Simultaneous determination of total plasma glutathione, homocysteine, cysteinylglycine, and methionine by high-performance liquid chromatography with electrochemical detection

We here describe an ion-exchange high-performance liquid chromatography technique with electrochemical detection for rapid quantification of glutathione, homocysteine, cysteinylglycine, and methionine. The analytical validation of the technique showed within-assay and between-assay coefficients of variation between 3.1 and 4.3%, and 3.7 and 8.6%, respectively. Percentages of recovery for overload and dilution tests were between 87 and 120%. Detection limits were 1 micromol/L for methionine and 0.5 micromol/L for other compounds. There was no interference with any physiological and pharmacological substances possessing a thiol function. Aminothiol concentrations determined in 100 control subjects (50 women and 50 men) showed no age- or sex-rated differences for except for homocysteine which was increased (+ 28%) in oldest subjects of both sexes. In 60 patients at risk (30 with chronic renal failure, 30 with diabetes), homocysteine concentration was significantly increased. No variation in other aminothiols was observed in diabetic subjects. Methionine was decreased and cysteinylglycine was increased in patients with chronic renal failure. The present technique-rapid, easy to use, and reliable-appears suitable for routine application in the exploration of aminothiol metabolic pathways including mechanisms of hyperhomocysteinemia.     

血清糖化白蛋白和同型半胱氨酸与冠状动脉病变的相关性

目的 探讨冠状动脉病变程度与血清糖化白蛋白（glycated albumin,GA）、同型半胱氨酸（homocysteine,Hcy）的关系及临床意义。方法 行冠状动脉造影检查患者151例,依据检查结果分为冠心病组121例和非冠心病组30例,冠心病组根据病变血管支数、Gensini积分、SYNTAX积分分为不同亚组,分析冠心病组和非冠心病组、冠心病不同亚组GA、Hcy水平差异。结果 冠心病组血清GA、Hcy、高密度脂蛋白胆固醇、陈旧性心肌梗死构成比分别为（15.38±3.68）%、（15.55±7.23）μmol/L、（0.93±0.22）mmol/L、18.2%,非冠心病组分别为（13.88±2.0）%、（12.73±4.42）μmol/L、（1.06±0.21）mmol/L、3.3%,2组比较差异有统计学意义（P〈0.05）;不同病变血管支数冠心病患者GA、Hcy水平比较差异无统计学意义（P〉0.05）;冠心病组Gensini积分、SYNTAX积分与血清GA、Hcy水平呈正相关（r值为0.4969~0.6731,P〈0.05）。结论 GA与Hcy联合检测对冠状动脉病变程度有预测价值。     

急性脑梗死患者血浆同型半胱氨酸、半胱氨酸、叶酸及维生素B_(12)水平的变化及其相关性

目的观察急性脑梗死(ACI)患者血浆同型半胱氨酸(Hcy)、半胱氨酸(Cys)、叶酸(FA)及维生素B12(Vit B12)水平的变化,并探讨其相关性。方法 80例ACI患者根据动脉狭窄情况分为观察组A(36例)和观察组B(44例),另选取同期健康体检者30例设为对照组,检测3组血浆Hcy、Cys、FA及Vit B12水平。结果血浆Hcy、Cys水平在观察组A、B及对照组中呈逐渐下降趋势,FA、Vit B12水平在3组中呈逐渐上升趋势,两两比较差异显著(P0.05或P0.01)。相关性分析表明,血浆Hcy水平与Cys显著正相关(r=0.7798,P0.01),与FA及Vit B12水平负相关(r=-0.623,0.593,P0.01)。结论 ACI患者血浆Hcy、Cys水平显著升高,FA、Vit B12水平显著降低,高Hcy血症可能参与了ACI的发病过程。     

Specific and sensitive imaging of basal cysteine over homocysteine in living cells

Biological thiols play important roles in maintaining appropriate redox status of organisms. Accepting the challenge to differentiate structurally similar cysteine (Cys) and homocysteine (Hcy), we have successfully developed a miniature synthetic turn-on fluorescent probe based on 6-(2-benzothiazolyl)-2-naphthalenol for Cys. This probe is able to specifically react with Cys to yield its naphthalenol derivative, accompanied by remarkable green fluorescence enhancement with a detection limit of 14.8 nM. Besides, this probe displays much greater selectivity for Cys over other biological thiols, including homocysteine (Hcy) and glutathione (GSH). Practically, good cell permeability and low cytotoxicity make it suitable for monitoring basal Cys in living cells.

A turn-on fluorescent probe conjugating with a reactive acrylate for visualization of basal Cys specifically in living cells was developed.     

