同型半胱氨酸对蛋白质的修饰作用研究进展

高同型半胱氨酸血症在心血管疾病、孕期并发症、认知障碍和骨质疏松症等疾病发生发展过程中是一个独立的危险因子,同型半胱氨酸的致病机制相关研究一直受到科研工作者的广泛关注.本综述主要介绍新近国际科研界热点,即以蛋白质同型半胱氨酸化为中心的假设理论.该理论以同型半胱氨酸的分子化学性质为基础,通过两种基本的蛋白质修饰途径,较好地解释了蛋白质的结构和功能损伤之间的关系,而这种对蛋白质的修饰所造成的血管性损伤也许正是引起上述多种不甚相关疾病的共同关键机制.     

同型半胱氨酸相关性慢性阻塞性肺疾病临床研究进展

在世界慢性疾病中,慢性阻塞性肺疾病(COPD)发病率和死亡率均较高,鉴于对患者生活造成严重伤害,越来越受到人们关注。同型半胱氨酸(Homocysteinemia,Hcy)对全身器官损害多有报道,随着研究的深入,Hcy目前对于肺部疾病的影响也受到重视。部分学者提出同型半胱氨酸可能是COPD发病机制中又一重要因素。Hcy在体内可以刺激产生大量的ROS和自由离子,并引起内皮细胞应激,还可降低肺脏内还原型谷胱甘肽含量。研究表明同型半胱氨酸水平在慢性阻塞性肺疾病中处于高表达状态,且其高表达水平与患者疾病的程度成相关性。本文将通过总结Hcy在肺脏及体内的代谢、各种应激反应等方面阑述同型半胱氨酸水平与COPD的相关性,并总结高水平的同型半胱氨酸相关的COPD治疗方法。     

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。     

重组BHMT对高同型半胱氨酸血症大鼠的影响

目的探讨重组BHMT对大鼠HHcy血症的防治作用。方法用含2%蛋氨酸(methionine,Met)饲料诱发HHcy的大鼠,经尾静脉注射BHMT后,观察模型大鼠血浆中同型半胱氨酸(homocysteine,Hcy)浓度、LDH酶活性及HDL/LDL的影响。结果重组BHMT能将HHcy组大鼠血浆中Hcy浓度(23.70±6.75)μmol/L显著降低为(14.61±2.80)μmol/L(P<0.05),LDH酶活性由(209.57±10.22)U/L降低为(225.04±30.47)U/L(P<0.05),对脂蛋白HDL/LDL基本没有影响。结论重组BHMT对高同型半胱氨酸血症有一定的治疗作用。     

组蛋白 H3K79同型半胱氨酸修饰位点在神经管畸形中的作用

目的:探讨人类胚胎脑组织中是否存在H3K79同型半胱氨酸修饰(H3K79Hcy)及其在神经管畸形(NTDs)中的作用。方法:通过质谱检测组蛋白H3K79是否存在同型半胱氨酸修饰位点。进一步合成包含组蛋白H3K79位点的同型半胱氨酸(Hcy)修饰的肽段,并与牛血清白蛋白(BSA)偶联后免疫兔子得到抗组蛋白H3K79Hcy多克隆抗体,并对抗体进行特异性检测;采用此抗H3K79Hcy抗体比较人类高Hcy NTDs样本和正常对照样本的H3K79Hcy水平。结果:(1)人胚胎组织组蛋白H3K79位点存在同型半胱氨酸修饰;(2)高Hcy水平NTDs脑组织中H3K79Hcy修饰水平高于正常对照(P0.05)。结论:人胚胎组织存在H3K79Hcy修饰,此修饰异常可能促进神经管畸形的发生。     

金属硫蛋白拮抗同型半胱氨酸对血管内皮细胞的脂质过氧化作用

目的:观察金属硫蛋白(metallothionein,MT)对同型半胱氨酸(homocysteine Hcy)损伤血管内皮细胞的拮抗作用及机制.方法:培养液中分别加入Hcy及Hcy MT孵育血管内皮细胞,自动生化分析仪测培养液乳酸脱氢酶(LDH)活性,硫代巴比妥酸法测细胞丙二醛(MDA)含量.结果:同型半胱氨酸增加内皮细胞LDH及蛋白漏出,并使细胞MDA含量明显升高.MT呈浓度依赖性地抑制Hcy所致的血管内皮细胞损伤及MDA增高.结论:MT拮抗Hcy对血管内皮细胞的脂质过氧化损伤.     

