Determination of in vivo protein binding of homocysteine and its relation to free homocysteine in the liver and other tissues of the rat

Low concentrations (0.5-6 nmol/g) of homocysteine (Hcy) have recently been demonstrated in acid extracts of various tissues of the mouse and rat (Ueland, P.M., Helland, S., Broch, O.-J., and Schanche, J.-S. (1984) J. Biol. Chem. 259, 2360-2364). This is referred to as free Hcy in tissues. This paper describes a method for the determination of protein-bound Hcy, which involves precipitation and washing of tissue protein with ammonium sulfate, release of Hcy from native proteins in the presence of dithioerythritol, and determination of free Hcy by a sensitive radioenzymic assay. Both free and bound Hcy decreased markedly in rat tissues within a few seconds following death of the animal. The amount of protein-bound Hcy was highest in liver, somewhat lower in kidney, brain, heart, lung, and spleen. The ratio between free and bound Hcy was between 1 and 2 in most tissues, except in cerebellum, containing a large excess of free Hcy (free/bound ratio of 18). Free Hcy was almost exclusively localized to the soluble fraction of rat liver, whereas protein-bound Hcy was about equally distributed between this fraction and the microsomes. Isolated rat hepatocytes contained free and protein-bound Hcy in proportions observed in whole liver, but a large amount of Hcy was exported into the extracellular medium. The half-lives, as determined from pulse-chase experiments with [35S] methionine, were 53 s for S-adenosylmethionine, 2 s for S-adenosylhomocysteine and 3 s for Hcy (free and bound regarded as a single pool). Furthermore, isotope equilibrium between these metabolites and between free and bound Hcy throughout the rapid chase period suggests the turnover rates of S-adenosylhomocysteine and Hcy to be production rate limited, and the dissociation rate of the Hcy-protein complex may greatly exceed the turnover rate of Hcy. Thus, the half-lives of Hcy are such that participation of both free and bound Hcy in metabolic regulation is feasible.     

Homocysteine thiolactone-induced hyperhomocysteinemia does not alter concentrations of cholesterol and SREBP-2 target gene mRNAS in rats

The present rat study was conducted to test whether hyper-homocysteinemia induced by dietary homocysteine (Hcy) alters the cholesterol concentration in plasma and tissue and the gene expression of genes involved in cholesterol biosynthesis and uptake. Therefore, rats were fed 100 or 200 mg Hcy per kilogram body mass per day (Hcy100 group and Hcy200 group, respectively) as dl-homocysteine thiolactone, or an Hcy-free diet, which served as control, over 14 days. Rats from the Hcy100 group and the Hcy200 group had higher plasma Hcy concentrations (34.4 +/- 4.6 and 69.4 +/- 11.5 microM, respectively) than rats fed an Hcy-free diet (9.5 +/- 1.7 microM). The concentration of Hcy in liver was 2.6 and 3.8 times higher, and in small intestine was 2.6 and 5.1 times higher, in the Hcy100 group and the Hcy200 group, respectively, than in control rats (P < 0.05). The concentrations of cholesterol in plasma, lipoproteins, liver, and small intestine and the relative mRNA concentrations of sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and low-density lipoprotein (LDL) receptor in liver and small intestine were not influenced by dl-homocysteine thiolactone supplementation. In conclusion, in view of the experimental conditions used here, increased plasma and tissue concentrations of Hcy do not alter cholesterol metabolism of liver and intestine.     

Homocysteine in tissues of the mouse and rat

A method for the determination of L-homocysteine (Hcy) in tissues is described, which involves adsorption of adenosine and S-adenosyl-L-homocysteine (AdoHcy) in the tissue extract to dextran-coated charcoal, while leaving Hcy in solution. Sufficient dilution of the tissue homogenates and the presence of a reducing agent during the adsorption step are required to obtain high recovery of Hcy. Hcy is condensed with radioactive adenosine, and labeled AdoHcy is quantified by high performance liquid chromatography on a 3-micron reversed phase column. The amount of Hcy was determined in several tissues (liver, kidney, brain, heart, lung, and spleen) of mice and rats, and the concentrations of Hcy were in the range 0.5-6 nmol/g, wet weight. Hcy concentration was about 1 microM in mouse plasma. In mice, liver contained the highest amount of Hcy, and kidneys were also rich in Hcy. Similar concentrations were found in rat tissues. S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), the enzyme which is believed to catalyze the only pathway leading to Hcy formation in vertebrates, was nearly completely inactivated in mice injected with the drug combination 9-beta-D-arabinofuranosyladenine plus 2'-deoxycoformycin. This treatment induced a massive accumulation of AdoHcy in all tissues (Helland, S., and Ueland, P. M. (1983) Cancer Res. 43, 1847-1850). The amount of Hcy increased several-fold in kidney, whereas no change was observed in liver, heart, brain, lung, and spleen.     

