HHcy Group Research Articles

Homocysteine induces blood vessel global hypomethylation mediated by LOX-1.

Homocysteine (Hcy) is an independent risk factor of atherosclerosis through its involvement with the methionine cycle. In this study, we aimed to determine the blood vessel global methylation rate in Hcy-induced atherosclerosis in apolipoprotein-E-deficient (ApoE-/-) mice, and to explore the possible mechanism of this change in endothelial cells. ApoE-/- mice were divided into a hyperlipidemia (HLP) group, a hyperhomocysteinemia (HHcy) group, and an HHcy + folate + vitamin B12 (HHcy+FA+VB) group. Wild-type C57BL/6J mice were prepared as controls. Total Hcy, lipids, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) contents in serum were measured with an automatic biochemistry analyzer and high-performance liquid chromatography. Methylation of B1 repetitive elements in blood vessels was tested using nested methylation-specific-polymerase chain reaction (nMS-PCR). Endothelial cells (ECs) were pretreated with Hcy or by adding FA and VB. Lectin-like oxidized LDL receptor-1 (LOX-1) expressions were determined by quantitative PCR, Western blot, and nMS-PCR. The HHcy group displayed severe HLP and HHcy. SAM and SAH contents were also elevated in the HHcy group compared with other groups. Methylation of B1 repetitive elements was significantly increased in the HHcy group (0.5050 ± 0.0182) compared to the HLP (0.5158 ± 0.0163) and control (0.5589 ± 0.0236) groups. mRNA and protein expressions of LOX-1 increased (0.2877 ± 0.0341, 0.6090 ± 0.0547), whereas methylation expression decreased (0.5527 ± 0.0148) after 100 μM Hcy stimulation in ECs. In conclusion, Hcy-induced atherosclerosis was closely associated with induced hypomethylation status in the blood vessel, and this process was partially mediated by LOX-1 DNA methylation.

Validity and reliability of the 13C-methionine breath test for the detection of moderate hyperhomocysteinemia in Mexican adults

Hyperhomocysteinemia (Hhcy) is considered an independent risk factor for vascular diseases and, more recently, for dementia. The methionine loading test (MLT) is useful for diagnosing additional subjects with moderate Hhcy. However, it is a complex and time-consuming procedure. A noninvasive test for the diagnosis of moderate Hhcy is desirable. The study protocol consisted of three consecutive visits. During the first visit, we performed an MLT to characterize the Hhcy status of 75 healthy adult subjects. For the breath test protocol, we selected a subsample and assigned to the control group 17 subjects with fasting and post-loading homocysteine (Hcy) ≤12 and <42.3 μmol/L, respectively, and to the Hhcy group 16 subjects with fasting Hcy ≤12 and >42.3 μmol/L after loading. Selected subjects were requested to have a second visit to perform a breath test within 1-4 weeks following the MLT test and received an oral dose of 2.5 mg/kg of 1-13C-methionine dissolved in water. Breath samples were collected at basal, 20, 40, 60, 80, 100 and 120 min (test 1). The same procedure was repeated within 1 week (test 2). MLT was useful for diagnosing almost twice the number of individuals with Hhcy (24%) in comparison with the fasting determination alone (13.3%). The 13C-methionine breath test reported a sensitivity of 81.3% and a specificity of 64.7% against the MLT. The coefficient of variation between breath test 1 and breath test 2 was 9.0±5.4%. The 13C-methionine breath test is a valid and reliable method for identifying subjects with moderate Hhcy.