Testosterone-binding globulins in human plasma: studies on sex distribution and specificity

When human plasma is mixed with testosterone-(3)H and subjected to electrophoresis on paper in glycine acetate buffer at pH 8.6, at least two proteins other than albumin bind the testosterone. In normal women 80.5 +/- 1.9% (SEM) of the recovered radioactivity migrates with the beta-globulins, 7.3 +/- 0.80% with the inter-alpha-globulins, and 4.3 +/- 0.40% with albumin. In normal men the percentages are 69.3 +/- 3.0%, 14.3 +/- 1.6%, and 6.2 +/- 1.1%, respectively. These differences between men and women in binding among the beta-globulins and inter-alpha-globulins are statistically significant (P < 0.001). The highest percentages of radioactivity associated with the beta-globulins are seen in infants of both sexes, men receiving diethylstillbestrol, and pregnant women. These same subjects have the lowest percentages of radioactivity associated with the inter-alpha-globulins. Experiments with carrier testosterone indicate that at least some of the differences between the normal men and women and infants can be explained by differences in the concentration of endogenous testosterone. This factor alone, however, cannot explain the increased binding among the beta-globulins in the men receiving diethylstilbestrol or in the pregnant females. In this system estrone, estradiol, dehydroisoandrosterone, androsterone, 17alpha-hydroxyprogesterone, and 19-nortestosterone compete with testosterone for binding sites on the proteins. None is as potent as testosterone itself.     

同型半胱氨酸与脑梗死大小的相关性研究

目的探讨血浆同型半胱氨酸水平与脑梗死及其不同亚型之间的关系。方法选取105 例脑梗死患者,参照TOAST标准分为大梗死(56例)和小梗死(49例)2个亚组。对照组为健康体检者50名。采用酶联免疫吸附法测定空腹血浆总同型半胱氨酸水平。结果脑梗死组血浆同型半胱氨酸水平为(24. 85±24. 56)μmol/L,高于对照组(16 .18±6. 97)μmol/L(P<0. 05);大梗死组血浆同型半胱氨酸水平为(30 .46±31. 16)μmol/L,高于小梗死组(18 .43±10. 73)μmol/L和对照组(均P<0. 05);小梗死组和对照组之间无显著性差异。结论大梗死组血浆同型半胱氨酸水平升高,提示同型半胱氨酸水平升高是动脉粥样硬化性脑梗死的独立危险因素。     

The colorimetric and microfluidic paper-based detection of cysteine and homocysteine using 1,5-diphenylcarbazide-capped silver nanoparticles

We have prepared a microfluidic paper-based analytical device (μPAD) for the determination of cysteine and homocysteine based on 1,5-diphenylcarbazide-capped silver nanoparticles. The μPAD was developed to identify and quantify the levels of cysteine and homocysteine. The proposed μPAD enabled the detection of cysteine and homocysteine using a colorimetric reaction based on modified silver nanoparticles. The color of the modified AgNPs in the test zone immediately changed after the addition of cysteine and homocysteine. Based on this change, the quantification of these two amino acids was achieved using an RGB color model and ImageJ software. Under optimized conditions, the proposed device enabled the determination of cysteine in the 0.20–20.0 μM concentration range with a limit of detection (LOD) of 0.16 μM. In addition, the LOD of homocysteine was calculated to be 0.25 μM with a linear range of 0.50–20.0 μM. In this work, we focused on the use of the μPAD for the analysis of a series of human urine samples.

A simple and novel portable method for the quantitative measurement of cysteine and homocysteine in human urine samples is presented.     

脑卒中与血浆同型半胱氨酸水平的关系

背景很多研究表明高同型半胱氨酸血症可能是导致脑卒中发病的独立危险因素.目的探讨高同型半胱氨酸血症与脑梗死和脑出血发生之间的关系,并分析可能影响同型半胱氨酸水平的相关因素.设计病例-对照实验.单位浙江大学医学院附属第二医院神经内科.对象选择2003-01/11在浙江大学医学院附属第二医院的住院患者57例,其中脑卒中57例(脑出血组21例,脑梗死组36例),并以同期到本院作体检的健康人28例作为对照组.方法所有被试者均晨起空腹抽取静脉血2 mL,测定血浆同型半胱氨酸、维生素B12、叶酸、肌酐等含量,并对所有患者进行临床神经功能缺损程度评分,脑出血组患者根据CT摄片计算出每例患者的血肿体积.主要观察指标①各组被试者血浆同型半胱氨酸水平.②血浆同型半胱氨酸水平与叶酸,维生素B12,临床神经功能缺损程度评分及血肿体积的相关性.结果57例脑卒中患者和28例健康者全部进入结果分析.①脑梗死组、脑出血组男性和女性同型半胱氨酸的水平均高于同性别对照组[(25.2±21.4),(18.3±10.9),(11.5±2.9)μ mol/L;(22.8±18.9),(14.7±7.4),(10.8±2.6)μ mol/L,P＜0.05-0.01].脑梗死组和脑出血组基本接近(P＞0.05).②脑梗死组、脑出血组患者同型半胱氨酸水平与叶酸水平呈极显著负相关(r=-0.442,-0.531,P＜0.05),与维生素B12水平无显著相关性(r=-0.086,-0.111,P＞0.05).脑梗死组同型半胱氨酸水平与临床神经功能缺损程度评分无显著相关性(r=-0.139,P＞0.05),脑出血组同型半胱氨酸水平与临床神经功能缺损程度评分和血肿体积无显著相关性(r=0.225,0.425,P＞0.05).结论高同型半胱氨酸血症是脑梗死和脑出血的危险因素.血浆同型半胱氨酸水平与叶酸水平呈负相关,与维生素B12、临床神经功能缺损程度评分、血肿体积无显著相关.     