同型半胱氨酸对缺血性脑卒中的影响

同型半胱氨酸是引起缺血性脑卒中的重要风险因子,并且参与缺血后引起的的一系列损伤,其机制包括氧化应激、血管内皮损伤、炎症反应、表观遗传学改变等。同时,同型半胱氨酸也对卒中的复发、血管性痴呆、脑出血、卒中后肾功能不全等卒中后相关疾病有着广泛的影响。本文将分别从同型半胱氨酸参与卒中分子神经功能损伤机制以及卒中预后两个方面阐述同型半胱氨酸在脑缺血中的作用。     

硫化氢减轻同型半胱氨酸诱导大鼠海马CA1区神经元损伤

目的探讨硫化氢(H2S)对同型半胱氨酸(Hcy)诱导大鼠海马CA1区神经元损伤的改善作用。方法以侧脑室注射同型半胱氨酸的SD大鼠为同型半胱氨酸神经毒性动物模型,采用Tunel染色法分析大鼠海马CA1区神经元的凋亡情况,采用Elisa法分析大鼠海马组织MDA含量。结果 0.6μmol和2.0μmol的同型半胱氨酸使大鼠海马CA1区神经元发生大量的凋亡(P0.01),而NaHS(100μmol/kg)显著抑制同型半胱氨酸(0.6μmol)诱导大鼠海马CA1区神经元的凋亡(P0.05);同型半胱氨酸(0.2,0.6μmol)可增加大鼠海马CA1区丙二醛含量(P0.001),而NaHS(100μmol/kg)可抑制同型半胱氨酸(0.6μmol/L)诱导大鼠海马CA1区丙二醛水平的增加(P0.001)。结论硫化氢可减轻同型半胱氨酸诱导大鼠海马CA1区神经元的损伤。     

高效液相测定同型半胱氨酸方法的建立

为测量包括同型半胱氨酸在内的 1 8种氨基酸 ,用高效液相紫外检测法 ,在氨基酸混合标样中 ,加进Hcy标准品 ,仍用 4 5min程序分析 ,对AccqTag方法进行了修改 ,衍生温度改为常温 ,衍生后用醋酸酸化 ,并保存于 0～ 1℃ ,同型半胱氨酸出峰的时间为第 33min左右 ,结果得到了分离良好的 1 8种氨基酸图谱。     

同型半胱氨酸与不同类型卒中的相关性研究

目的：探讨同型半胱氨酸（Hcy）与不同类型脑卒中的关系,并对高Hcy血症成因作初步分析。方法：测定225例缺血性脑卒中和40例出血性脑卒中患者以及85例同龄健康受试者的血浆Hcy水平以及叶酸、维生素B12的浓度,将缺血性卒中按照TOAST分型分为不同临床亚组--动脉粥样硬化性脑血栓形成组,腔隙性脑梗死组,心源性脑栓塞组以及其他或不明原因脑梗死组,并分别与健康组进行对照研究。结果：血浆同型半胱氨酸平均水平在动脉粥样硬化性脑梗死组患者为（16.19±4.35）μmol/L,腔隙性脑梗死患者为（16.89±6.41）μmol/L,心源性脑栓塞组为（18.23±4.83）μmol/L,其他或不明原因脑梗死患者为（17.31±2.56）μmol/L,脑出血组患者为（14.91±4.54）μmol/L,均高于对照组（7.20±7.91）μmol/L,P〈0.05;各缺血性卒中组间同型半胱氨酸水平差异无显著性（P〈0.05）;缺血性卒中组患者血浆同型半胱氨酸水平高于出血性卒中组（P〈0.05）。卒中各组叶酸和维生素B12浓度均显著低于对照组（P〈0.05）。结论：血浆同型半胱氨酸在不同类型卒中中均升高,高血浆Hcy水平可能是脑卒中的独立危险因素,叶酸和VitB12缺乏可能是导致高Hcy血症的重要原因。     

Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys³⁴ of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys³⁴ of albumin and homocysteine attached to lysine residues.     

歼击飞行员飞行应激对同型半胱氨酸和皮质醇的影响

目的:探讨南部战区某旅歼击飞行员血清皮质醇(COR)和同型半胱氨酸(Hcy)联合检测的意义。方法:对歼击飞行员(A组)、地方健康体检人员(B组)、部队考学战士(C组)、空军地勤人员(C组)的血清COR、Hcy水平进行两两比较,通过Kruskal-Wallis统计学方法处理分析。结果:B组人员COR水平比A组、C组、D组人员低,差异有统计学意义(P<0.05);C组人员Hcy水平比A组、B组、D组人员高,差异有统计学意义(P<0.05)。结论:经常性的飞行训练可增强飞行员分泌激素的适应性,在航空保健工作中,COR联合Hcy监测及动态观察对保障飞行员的健康和飞行安全,提高部队战斗力具有一定的意义。     

Long-term prognostic significance of homocysteine in middle-aged and elderly

Objective: We investigated the association among increased levels of plasma homocysteine (Hcy), all-cause mortality, and cardiovascular events. Methods: Hcy was measured in 670 middle-aged and elderly subjects with no previous manifest cardiovascular disease. The follow-up period was 15 years. Results: Subjects with Hcy?≥?10.8?μmol/l (n?=?231) had a significant higher incidence of all-cause mortality (p?<?0.001) and CV events (p?<?0.001) compared with subjects with Hcy?<?10.8?μmol/l (n?=?439). However, there was no association on high levels of Hcy and VTE events or stroke. Conclusion: Increased levels of Hcy are associated with all-cause mortality and CV events.     