Effects of folic acid supplementation on bone turnover markers in male rats subjected to experimental hyperhomocysteinemia

Objective The objective of this study was to examine whether lowering homocysteine (HCY) with folate has beneficial effects on altered structural and biomechanical properties of bone, induced by experimental hyperhomocysteinemia (HHCY). Methods Thirty adult male Wistar albino rats were randomized into three equal groups: a control group, an HHCY group, and an HHCY+folate group. After 8 weeks of treatment, blood was collected to measure the plasma concentrations of HCY and some bone markers including osteocalcin, bone-specific alkaline phosphatase, and tartrate-resistant acid phosphatase (TRAP5b). Also, histopathological examination of bone was carried out. Results TRAP5b showed higher plasma concentrations, whereas plasma concentrations of osteocalcin and bone-specific alkaline phosphatase were significantly lower in HHCY rats compared with the control rats. Feeding of rats with a folate-enriched diet showed a significant reduction in the plasma HCY level and induced a significant improvement in the disturbances resulting from HHCY on the bone structure and turnover markers. Conclusion The results of this study indicate that HHCY is associated with altered bone turnover. Also, folate supplementation may have beneficial effects to avoid osteoporosis and fractures.

FOLIC ACID PREVENTS LINE-1 HYPOMETHYLATION IN RATS WITH HYPERHOMOCYSTEINEMIA

ObjectivesLINE-1 (Long Interspersed Nuclear Elements-1) hypomethylation has been associated with risk of cardiovascular disease. Increased plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. However, it is still...

The effects of ginsenoside Rb1 on endothelial damage and ghrelin expression induced by hyperhomocysteine

Studies have indicated that ginsenoside Rb1 and ghrelin could both prevent homocysteine (Hcy)-induced endothelial dysfunction through the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) mechanism. This study investigated whether endogenous ghrelin mediates the endothelial protection of ginsenosidee Rb1 through in vitro and in vivo experiments. Rats were randomized into a control group, a hyperhomocysteine (HHcy) model group with a high methionine diet, a ginsenosides (GS) group, and HHcy plus GS group. Plasma ghrelin was detected by enzyme-linked immunosorbent assay. Aortic rings for control and HHcy groups were treated with ghrelin or not. Endothelium-dependent vasodilatation function was evaluated by the aortic ring assay, and the structural changes were visualized by hematoxylin and eosin staining. Human umbilical vein endothelial cells (HUVECs) were cultured, and the experimental conditions were optimized according to NO production. After treatment, the NO, ghrelin, and von Willebrand factor (vWF) levels in the media were detected and analyzed with linear regression. Ghrelin and eNOS expression were observed by cell immunohistochemical staining. Ghrelin receptor antagonist was used to detect the mechanism of ginsenoside Rb1 on NO production, which was reflected by diacetylated 4,5-diaminofluorescein-2 diacetate fluorescence. In vivo experiments demonstrated that plasma ghrelin levels in the HHcy group were significantly elevated vs controls (P < .05) and were significantly increased in the HHcy plus GS group (P < .01). Compared with control, endothelium-dependent vasodilatation function was greatly reduced in the HHcy group (P < .01), which was significantly increased in HHcy plus ghrelin group compared with HHcy group (P < .01). The arterial walls of HHcy group exhibited characteristic pathologic changes, which were repaired in HHcy plus ghrelin group. In vivo, compared with Hcy (200 μM) group, HUVECs pretreated with ginsenoside Rb1 (10 μM) for 30 minutes showed significant increases in NO and ghrelin levels and evident reduction in vWF levels. Linear regression analysis demonstrated that ghrelin levels were significantly positively correlated with NO levels and significantly negatively correlated with vWF levels. The addition of Rb1 to Hcy also greatly reversed Hcy-induced downregulation of ghrelin and eNOS expression. Ghrelin inhibition significantly abolished the upregulation of NO levels induced by Rb1. Ghrelin can prevent Hcy-induced vascular endothelial dysfunction and structural damage. The compensatory elevation of plasma ghrelin levels in an Hcy-induced endothelial injury model may be a protective response. Ginsenoside Rb1 can significantly stimulate the ghrelin endocrine to inhibit endothelial injury. Ginsenoside also upregulates the NO signaling pathway reduced by Hcy through the ghrelin molecular mechanism.