瑞舒伐他汀对同型半胱氨酸诱导的小鼠血管平滑肌细胞去分化和内质网应激的影响及其机制研究

目的：探讨瑞舒伐他汀（Rsv）对同型半胱氨酸（Hcy）诱导的小鼠血管平滑肌细胞（VSMCs）去分化及内质网应激（ERS）的影响。方法：Hcy和不同浓度瑞舒伐他汀（0.1,1.0,10 μmol/L）干预VSMCs,48 h后检测细胞骨架及表型蛋白（α-SMA）、钙调节蛋白（calponin）和骨桥蛋白（OPN）变化,并检测ERS相关mRNA （Herpud1,XBP1s和GRP78）在不同时间点的水平;再在Hcy及Rsv干预基础上给予ERS抑制剂4-苯基丁酸（4-PBA）或诱导剂衣霉素来调控细胞ERS水平,检测细胞增殖、迁移和表型蛋白表达,明确ERS在Rsv表型保护中的作用;在Hcy及Rsv干预基础上给予雷帕霉素靶蛋白（mTOR）-P70S6激酶（P70S6K）信号抑制剂雷帕霉素或激活剂磷脂酸,检测mTOR-P70S6K磷酸化和ERS水平,明确mTOR-P70S6通路在Rsv调控ERS中的作用。结果：与Hcy组相比,Hcy+中Rsv组（1 μmol/L）和Hcy+高Rsv组（10 μmol/L）细胞骨架极性明显增强,α-SMA、calponin表达升高,而OPN及ERS相关mRNA水平显著降低（P<0.01）;与Hcy组比较,Hcy+Rsv组和Hcy+4-PBA组增殖、迁移水平降低（P<0.01）,收缩蛋白表达增强,但衣霉素干预则逆转了Rsv的上述作用;与Hcy组相比,Hcy+Rsv组和Hcy+雷帕霉素组的mTOR-P70S6K磷酸化及ERS水平降低（P<0.01）,但磷脂酸干预抑制了Rsv对mTOR-P70S6K通路和ERS的影响。结论：瑞舒伐他汀可能通过mTOR-P70S6K通路抑制ERS水平,抑制Hcy诱导的小鼠VSMCs去分化改变。     

GABA receptors ameliorate Hcy-mediated integrin shedding and constrictive collagen remodeling in microvascular endothelial cells

Mammalian endothelial cells are deficient in cystathionine β synthetase (CBS) activity, which is responsible for homocysteine (Hcy) clearance. This deficiency makes the endothelium theprime target for Hcy toxicity. Hcy induces integrin shedding in microvascular endothelial cells (MVEC) by increasing matrix metalloproteinase (MMP). Hcy competes with inhibitory neurotransmitter γ aminobutyric acid (GABA)-A receptor. We hypothesized that Hcy transduces MVEC remodeling by increasing metalloproteinase activity and shedding β-1 integrin by inactivating the GABA-A/B receptors, thus behaving as an excitatory neurotransmitter. MVEC were isolated from mouse brain. The presence of GABA-A receptor was determined by immunolabeling. It was induced by muscimol, an agonist of GABA-A receptors as measured by Western blot analysis. Hcy induced MMP-2 activity in a dose- and time-dependent maner, measured by zymography. GABA-A/B receptors ameliorated the Hcy-mediated MMP-2 activation. Hcy selectively increased the levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 but decreased the levels of TIMP-4. Treatment with muscimol decreased the levels of TIMP-1 and TIMP-3 and increased the levels of TIMP-4 to control. Hcy caused a robust increae in the levels of a disintegrin and metalloproteinase (ADAM)-12. In the medium of MVEC reated with Hcy, the levels of β-1 integrin were significantly increased. Treatment with muscimol or baclofen (GABA-B receptor agonist) ameliorated the levels significantly increased. Treatment with muscimol or baclofen (GABA-B receptor agonist) ameliorated the levels of β-1 integrin in the medium. These results suggested that Hcy induced DAM-12. Significantly, Hcy facilitated the β-1 integrin shedding. Treatment of MVEC with muscimole or baclofen during Hcy administration ameliorated the expression of metalloproteinase, integrin-shedding, and constrictive collagen remodeling, suggesting a role of Hcy in GABA receptor-mediated cerebrovascular remodeling.     