脑卒中与血同型半胱氨酸水平的关系

目的探讨脑卒中与同型半胱氨酸 (Hcy)的关系。 方法选取脑卒中患者 5 7例 ,其中脑出血 (CH)组 2 1例 ,脑梗死(CI组 ) 3 6例 ,另选正常对照 2 8例 ,分别测定血浆Hcy、VitB12 、叶酸、肌酐浓度 ,对所有患者进行临床神经功能缺损指数 (CNDS)评定 ,并测定CH组的血肿体积 (HV)。结果CI组和CH组的Hcy水平均比对照组高 (P <0 0 5或P <0 0 1) ,而CI组和CH组之间比较无显著性差异。CI组和CH组Hcy与叶酸水平呈高度显著负相关性 (r =-0 44 2 ,P =0 0 0 7)或显著负相关 (r =-0 5 3 1,P= 0 0 13 ) ,均与VitB12 水平无显著相关性。结论高Hcy血症与脑卒中关系密切 ;血浆Hcy水平与叶酸水平呈负相关 ,与VitB12 相关不显著。     

脑卒中与血浆同型半胱氨酸水平的关系

背景：很多研究表明高同型半胱氨酸血症可能是导致脑卒中发病的独立危险因素。目的：探讨高同型半胱氨酸血症与脑梗死和脑出血发生之间的关系，并分析可能影响同型半胱氨酸水平的相关因素。设计：病例-对照实验。单位：浙江大学医学院附属第二医院神经内科。对象：选择2003—01/11在浙江大学医学院附属第二医院的住院患者57例，其中脑卒中57例（脑出血组21例，脑梗死组36例），并以同期到本院作体检的健康人28例作为对照组。方法：所有被试者均晨起空腹抽取静脉血2mL，测定血浆同型半胱氨酸、维生素B12、叶酸、肌酐等含量，并对所有患者进行临床神经功能缺损程度评分，脑出血组患者根据CT摄片计算出每例患者的血肿体积。主要观察指标：①各组被试者血浆同型半胱氨酸水平。②血浆同型半胱氨酸水平与叶酸，维生素B12，临床神经功能缺损程度评分及血肿体积的相关性。结果：57例脑卒中患者和28例健康者全部进入结果分析。①脑梗死组、脑出血组男性和女性同型半胱氨酸的水平均高于同性别对照组[（25．2&;#177;21．4），（18．3&;#177;10．9），（11．5&;#177;2．9）μmol/L：（22．8&;#177;18．9），（14．7&;#177;7．4），（10．8&;#177;2．6）μmol/L，P〈0．05～0．01]。脑梗死组和脑出血组基本接近（P〉0．05）。②脑梗死组、脑出血组患者同型半胱氨酸水平与叶酸水平呈极显著负相关（r=-0．442，-0．531，P〈0．05），与维生素B12,水平无显著相关性（r=-0．086，-0．111，P〉0．05）。脑梗死组同型半胱氨酸水平与临床神经功能缺损程度评分无显著相关性（r=-0．139，P〉0．05），脑出血组同型半胱氨酸水平与临床神经功能缺损程度评分和血肿体积无显著相关性（r=0．225，0．425，P〉0．05）。结论：高同型半胱氨酸血症是脑梗死和脑出血的危险因素。血浆同型半胱氨酸水平与叶酸水平呈负相关，与维生素B12、临床神经功能缺损程度评分、血肿体积无显著相关。     

Effects of some pre-analytical conditions on the measurement of homocysteine and cysteine in plasma.

