent endothelial dysfunction. Mesenteric and coronary arteries isolated from normal rats were incubated for 3 days with or without exogenous methionine (Met, 0.1-0.3 mM), a precursor to Hcy. Incubation of arteries in Met-free media resulted in time-dependent decreases in vascular Hcy formation. By contrast, vessels incubated with Met produced Hcy in a dose-dependent manner. There was a notably greater de novo synthesis of Hcy from endothelial than from smooth muscle cells. Enhanced levels of Hcy production significantly impaired shear stress-induced dilation and release of nitric oxide, events that are associated with elevated production of vascular superoxide. Each of these processes was attenuated by ANG II type I receptor blocker or ACE and NADPH oxidase inhibitors. In addition, in vitro exposure of purified ACE to Hcy-S-S-Hcy/HCTL resulted in formation of homocysteinylated ACE and an enhanced ACE activity. The enhanced ACE activity was confirmed in isolated coronary and mesenteric arteries that had been exposed directly to Hcy-S-S-Hcy/HCTL or after Met incubation. In conclusion, vasculature-derived Hcy initiates endothelial dysfunction that, in part, may be mediated by ANG II-dependent activation of NADPH oxidase in association with homocysteinylation of ACE.

Suitability of methylmalonic acid and total homocysteine analysis in dried bloodspots.

Methylmalonic acid (MMA) and total homocysteine (tHCYS) concentrations are used to detect acquired and inborn errors of cobalamin (vitamin B12, Cbl) metabolism and to evaluate the effect of therapeutic interventions. Dried blood spot sampling offers a patient-friendly and easy alternative to plasma sampling. However, dried blood spot concentrations are not necessarily equal to plasma concentrations. Therefore, the objective of this work was to establish the relationship between MMA and tHYS dried blood spot and plasma concentrations to facilitate clinical implementation of dried blood spot sampling. MMA and tHCYS in both plasma and DBS were validated on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). While position of the punch (in DBS) did affect tHCYS concentration, no influence of hematocrit (Ht) and blood volume on both MMA and tHCYS concentrations was observed. The plasma assay performed better than the DBS assay by most criteria. However, the DBS matrix was superior for tHCYS stability. Paired plasma and DBS samples were obtained from patients suspected for Cbl deficiency and from patients with a known inborn error of metabolism affecting MMA or tHCYS concentration. Based on the strong correlation of tHCYS in both matrices (y=0.46±1.12 (r(2)=0.91)), determination of tHCYS in plasma can be replaced by tHCYS in DBS. However, for MMA, a correlation in the higher (pathological) range of MMA exist, but no correlation was observed in the lower ranges. Therefore the added value of MMA concentrations in DBS is currently unknown and should be further investigated.

An alternative mechanism for guanidinoacetic acid to affect methylation cycle.

Guanidinoacetic acid (also known as glycocyamine; GAA) is an endogenous substance which occurs in humans and plays a central role in the biosynthesis of creatine. The formation of creatine from GAA consumes methyl groups, and increases production of homocysteine. GAA may have the potential to stimulate insulin secretion. Insulin reduces plasma homocysteine and raises methyl group supply. It is possible that the ability of GAA to trigger the insulin secretion modulates methyl group metabolism, and comparatively counterbalance for the direct effect of GAA on increased methylation demand. Possible insulinotropic effect of GAA may contribute to total in vivo methylation demand during biotransformation.

Vitamin B-6 restriction reduces the production of hydrogen sulfide and its biomarkers by the transsulfuration pathway in cultured human hepatoma cells.