Homocysteine-induced decrease in endothelin-1 production is initiated at the extracellular level and involves oxidative products.

The increased cardiovascular risk associated with hyperhomocysteinemia has been partly related to homocysteine (Hcy)-induced endothelial cell dysfunction. However, the intra or extracellular starting point of the interaction between Hcy and endothelial cells, leading to cellular dysfunction, has not yet been identified. We investigated the effects of both intracellular and extracellular Hcy accumulation on endothelin-1 (ET-1) synthesis by cultured human endothelial cells. Incubation of cultures with methionine (1.0 mmol x L(-1)) for 2 h induced a slight increase in cellular Hcy content but no change in ET-1 production. Incubation of cells with Hcy (0.2 mmol x L(-1)) led to a significant fall in ET-1 generation, accompanied by a significant increase in cellular Hcy content. Addition of the amino-acid transport system L substrate 2-amino-2-norbornane carboxylic acid had no effect on the Hcy-induced decrease in ET-1 production but significantly inhibited the Hcy-induced increase in the cellular Hcy content. Incubation of cells with a lower Hcy concentration (0.05 mmol x L(-1)) also reduced ET-1 production without increasing the cellular Hcy content. Co-incubation with extracellular free-radical inhibitors (superoxide dismutase, catalase and mannitol) markedly reduced the effect of Hcy on ET-1 production. Thus, it is extracellular Hcy accumulation that triggers the decrease in ET-1 production by endothelial cells through oxidative products.     

Isolation of a homocysteine gamma-lyase-producing bacterium and study of its enzyme production conditions

Aim: To investigate the possibility of finding a new homocysteine (Hcy) γ‐lyase with the desired properties for Hcy measurement in bacteria. Methods and Results: Through a process of enrichment, the Hcy γ‐lyase‐producing bacterium strain N2‐1 was isolated from soil. Based upon its morphological, physiological, and biochemical characteristics, as well as its 16S rDNA sequence and phylogenetic tree analysis, this isolate belongs to the genus Serratia. The effects of pH, aeration, inducers, carbon (C) and nitrogen (N) sources on enzyme production were studied. Methionine, yeast extract, and glucose were selected as the optimal inducer, C and N sources, respectively. Maximum production of Hcy γ‐lyase was obtained when the isolate was cultured at 30°C at pH 6·5 for about 36 h in the optimum medium. Results also showed that this Hcy γ‐lyase has relatively high specificity towards Hcy. Conclusions: Because of its high specificity for Hcy, this bacterial Hcy γ‐lyase has the potential application in Hcy determination. Significance and Impact of the Study: In addition to isolating a bacterium that produces Hcy γ‐lyase suitable for Hcy determination, this study also indicates that the bacterium could be a source for production of Hcy γ‐lyase for clinical applications.     

Effects of S‐adenosylhomocysteine and homocysteine on DNA damage and cell cytotoxicity in murine hepatic and microglia cell lines

Limited research has been performed on S‐adenosylhomocysteine (SAH) or homocysteine (Hcy)‐evoked cell damage in hepatic and neuronal cells. In this study, we assessed effects of SAH or Hcy on cell cytotoxicity and DNA damage in hepatic and neuronal cells and attempted to find the underlying mechanism. Cell cytotoxicity and DNA damage were evaluated in murine hepatic cells (BNL CL.2 cell line) and microglia cells (BV‐2 cell line) with SAH or Hcy treatment for 48 h. The influences of SAH or Hcy on lipid peroxidation and DNA methylation were also measured in both cell lines. SAH (5–20 μM) or Hcy (1–5 mM) dose dependently inhibited cell cytotoxicity and enhanced DNA damage in both types of cells. Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, whereas Hcy caused minimal effects on these two parameters at much higher concentrations. Hcy significantly induced lipid peroxidation, but not SAH. The present results show that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA hypomethylation, resulting in irreversible DNA damage and increased cell cytotoxicity. In addition, higher Hcy could induce cellular DNA damage through increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is a better marker of cell damage than Hcy in hepatic and microglia cells. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:349–356, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20298     