Hydrogen sulfide: Regulatory role on blood pressure in hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is a metabolic disorder marked by an excess amount of the amino acid homocysteine (Hcy) in the blood stream. Hcy is a H2S precursor—formed from the metabolism of methionine. Elevated Hcy levels have been associated with higher blood pressure. However, the precise contribution of H2S to blood pressure in HHcy is not known. In the current study, we have examined a novel link between H2S, blood pressure and HHcy. Male Sprague–Dawley rats were injected with PAG, NaHS, L-NAME+PAG and saline. HHcy condition was induced by providing methionine (1g/kg) in drinking water for 8weeks. After 8weeks, plasma Hcy and H2S were measured. The treated rats were anaesthetized with a mixture of ketamine hydrochloride and medetomidine. Blood pressures were measured by intra-carotid artery catheterization and to further investigate the immediate effect of NO and H2S, exogenous drugs namely NaHS, SNP, Ach and NA were administered. Plasma Hcy levels were higher in HHcy groups and this group exhibited hypertension. We observed high blood pressure at low levels of H2S and vice versa. Endogenous H2S in HHcy condition facilitated a mild decrease in MAP (Mean Arterial Pressure). Exogenous SNP (NO donor) showed a greater pressure decrease in HHcy group. The underlying mechanism is yet to be exploited. High levels of Hcy play an important role in the pathogenesis of hypertension. The results suggest that both endogenous and exogenous H2S may play a vital role in regulating blood pressure in HHcy.

Serum folate is a reliable indicator of hyperhomocysteinemia and borderline hyperhomocysteinemia in young adults

Serum folate has been shown to correlate well with fasting plasma homocysteine; however, erythrocyte folate concentration is a better index of tissue folate stores and probably could be a more reliable indicator for reflecting long-term supply of the vitamin and homocysteine status. The present study was undertaken to test the hypothesis that serum folate and erythrocyte folate levels had a different degree of correlation to fasting plasma homocysteine in young Taiwanese adults. This study had a cross-sectional design. Healthy young adults were divided into either a hyperhomocysteinemia (HHcy; > or =14.9 micromol/L; n = 13), borderline HHcy (BHcy; fasting homocysteine, 14.9-10.2 micromol/L; n = 52), or normohomocysteinemia (fasting homocysteine, <10.2 micromol/L; n = 65) groups based on fasting homocysteine levels. The concentrations of plasma fasting homocysteine, serum folate, erythrocyte folate, vitamin B(12), and plasma pyridoxal 5'-phosphate were measured. Fasting homocysteine was only significantly and inversely affected by serum folate (beta = -0.21, P < .05) concentration after adjusting for potential confounders. Only serum folate concentration remained to decrease the risk of fasting HHcy (odds ratio, 0.73; confidence interval, 0.56-0.95) after the other B vitamins were additionally adjusted. Serum folate also had the highest area under the receiver operating characteristic (AUC) curve to predict the risk of HHcy (AUC, 0.81) and BHcy (AUC, 0.77). Serum folate is a reliable indicator of fasting hyperhyperhomocysteinemia and BHcy in young adults.

Poly (ADP-ribose) Polymerase Inhibition Improves Endothelial Dysfunction Induced by Hyperhomocysteinemia in Rats

We investigated the possible protective effect of poly (ADP-ribose) polymerase (PARP) inhibition in preventing endothelial dysfunction induced by hyperhomocysteinemia (Hhcy). Sprague-Dawley rats were divided into Hhcy group, Hhcy + 3-aminobenzamide(3-AB) group, control group and control + 3-AB group. A high-methionine diet was given to induce hyperhomocysteinemia. In Hhcy + 3-AB and control + 3-AB groups, rats were injected intraperitoneally with 3-AB (inhibitor of PARP). After 45 days, ultrastructural changes of aortas were observed by transmission electron microscope. Vascular reactivity of thoracic aortic rings was measured in organ chambers. PARP activity was detected. The levels of plasma total homocysteine, nitrite/nitrate, endothelin (ET)-1 and malondialdehyde were assayed. Rats in Hhcy group developed severe hyperhomocysteinemia and significant loss of endothelial function as measured by both vascular rings and levels of nitrite/nitrate and ET-1. Malondialdehyde levels increased significantly in Hhcy rats compared with control rats. 3-AB improved Ach-induced, NO-mediated vascular relaxation and stabilized the level of nitrite/nitrate and ET-1. Obvious improvement of ultrastructure can be observed in Hhcy + 3-AB group. These results suggest that pharmacological inhibition of PARP prevents the development of endothelial dysfunction in rats with hyperhomocysteinemia which may represent a novel approach to improve vascular dysfunction associated with hyperhomocysteinemia.