BACKGROUND: Pyridoxal 5'-phosphate (PLP) functions as a coenzyme in many cellular processes including one-carbon metabolism and the interconversion and catabolism of amino acids. PLP-dependent enzymes, cystathionine ß-synthase and cystathionine γ-lyase, function in transsulfuration but also have been implicated in the production of the endogenous gaseous signaling molecule hydrogen sulfide (H2S) concurrent with the formation of the biomarkers lanthionine and homolanthionine. OBJECTIVE: Our objective was to determine if H2S production and concurrent biomarker production is affected by vitamin B-6 restriction in a cell culture model. METHODS: We used cultured human hepatoma cells and evaluated static intracellular profiles of amino acids and in vivo kinetics of H2S biomarker formation. Cells were cultured for 6 wk in media containing concentrations of pyridoxal that represented severe vitamin B-6 deficiency (15 nmol/L pyridoxal), marginal deficiency (56 nmol/L pyridoxal), adequacy (210 nmol/L pyridoxal), and standard medium formulation providing a supraphysiologic pyridoxal concentration (1800 nmol/L pyridoxal). RESULTS: Intracellular concentrations of lanthionine and homolanthionine in cells cultured at 15 nmol/L pyridoxal were 50% lower (P < 0.002) and 47% lower (P < 0.0255), respectively, than observed in cells cultured at 1800 nmol/L pyridoxal. Extracellular homocysteine and cysteine were 58% and 46% higher, respectively, in severely deficient cells than in adequate cells (P < 0.002). Fractional synthesis rates of lanthionine (P < 0.01) and homolanthionine (P < 0.006) were lower at 15 and 56 nmol/L pyridoxal than at both higher pyridoxal concentrations. The rate of homocysteine remethylation and the fractional rate of homocysteine production from methionine were not affected by vitamin B-6 restriction. In vitro studies of cell lysates using direct measurement of H2S also had a reduced extent of H2S production in the 2 lower vitamin B-6 conditions. CONCLUSION: In view of the physiologic roles of H2S, these results suggest a mechanism that may be involved in the association between human vitamin B-6 inadequacy and its effects on human health.

Sinefungin, a natural nucleoside analogue of S-adenosylmethionine, inhibits Streptococcus pneumoniae biofilm growth.

Pneumococcal colonization and disease is often associated with biofilm formation, in which the bacteria exhibit elevated resistance both to antibiotics and to host defense systems, often resulting in infections that are persistent and difficult to treat. We evaluated the effect of sinefungin, a nucleoside analogue of S-adenosylmethionine, on pneumococcal in vitro biofilm formation and in vivo colonization. Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferation and structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms. Sinefungin significantly reduced pneumococcal colonization in rat middle ear. The quorum sensing molecule (autoinducer-2) production was significantly reduced by 92% in sinefungin treated samples. The luxS, pfs, and speE genes were downregulated in biofilms grown in the presence of sinefungin. This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonization in vivo, decreases AI-2 production, and downregulates luxS, pfs, and speE gene expressions. Therefore, the S-adenosylmethionine (SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci.

The effect of taurine on the relationship between NO, ADMA and homocysteine in endotoxin-mediated inflammation in HUVEC cultures.

The aim of our study was to investigate the effect of taurine on the relationship between nitric oxide (NO), asymmetric dimethylarginine (ADMA) and homocysteine (Hcy) in endotoxin-induced human umblical vein endothelial cell (HUVEC) cultures. For this reason, four groups were formed (n=12). Control group consists of HUVEC cultures without any treatment. Lipopolysaccharide (LPS) and LPS+taurine groups were treated with 10 µg/mL endotoxin, 5 µg/mL taurine and endotoxin+taurine (same doses), respectively. Nitrite/nitrate (NOx), ADMA and Hcy levels were measured. There was a significant increase of NOx, ADMA and Hcy in endotoxemia (p<0.05). Taurine treatment elevated NOx levels significantly (p<0.01) in taurine and LPS + taurine group compared to control group, while it reduced NOx levels compared to LPS group. In contrast, taurine decreased ADMA levels to the control level both in taurine and taurine+LPS group compared to LPS. Hcy levels increased significantly compared to taurine group (p<0.05) and did not change compared to LPS group. Taurine was effective on ADMA-NO relationship whereas no beneficial effect was observed in Hcy levels (p<0.05).

B-vitamins intake, DNA-methylation of One Carbon Metabolism and homocysteine pathway genes and myocardial infarction risk: the EPICOR study.

BACKGROUND AND AIMS: Several epidemiological studies highlighted the association between folate and B-vitamins low intake and cardiovascular diseases (CVD) risk. Contrasting results were reported on the relationship between folate intake and DNA-methylation. Folate and B-vitamins may modulate DNA-methylation of specific enzymes which are included in the One-Carbon Metabolism (OCM) and in the homocysteine (Hcy) pathways. The aim of the study was to evaluate whether DNA-methylation profiles of OCM and Hcy genes could modulate the myocardial infarction (MI) risk conferred by a low B-vitamins intake. METHODS AND RESULTS: Study sample (206 MI cases and 206 matched controls) is a case-control study nested in the prospective EPIC cohort. Methylation levels of 33 candidate genes where extracted by the whole epigenome analysis (Illumina-HumanMethylation450K-BeadChip). We identified three differentially methylated regions in males (TCN2 promoter, CBS 5'UTR, AMT gene-body) and two in females (PON1 gene-body, CBS 5'UTR), each of them characterized by an increased methylation in cases. Functional in silico analysis suggested a decreased expression in cases. A Recursively Partitioned Mixture Model cluster algorithm identified distinct methylation profiles associated to different MI risk: high-risk vs. low-risk methylation profile groups, OR = 3.49, p = 1.87 × 10(-)(4) and OR = 3.94, p = 0.0317 in males and females respectively (multivariate logistic regression adjusted for classical CVD risk factors). Moreover, a general inverse relationship between B-vitamins intake and DNA-methylation of the candidate genes was observed. CONCLUSIONS: Our findings support the hypothesis that DNA-methylation patterns in specific regions of OCM and Hcy pathways genes may modulate the CVD risk conferred by folate and B-vitamins low intake.