Vascular oxidative stress increases dendritic cell adhesion and transmigration induced by homocysteine

Atherosclerosis is a long-term chronic inflammatory and immunological disease. Endothelial dysfunction and the dendritic cell (DC) immune response are pivotal early events in atherogenesis. This study investigated the effects and possible mechanisms of action of homocysteine (Hcy) on DC adhesion to and transmigration between endothelial cells (ECs), and indicated a novel immunoregulatory mechanism by which Hcy induces atherogenesis. When ECs were stimulated with increasing concentrations of Hcy, immunofluorescence showed that endothelial reactive oxygen species (ROS) generation strikingly increased, while nitrite assay showed that nitric oxide (NO) release markedly decreased. Furthermore, DC adhesion and transmigration were significantly increased when ECs were activated by Hcy. However, pretreatment of ECs with antioxidant before Hcy markedly attenuated the induction of DC adhesion and transmigration, dependent on the intracellular ROS decrease and endothelial NO increase. In conclusion, DC adhesion and transmigration are significantly increased by vascular oxidative stress under conditions of elevated Hcy levels. These findings provide insight into the inflammatory processes and immune responses occurring in atherosclerosis induced by Hcy.     

Effects of Homocysteine on ERK Signaling and Cell Proliferation in Fetal Neural Stem Cells In Vitro

The aim of the present study was to determine if the excitatory amino acid homocysteine (Hcy) alters ERK signaling and cell proliferation in fetal neural stem cells (NSCs) in vitro. NSCs were isolated from fetal rats and grown in serum-free suspension medium. The cells were identified as NSCs by their expression of immunoreactive Sox2. NSCs were assigned to one of four treatment groups: vehicle control, low-dose Hcy group (Hcy-L, medium contained 30 μmol/L Hcy), middle-dose Hcy group (Hcy-M, 100 μmol/L Hcy) and high-dose Hcy group (Hcy-H, 300 μmol/L Hcy). Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Protein expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by Western blot. The effects of Hcy on NSC death, including apoptosis, were assessed by using flow cytometry and trypan blue exclusion. The results showed that NSCs grew as neurospheres in the serum-free medium. Hcy decreased ERK1/2 protein phosphorylation and NSC proliferation, but it did not induce cell death or apoptosis within the concentration from 30 to 300 μmol/L. The above results are consistent with the hypothesis that Hcy decreases fetal NSC proliferation by inhibiting ERK signaling.     

Arrhythmia and neuronal/endothelial myocyte uncoupling in hyperhomocysteinemia

Elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with arrhythmogenesis and sudden cardiac death (SCD). Hcy decreases constitutive neuronal and endothelial nitric oxide (NO), and cardiac diastolic relaxation. Hcy increases the iNOS/NO, peroxynitrite, mitochondrial NADPH oxidase, and suppresses superoxide dismutase (SOD) and redoxins. Hcy activates matrix metalloproteinase (MMP), disrupts connexin-43 and increases collagen/elastin ratio. The disruption of connexin-43 and accumulation of collagen (fibrosis) disrupt the normal pattern of cardiac conduction and attenuate NO transport from endothelium to myocyte (E-M) causing E-M uncoupling, leading to a pro-arrhythmic environment. The goal of this review is to elaborate the mechanism of Hcy-mediated iNOS/NO in E-M uncoupling and SCD. It is known that Hcy creates arrhythmogenic substrates (i.e. increase in collagen/elastin ratio and disruption in connexin-43) and exacerbates heart failure during chronic volume overload. Also, Hcy behaves as an agonist to N-methyl-D-aspartate (NMDA, an excitatory neurotransmitter) receptor-1, and blockade of NMDA-R1 reduces the increase in heart rate-evoked by NMDA-analog and reduces SCD. This review suggest that Hcy increases iNOS/NO, superoxide, metalloproteinase activity, and disrupts connexin-43, exacerbates endothelial-myocyte uncoupling and cardiac failure secondary to inducing NMDA-R1.     