Effect of Folic Acid and Vitamins B6 and B12 on Microcirculatory Vasoreactivity in Patients With Hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) has been identified as an independent risk factor for atherosclerotic vascular disease. The effect of high-dose folic acid or combination vitamin therapy for the treatment of HHcy on the microcirculation is unknown. The purpose of this study was to evaluate the effect of a combination of folic acid, vitamin B6, and vitamin B12 on endothelium-dependent and endothelium-independent vasoreactivity in patientswith HHcy. Baseline cutaneous microvascular vasoreactivity was measured in 20 patients with HHcy and 18 patients with normohomocysteinemia (NHcy). Laser Doppler scan imaging before and after iontophoresis of 1% acetylcholine chloride (endothelium-dependent response) and 1% sodium nitroprusside (endothelium-independent response) was performed for the measurement of forearm skin vasodilatation. Patients were then treated with 10 mg folic acid, 100 mg vitamin B6, and 1 mg vitamin B12 orally once a day for 6 months. Follow-up fasting serum homocysteine and cutaneous Laser Doppler scan imaging before and after iontophoresis were performed at 1, 2, 3, and 6 months. Statistical analysis was performed using Fisher's exact test, paired t test, and Wilcoxon matched-pairs signed-ranks test, with significance set at P < .05. The HHcy group was older than the NHcy group (70.89 +/- 1.95 vs 61.78 +/- 2.73 years, P = .02). Otherwise the groups were similar in terms of race, tobacco use, comorbid diseases, and serum lipoproteins. Over the 6-month period, fasting serum homocysteine levels decreased significantly in both the NHcy group (10.40 +/- 0.59 micromol/L vs 8.97 +/- 0.84 micromol/L, P = .01) and the HHcy group (19.80 +/- 1.06 micromol/L vs 13.40 +/- 0.86 micromol/L, P = .0002). There were no statistically significant changes in endothelium-independent vasoreactivity (voltage change from baseline) in either group. Endothelium-independent vasore activity decreased over the 6-month period in the HHcy group (0.20 +/- 0.04 V vs 0.11 +/- 0.03 V, P = .03). Subanalysis of HHcy with diabetes or age greater than 65 years both showed worsening trends in endothelium-independent vasoreactivity (P = .05 for both groups). There were no statistically significant changes in endothelium-independent vasoreactivity in the NHcy group. High doses of folic acid and vitamins B6 and B12 lower fasting serum homocysteine levels in patients with HHcy. Older and diabetic patients with HHcy tend to do worse possibly because of long-term fixed microvascular insult secondary to multiple sustained comorbidities.

Experimental Hyperhomocysteinemia Reduces Bone Quality in Rats

Recently, hyperhomocysteinemia (HHCY) has been suggested as a new risk factor for osteoporosis. This study investigated if HHCY is a causal osteoporotic factor in vivo. We used 3 groups of rats: a control group (n = 20), a moderate HHCY group (induced by a 2.4% methionine-enriched diet, n = 10), and an intermediate HHCY group (induced by a 2% homocystine-enriched diet, n = 10). We measured bone fragility [maximum force of an axial compression test (F(max))], bone area as percentage of total area (BAr/TAr, histomorphometry), and biochemical bone turnover markers [osteocalcin (OC) and collagen I C-terminal crosslaps (CTx)]. Compared with controls, 3 months of moderate or intermediate HHCY increased mean (SD) bone fragility at the femoral neck by 18% (6%) in methionine-fed (P = 0.001) and 36% (13%) in homocystine-fed rats (P <0.001). Mean (SD) BAr/TAr at the distal femur in methionine and homocystine groups was decreased by 45% (21%; P = 0.001) and 93% (9%; P = 0.001), respectively. At the femoral neck, BAr/TAr was decreased by 19% (11%; P <0.001) and 55% (19%; P <0.001). At the lumbar spine, the reduction of BAr/TAr was 17% (23%; P = 0.099) and 44% (19%; P <0.001). Plasma OC (bone formation marker) was decreased by 23% (20%; P = 0.006) and 34% (21%; P <0.001). Plasma CTx (bone resorption marker) did not differ between groups. Bone quality is consistently decreased in the presence of increased circulating homocysteine. The results provide evidence that HHCY is a causal osteoporotic factor.