Conversion of a disulfide bond into a thioacetal group during echinomycin biosynthesis.

Echinomycin is a nonribosomal depsipeptide natural product with a range of interesting bioactivities that make it an important target for drug discovery and development. It contains a thioacetal bridge, a unique chemical motif derived from the disulfide bond of its precursor antibiotic triostin A by the action of an S-adenosyl-L-methionine-dependent methyltransferase, Ecm18. The crystal structure of Ecm18 in complex with its reaction products S-adenosyl-L-homocysteine and echinomycin was determined at 1.50 Å resolution. Phasing was achieved using a new molecular replacement package called AMPLE, which automatically derives search models from structure predictions based on ab initio protein modelling. Structural analysis indicates that a combination of proximity effects, medium effects, and catalysis by strain drives the unique transformation of the disulfide bond into the thioacetal linkage.

Mass spectrometry evidence for formation of estrogen-homocysteine conjugates: estrogens can regulate homocysteine levels.

Homocysteine (HCys), a sulfur-containing amino acid, is formed during the metabolism of methionine. An imbalance between the rate of production and the use of HCys during methionine metabolism can result in an increase in the plasma and urinary levels of HCys. HCys has been shown to be toxic to vascular endothelial cells through several pathways. Many earlier clinical studies have revealed an association between plasma HCys and cardiovascular and other diseases. In contrast, estrogens are suggested to lower the risk of cardiovascular disease. Several studies indicate that estrogen metabolites could be responsible for cardiovascular protection. It has been demonstrated that electrophilic estrogen quinones, E1(E2)-2,3-Q and E1(E2)-3,4-Q, can alkylate DNA as well as form conjugates with glutathione. I hypothesize that estrogen quinones generated in situ by oxidative enzymes, metal ions, or molecular oxygen can interact with HCys to form conjugates. This in turn could lower the levels of toxic HCys as well as quenching the reactive estrogen quinones, resulting in cardiovascular protective effects. To test the feasibility of a protective estrogen-HCys pathway, estrogen quinones were treated with HCys. Tandem mass spectrometry analysis of the assay mixture shows the formation of estrogen-HCys conjugates. Furthermore, incubation of catechol estrogens with myeloperoxidase (MPO) in the presence of HCys resulted in the formation of respective estrogen-HCys conjugates. The identities of estrogen-HCys conjugates in MPO assay extracts were confirmed by comparing them to pure synthesized estrogen-HCys standards. I propose that through conjugation estrogens could chemically regulate HCys levels; moreover these conjugates could be used as potential biomarkers in determining health.

[Association study for gene polymorphism of folic acid/homocysteine metabolic pathway and nonsyndromic cleft lip with or without cleft palate in Chinese populations].

OBJECTIVE: To explore the association between 18 candidate genes encoding enzymes on the folate/homocysteine metabolism pathway and non-syndromic cleft lip with or without cleft palate (NSCL/P) in Chinese populations. METHODS: A total of 806 NSCL/P trios were drawn by an international consortium, which conducted a genome-wide association study using a case-parent trio design to investigate genes affecting risks to NSCL/P. The transmission disequilibrium test (TDT) was used for deviation from Mendelian expectations for 257 SNPs in 18 folate/homocysteine metabolism-related genes. The interactions between markers in these gene and environmental risk factors were also tested using conditional Logistic regressions. RESULTS: Although four SNPs (rs6428977, rs12060264, rs7730643 and rs4920037) showed nominal significant association with NSCL/P in the TDT on 806 NSCL/P trios (P<0.05), no significant evidence of linkage and association remained in all the SNPs after Bonferroni correction. Similar tests for interactions between genes and maternal smoking, environmental tobacco smoke, alcohol consumption and multi-vitamin supplementation during pregnancy did not attain statistical significance after correction for multiple comparisons. CONCLUSION: Folate/homocysteine metabolism-related genes could not influence the risk of NSCL/P.