Homocysteine enhances clot-promoting activity of endothelial cells via phosphatidylserine externalization and microparticles formation

Total elevated plasma homocysteine (Hcy) is a risk factor for thromboembolism. Vascular endothelium is important to regulate coagulation, but the impact of Hcy on the clot-promoting activity (CPA) of endothelial cells has not been fully understood. In our study, human umbilical vein endothelial cells (HUVECs) were treated with Hcy (8, 20, 80, 200, 800?μmol/L) for 24?h. Annexin V was utilized to detect phosphatidylserine (PS) externalization and endothelial microparticles (MPs) formation. CPA was assessed by recalcification time and purified clotting complex tests. We found that Hcy enhanced the externalized PS and consequent CPA of HUVECs in a dose-dependent fashion, effect of Hcy had statistical significance at 800?μmol/L. In addition, Hcy also increased the shedding of procoagulant endothelial MPs. Blocking of PS with 128?nmol/L annexin V reduced approximately 70% CPA of HUVECs and endothelial MPs, but human anti-tissue factor antibody had little inhibitive effect. Our results showed that Hcy increased CPA of HUVECs via PS externalization and MPs release. Our present study has implications for hyperhomocysteinemia-related hypercoagulability.     

血浆同型半胱氨酸水平升高与动脉粥样硬化

心血管疾病已成为当今全球性致残与致死的最重要原因之一。目前确定的冠心病的危险因素主要包括高龄、血脂异常、高血压、糖尿病、吸烟、肥胖症。大量的临床试验及流行病学研究已经证实血浆同型半胱氨酸水平升高是心血管疾病的一个独立的危险因素。健康人的血浆同型半胱氨酸水平为5～10μmol／L。血浆同型半胱氨酸水平严重升高的主要原因是胱硫醚-β-合成酶(cystathionine-β-synthase,CBS)基因的缺陷。CBS基因缺陷的纯合体可导致血浆同型半胱氨酸水平升高至100～500μmol／L。血浆同型半胱氨酸水平严重升高的病人通常伴随神经系统异常、早发性的动脉粥样硬化。叶酸、维生素B6和B12治疗能降低血浆同型半胱氨酸的水平并改善血管内皮功能、减少经皮冠状动脉腔内成形术(percutaneou stransluminal coronary angioplasty,PTCA)术后并发症。迄今为止,血浆同型半胱氨酸水平升高引起心血管疾病的发病机制并未完全明了,目前认为主要与以下几个方面有关：(1)内皮细胞损伤及功能障碍。我们实验室在CBS基因敲除的小鼠模型上证实血浆同型半胱氨酸水平升高能抑制eNOS的活性,导致主动脉内皮功能的障碍。我们还在细胞模型上证实了同型半胱氨酸能显著抑制内皮细胞的增殖。(2)KH固醇和甘油三脂生物合成代谢异常。我们实验室在apoE、CBS双基因敲除的小鼠模型上证实血浆同型半胱氨酸水平升高能改变肝脏的脂肪代谢,增加巨噬细胞对修饰LDL的摄取,从而导致胆固醇脂和甘油三脂在血管壁的堆积,促进主动脉粥样斑块的形成。(3)刺激血管平滑肌细胞增殖。此外还发现同型半胱氨酸能激活蛋白激酶C信号途径,促进胶原蛋白的合成,抑制弹性蛋白和胶原蛋白的交联。(4)激活血栓形成。(5)激活单核细胞。目前认为同型半胱氦酸主要通过以下几个化学机制致病;(1)自氧化产生活性氧。同型半胱氨酸在自氧化的过程中能产生大量的活性氧,从而引起血液中脂蛋白和细胞膜脂质的过氧化损伤,并进一步引起内皮功能的障碍。(2)在腺苷的参与下形成SAH,一种甲基转移抑制剂,导致细胞内的低甲基化。(3)与一氧化氮结合形成亚硝酰物。(4)参与蛋白质的合成。总之,我们和其他实验室的研究结果均表明同型半胱氨酸不仅与动脉粥样硬化相关,而且具有致病效应。尽管补充叶酸、维生素B6和B12等治疗能降低血浆同型半胱氨酸的水平,但是否能降低心血管疾病的风险仍有待于大量的动物研究及临床试验。     