Folic acid reduces chemokine MCP-1 release and expression in rats with hyperhomocystinemia

This study aimed to investigate the effects of folate on the monocyte chemoattractant protein-1 (MCP-1) expression and release in rats with hyperhomocystinemia induced by ingestion of excess methionine. Thirty male Sprague-Dawley rats (200+/-20 g) were randomly divided into three groups (n=10 for each group): control group (Control), high-homocystinemia (Hhcy) group, and folate treatment (FA) group. They were fed with a normal regular diet, enriched by 1.7% methionine plus 1.7% methionine and 0.006% folate for 45 days. Our study showed the following: (a) A high methionine diet for 45 days is sufficient to induce hyperhomocystinemia; folate supplementation to the rats fed the high-methionine diet prevented an elevation homocysteine (Hcy) levels in the blood (P<.01). (b) Compared with the Control group, the Hhcy group had elevated plasma levels of MCP-1, and Hcy was significantly correlated with MCP-1 (P<.05). (c) The protein and mRNA expression of MCP-1 in the aorta was higher in rats from the Hhcy group than in rats from the Control group. (d) Most important, after folic acid supplementation, the lowering of Hcy levels was accompanied by a marked reduction of MCP-1 expressed in aortae and released from plasma and peripheral blood mononuclear cells (PBMCs) stimulated by oxidized low-density lipoprotein (P<.05, P<.01). Folic acid supplementation not only can blunt the rise in Hcy and reduce MCP-1 released from both plasma and PBMCs of rats with hyperhomocystinemia but also can downgrade MCP-1 expression in the aorta of rats with hyperhomocystinemia.

Genetic determinants of hyperhomocysteinemia in atherosclerosis.

Hyperhomocysteinemia (Hhcy) is an independent risk factor for the development of atherosclerosis. The mechanisms by which HHcy promotes cardiovascular disease may be due to activation of pro-inflammatory factors, endoplasmic reticulum (ER) stress and oxidative stress. We aimed to study (i) gene mutations that cause HHcy. (ii) Estimation of inflammatory marker like ultrasenitive C-reactive proteins (hs-CRP) and total antioxidant levels (iii) determination of Hcy- dependent gene expression in vivo. 25 HHcy patients and 25 healthy controls were taken for this study. Mutation detection in MTHFR, CBS, MS and eNOS gene was by PCR-based restriction enzyme analysis and subsequently expression study was carried out by Reverse Transcriptase PCR and cloning technique. A significant association of HHcy with MTHFR (C677T) and MS (A2756G) genotype was observed (p<0.05). There was no association of Hhcy and eNOS genotype. The Hhcy patients, showed no expression of the ER stress gene, GRP78 in lymphocytes. Our study showed no effect of Hcy on the CD18 gene (pro-inflammatory pathway) expression, but a significant association of tHcy and hs-CRP levels in HHcy grp (t=2.28, p<0.05). This shows that HHcy induces inflammatory response, which could lead to tissue injury in the pathogenesis of the atherosclerotic process. Our findings show higher mRNA expression of manganese superoxide dismutase (Mn SOD) in HHcy group as compared to the control group. The Total Antioxidant Status (TAS) estimated was found to be significantly lower in the HHcy group as compared to healthy normals (t=4.8, p<0.01). Taken together these findings strongly suggest that the adverse effects of homocysteine are at least partly mediated by oxidative stress. Our study supports the hypothesis that Hcy evokes adverse vascular effects by promoting oxidative damage to endothelial cells.