Comparative Study on in Vitro Effects of Homocysteine Thiolactone and Homocysteine on HUVEC Cells: Evidence for a Stronger Proapoptotic and Proinflammative Homocysteine Thiolactone

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However the underlying mechanisms responsible for endothelial cell injury with increased plasma concentration of homocysteine or homocysteine derivatives remains still incompletely elucidated. In this study, we investigated the ability of homocysteine (Hcy) and homocysteine thiolactone (HcyT) to induce cell death and IL-8 secretion in primary human umbilical vein endothelial cells (HUVEC). Hcy and HcyT were both cytotoxic and capable of promoting cell death, as measured by caspase-3 activation and DNA fragmentation. ELISA assays clearly demonstrated that Hcy and HcyT strongly activated IL-8 release. Furthermore, our results showed that HcyT was much more efficient than Hcy in activating caspase-3 or in inducing IL-8 secretion. The use of antioxidants such as vitamin C and vitamin E strongly but not completely reduced programmed cell death and chemokine release suggesting that other pathways different than reactive oxygen species are also involved. This study suggests that Homocysteine derivatives like HcyT might possess stronger cytotoxicity and pro-inflammatory properties and that Hcy derivatives levels should therefore be more taken into account during diagnostics.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Sustained Weight Loss in Obese Subjects Has Benefits That Are Independent of Attained Weight

Objective: To explore the hypothesis that sustained weight loss in severely obese patients may have benefits that are independent of their attained BMI. Research Methods and Procedures: We conducted a comparison of two weight‐stable groups with BMI in the 30 to 35 kg/m² range. Subjects (n = 79) were selected obese patients 3 years after laparoscopic adjustable gastric band surgery, and controls (n = 79) were obese patients seeking weight loss therapy. Subjects were selected in a de‐identified manner from our database to best match the control group. A range of clinical, biochemical, and questionnaire measures were obtained to assess obesity‐related health status Results: Subjects maintained a mean weight loss of 32.8 ± 18 kg after surgery. The weight loss subjects had significantly lower fasting plasma glucose, insulin, and triglyceride concentrations, along with higher high‐density lipoprotein‐cholesterol levels and better indirect measures of insulin sensitivity when compared with controls (p < 0.05 for all). In addition, aminotransferase levels, neutrophil counts, and globulin levels were also significantly lower in weight loss subjects. All differences in laboratory variables remained significant after controlling for BMI. The subjects also reported better health‐related quality of life, fewer symptoms of depression, and greater satisfaction with their appearance than controls. Discussion: These findings suggest that the post‐weight loss state conveys benefits that are greater than predicted by the attained BMI. These findings may have important implications regarding the expectations of weight loss therapy, and mechanisms for this effect should be carefully sought.     

Correlation between maternal plasma homocysteine and zinc levels in preeclamptic women

Women with preeclampsia have been shown to have elevated blood levels of the metabolite homocysteine, and alterations in blood levels of zinc and copper have also been reported. This study measured plasma levels of zinc, copper, and homocysteine in women with preeclampsia and in women with healthy, normotensive pregnancies. For the patients with preeclampsia compared with controls, significantly higher mean plasma levels were found of homocysteine (16.39 vs 9.45 nmol/mL; p≤0.001), zinc (15.53 vs 11.93 μg/g protein; p < 0.05), and copper (47.90 vs 31.60 μg/g protein; p=0.001). The ratio of plasma Cu/Zn levels tended to be higher in preeclamptic women and could be taken as an index of inflammatory reaction, but the difference was not significant. Homocysteine concentrations correlated positively with plasma zinc concentrations in women with preeclampsia (r=0.588, p=0.003) but not in women with healthy pregnancies. No correlations were observed between plasma levels of homocysteine and copper. Thus, the present study found evidence that preeclampsia might be associated with hyperhomocysteinemia and elevated blood levels of zinc and copper. Furthermore, elevated blood levels of zinc were significantly associated with hyperhomocysteinemia in preeclampsia. More studies are warranted to investigate further any relationship between altered homocysteine metabolism and levels of zinc and copper in preeclampsia.     

Rapid high‐yield N‐acylation of aminothiols: N‐acetylglutathione and N‐acetylhomocysteine and their thiol pKa values

Methodology for the rapid N‐acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN‐acetylated in ~100% yield in 0.1 M aqueous NaHCO₃ (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N‐hydroxysulfosuccinimidyl acetate, an efficient water‐soluble acetylating reagent. Following acetone precipitation, diN‐acetylGSSG was further purified and desalted on a strong anion‐exchange (SAX) cartridge. DiN‐acetylhomocystine was simultaneously purified and desalted on a C₁₈ cartridge. The N‐acetylated aminothiols were generated using gel‐immobilized tris(2‐carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N‐acetylglutathione, which was purified on the SAX cartridge. pH titrations of N‐acetylglutathione (8.99) and N‐acetylhomocysteine (9.66) as well as those of commercially available N‐acetylcysteine (9.53) and N‐acetylpenicillamine (10.21) yielded pK_a(SH) values of importance for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

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.