Folate and ApoE DNA methylation induced by homocysteine in human monocytes

Homocysteine (Hcy) is an important and independent risk factor for arteriosclerosis, and apolipoprotein E (ApoE) is an important gene of anti atherosclerosis, but the characteristics and their key links that are involved in their pathogenic mechanisms are still poorly understood. The objective of the present study was to investigate the effects of Hcy and folate on ApoE as well as the underlying mechanism of ApoE expression induced by Hcy in monocytes. When clinically relevant concentrations of Hcy and folate were added to the cultured monocytes for 4 days, we found that clinically relevant Hcy (100 microM) may increase the levels of total cholesterol (TC), free cholesterol (FC), and cholesteryl ester (CE), and also decrease ApoE mRNA, protein expressions, leading to 34.28%, 45.00% in cultured primary human monocytes in comparison to the positive group. The effects of Hcy were primarily mediated by C-5 MTase, because Hcy could upregulate the activity of C-5 MTase and then accelerate DNA methylation of ApoE. However, folate decreased the levels of TC, FC, and CE (p < 0.001) and increased the ApoE expression; as to say, folate primarily repressed the effects of DNA methylation induced by Hcy and reduced anti atherosclerosis. In conclusion, these results suggested that ApoE DNA methylation that is induced by Hcy may play a potential role for ApoE expression in atherosclerosis. Folate has beneficial effects for anti atherosclerosis, and it may become a therapeutic target for preventing Hcy-induced atherosclerosis.     

5,10‐Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms: genotype frequency and association with homocysteine and folate levels in middle‐southern Italian adults.

Two genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene (C677T and A1298C) can influence the plasma homocysteine (Hcy) levels, especially in the presence of an inadequate folate status. The aim of this study was to evaluate the frequencies of C677T and of A1298C MTHFR polymorphisms and their correlation with Hcy and serum folate concentrations in a population of blood donors living in a region of middle‐southern Italy (the Molise Region). One hundred ninety seven blood donors were studied for total plasma Hcy, serum folate and C677T and A1298C MTHFR genotypes. The frequency of C677T genotypes was 20.8% (CC), 49.8% (CT) and 29.4% (TT); for the A1298C genotypes: 48.7% (AA), 43.7% (AC) and 7.6% (CC). Hcy and serum folate concentrations were significantly different among genotypes of the C677T polymorphism (CC versus CT versus TT: <0.0001 both for Hcy and folate), with Hcy values increasing, and serum folate decreasing, from CC to TT subjects. Regarding to A1298C polymorphism, the difference among genotypes (AA versus AC versus CC; p: 0.026 for Hcy and 0.014 for serum folate), showed an opposite trend for both parameters, with Hcy higher in the wild‐type and lower in the homozygotes and serum folate higher in CC than in AA subjects. In conclusion, we found a high frequency of MTHFR allele associated with high level of Hcy and low levels of folate in an Italian southern population. Copyright © 2013 John Wiley & Sons, Ltd.     

Homocysteine alterations in experimental cholestasis and its subsequent cirrhosis

Homocysteine (Hcy), an intermediate in methionine metabolism, has been proposed to be involved in hepatic fibrogenesis. Impaired liver function can alter Hcy metabolism. The aim of the present study was to determine plasma Hcy alterations in acute obstructive cholestasis and the subsequent biliary cirrhosis. Cholestasis was induced by bile duct ligation and sham-operated and unoperated rats were used as controls. The animals were studied on the days 7th, 14th, 21st and 28th after the operation. Plasma Hcy, cysteine, methionine, nitric oxide (NO) and liver S-adenosyl-methionine (SAM), S-adenosyl-homocysteine (SAH), SAM to SAH ratio and glutathione were measured. Chronic L-NAME treatment was also included in the study. Plasma Hcy concentrations were transiently elevated by the day 14th after bile duct ligation (P < 0.01) and subsequently returned to control levels. Similar relative fluctuations in plasma Hcy were observed in BDL rats after intraperitoneal methionine overload. Plasma methionine, cysteine and nitrite and nitrate were significantly increased after bile duct ligation. SAM to SAH ratio was diminished by the 1st week of cholestasis and remained significantly decreased throughout the study. These events were accompanied by a decrease in GSH to GSSG ratio in the liver. Chronic L-NAME treatment improved SAM to SAH ratio and prevented the elevation of plasma Hcy and methionine (P < 0.05) while couldn't influence the other parameters. In conclusion, this study demonstrates alterations in plasma Hcy and liver SAM and SAH contents in precirrhotic stages and in secondary biliary cirrhosis, for the first time. In addition, we observed that plasma Hcy concentrations in BDL rats follow a distinct pattern of alteration from what has been previously reported in other models of cirrhosis. NO overproduction may contribute to plasma Hcy elevation and liver SAM depletion after cholestasis.     