Cavernosal dysfunction in a rabbit model of hyperhomocysteinaemia

To investigate the effect of sustained hyperhomocysteinaemia (HHCy) on cavernosal smooth muscle function in a rabbit model of HHCy, developed using a methionine-enriched diet in which cavernosal responses were characterized, as elevated plasma levels of homocysteine may be a risk factor for vasculogenic erectile dysfunction. Six New Zealand White rabbits were fed a diet supplemented with methionine (20 g/kg chow) for 4 weeks, while six control animals were fed a standard diet. Cavernosal strips were mounted in an organ bath and relaxation assessed when stimulated with carbachol, sodium nitroprusside (SNP), or noncholinergic, nonadrenergic (NANC)-mediated relaxation to electrical-field stimulation (EFS). Cavernosal tissue cGMP levels were assessed using an enzyme-linked immunosorbent assay, and superoxide (O(2) (.-)) production assessed using an assay of the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. The methionine-rich diet led to an early but sustained HHCy; cavernosal strips from animals after 4 weeks of HHCy had a significantly impaired relaxation response to carbachol, an index of endothelium-dependent nitric oxide (NO)-mediated relaxation. This impairment was reversed by incubating with either SOD or catalase. Relaxation with either SNP, an index of endothelium-independent NO-mediated relaxation, or NANC-mediated EFS-induced relaxation, was unaffected by HHCy. There was a corresponding significant reduction in cavernosal cGMP levels (index of NO activity) in the HHCy group, with a more than five-fold increase in cavernosal tissue O(2) (.-) production. Supplementing the diet of rabbits with methionine for 4 weeks caused an early and sustained HHCy and promoted a marked inhibitory effect on endothelium-dependent relaxation and NO formation in isolated corpus cavernosum, an effect mediated by reactive oxygen species.

FOLIC ACID AND VITAMIN B 12 SUPPLEMENTATION ATTENUATES ISOPRENALINE-INDUCED MYOCARDIAL INFARCTION IN EXPERIMENTAL HYPERHOMOCYSTEINEMIC RATS

Hyperhomocysteinemia (Hhcy) is an independent risk factor for cardiovascular disease. Oxidative stress may contribute to the deleterious effects of homocysteine (Hcy). The aim of the present study is to study the effect of folic acid and Vitamin B 12 supplementation on isoprenaline (ISO)-induced myocardial infarction (MI) in hyperhomocysteinemic rats. Hhcy was induced by daily intake of methionine (1 g kg −1 body weight) in the drinking water for 4 weeks. MI was then produced by a single subcutaneous injection of ISO (300 mg kg −1, s.c.). Electrocardiographic parameters, heart rate, ST segment, and blood pressure as well as serum marker enzymes, creatine kinase (CK) and lactate dehydrogenase (LDH) were measured. Lipid peroxidation measured as malondialdehyde (MDA) and reduced glutathione (GSH) concentrations in heart tissue were estimated as indices of oxidative stress. Hhcy resulted in significant blood pressure reduction, ST segment elevation and increase in heart rate and serum CK and LDH levels. Cardiac MDA was significantly increased, while GSH was decreased in Hhcy group compared to the normal control group. All the measured parameters were greatly exaggerated in Hhcy rats treated with ISO in comparison with Hhcy rats alone. Administration of folic acid (10 mg kg −1, orally via gavage) and Vitamin B 12 (500 μg kg −1, i.m.) concurrently for 4 weeks during the induction of Hhcy markedly reduced the increase in heart rate, ST segment elevation and blood pressure reduction as well as the increase in serum CK and LDH levels. Cardiac MDA content was decreased while cardiac GSH was elevated in the treated group compared to Hhcy+ISO group. Moreover, the severe cardiac histopathological changes observed in Hhcy+ISO group were attenuated by folic acid and Vitamin B 12. These results suggest that Hhcy aggravates MI via oxidative stress mechanisms and that lowering Hcy level with folic acid and Vitamin B 12 can ameliorate the detrimental effects of Hhcy and may reduce the risk of MI.