首发缺血性脑卒中患者血清Hcy和EPO水平的变化及临床意义

目的:探究首发缺血性脑卒中患者血清同型半胱氨酸(Hcy)和红细胞生成素(EPO)水平的变化和意义。方法:于2013年10月-2015年4月我科收治的首发缺血性脑卒中患者中随机选取98例作为观察组,另选取同期健康体检者98例作为对照组,检测患者的血小板、血浆纤维蛋白原(Fib)以及血白细胞水平,比较两组血清Hcy、EPO、血小板、Fib及血白细胞水平,使用Logistic回归分析法评价缺血性脑卒中病发的危险因素,采用Spearman法对血清Hcy与EPO间相关性进行分析。结果:观察组的Hcy(23.52±12.15)m IU/L与EPO(34.61±11.25)m IU/L水平显著高于对照组的(10.57±2.18)m IU/L、(17.54±5.83)m IU/L;观察组血小板、血浆纤维蛋白原(fibrinogen,Fib)及血白细胞水平均高于对照组;差异均有统计学意义(均P0.05)。经Logistic回归分析法分析可知,Hcy为缺血性脑卒中病发的独立因素,经Spearman相关性分析显示,首发缺血性脑卒中患者EPO水平与Hcy呈正相关。结论:缺血性脑卒中病发与血清Hcy和EPO水平升高密切相关,且Hcy是导致缺血性脑卒中病发的高危因素。     

Large-conductance Ca2+-activated K+ currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca(2+)-activated K(+) channels (BK(Ca)) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK(Ca) channel properties of smooth muscle cells. Whole-cell patch-clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 microM Hcy on BK(Ca), the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK(Ca) density (measured at +60 mV) was inhibited by about 24% (n=6, P<0.05). In rat artery smooth muscle cells, maximum BK(Ca) density was decreased by approximately 27% in the presence of 50 microM Hcy (n=8, P<0.05). In addition, when rat artery smooth muscle cells was treated with 50 microM Hcy for 24 h, maximum BK(Ca) density decreased by 58% (n=5, P<0.05). These data suggest that Hcy significantly inhibited BK(Ca) currents in isolated human and rat artery smooth muscle cells. BK(Ca) reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.     

Homocysteine altered ROS generation and NO accumulation in endothelial cells

Mild hyperhomocysteinemia (HHcy) is a risk factor for vascular disease and is closely associated with endothelial dysfunction. Oxidative stress and decreased nitric oxide (NO) bioavailability were reported in HHcy-induced vascular injury; however, the exact relationship is not understood. We thus directly determine the production of reactive oxygen species (ROS) and NO in cultured endothelial cells (HUVECs) to demonstrate the correlated variation between ROS and NO induced by Hcy (homocysteine), Cys (cysteine), another thiol compound, and Met (methionine), precursor of HHcy in animal study. HUVECs were treated with Hcy, Cys, or Met for 0.5 or 22-24 h; ROS generation was detected by DCF fluorescence with flow cytometry and NO by chemiluminescence. In non-cytotoxic (<1.0 mM) concentration ranges, Met exerted no effects on either ROS production or NO concentration, Cys decreased ROS production and increased NO in both short-term (0.5 h) and long-term (22-24 h) treatments; Hcy, however, induced a biphasic effect on ROS production, i.e., inhibitory at 0.5 h but stimulatory at 24 h. The maximal stimulation by Hcy (0.25 mM) was significantly reduced by co-incubation (12 h) with estrogen (1 microM). Hcy caused an early (0.5 h) increase of medium NO which was absent in long-term Hcy treatment. The oxidative stress caused by long-term Hcy incubation could be ameliorated by estrogen, consistent with earlier in vivo observations that estrogen prevents HHcy-induced injury.