The association of hyperhomocysteinemia and hypercysteinemia with the risk of arterial and venous thrombosis is well documented. While it is known that standardized pre-analytical conditions are necessary for reliable measurement of plasma total homocysteine, the effects of pre-analytical conditions on cysteine measurement are less well known. The aim of this study was to evaluate the effects of pre-analytical conditions on the measurement of homocysteine and cysteine. We observed that the concentration of total homocysteine in plasma increased significantly with time (38% after 6 h), whereas total cysteine decreased (5% after 2 h) when blood anticoagulated with ethylenediaminetetraacetic tripotassium salt was kept at room temperature. These changes were minimized when acidic citrate dextrose was used as an anticoagulant and were abolished when blood samples were immediately placed on crushed ice, independently of the anticoagulant. Storage of plasma for 72 h at room temperature induced a small (approximately equal to 6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72 h, independently of the anticoagulant used. In conclusion, if blood samples for plasma total homocysteine and cysteine measurement cannot be kept on ice, they should be collected in acidic citrate dextrose to minimize the artifactual changes.     

The influence of smoking on plasma homocysteine and cysteine levels in passive and active smokers.

Total plasma homocysteine (tHcy) and cysteine (tCys) levels are associated with cardiovascular diseases. One of the determinants that influence their levels is cigarette smoking. The aim of this study was to determine the relationship between plasma levels of both amino acids and urinary cotinine concentration as a reliable biomarker of tobacco smoke exposure. One hundred and seventeen volunteers (61 women and 56 men) aged 19-60 years (mean 40.3 +/- 11.0) were included in the study. The study subjects were qualified into non-smokers, passive smokers and active smokers based upon the urinary cotinine concentration. In each particular group, plasma tHcy and tCys levels were measured and evaluated in the whole population and separately in women and men. Statistically insignificant differences in plasma tHcy and tCys levels in the whole group of passive smokers in comparison with non-smokers were observed (11.47 vs. 10.94 micromol/l, p=0.414, and 253.0 vs. 266.9 micromol/l, p=0.163, respectively). However, statistically significant differences in plasma tHcy levels (13.29 vs. 10.94 micromol/l, p=0.011) and in plasma tCys levels (218.2 vs. 266.9 micromol/l, p<0.001) were found in the whole group of active smokers compared with non-smokers. The Pearson's coefficient (r) for the correlation between plasma tHcy level and urinary cotinine concentration was r=0.630 (p<0.001) in the whole group of active smokers and r=0.480 (p=0.003) in the whole group of passive smokers. The correlation between plasma tCys level and urinary cotinine concentration in both study groups was insignificant. Similar results were obtained when calculated separately for men and women. The results suggest that cigarette smoking is a strong determinant of plasma tHcy level, but it is not a determinant of plasma tCys level.     

脑血管病患者总同型半胱氨酸水平与血肌酐相关性及肾脏代偿调节的性别差异分析

目的探讨正常血肌酐(Scr)水平脑血管病患者总同型半胱氨酸(tHcy)水平、与血肌酐的相关性及肾脏代偿调节的性别差异。方法测定脑梗死(CI)组男278例,女160例,脑出血(CH)组男22例,女16例,短暂性脑缺血发作(TIAs)组男27例,女20例的空腹血清tHcy、Scr、甘油三酯(TG)、总胆固醇(TCHO)、高密度脂蛋白胆固醇(HDLC)、低密度脂蛋白胆固醇(LDLC)并对上述3组患者伴有高血压、糖尿病的情况进行调查。结果3组间比较:CI组血tHcy明显高于TIAs组(P<0.001),CI组、CH组、TIAs组高同型半胱氨酸血症(HHe)发生率依次为41.5%、32.5%、19.1%(P=0.008);Scr水平差异无统计学意义(P>0.05)。男、女组间(CI、CH、和TIAs组,男327例、女196例)比较:tHcy差异有统计学意义[(18.36±11.30)μmol/L,(13.11±6.21)]μmol/L,P<0.001],男性明显高于女性;男性HHe发生率明显高于女性(47.1%,25.0%P<0.001);男性Scr明显高于女性(P<0.001);女性TG、TCHO、HDLC、LDLC明显高于男性(P=0.002～P<0.001);伴高血压、糖尿病男女性别之间差异无统计学意义(P>0.05)。LgtHcy与LgScr经Pearson相关分析显示:男性组相关系数r=0.166(P=0.003);女性r=0.405(P<0.001),女性相关系数明显高于男性。按性别分组、Scr分层作图显示:男性在Scr水平位于70～100μmol/L区间内肾脏对血tHcy具有明显的代偿调节作用,女性则未表现出明显的肾代偿调节。结论血tHcy水平和HHe发生率在脑血管病的不同类型中差异有统计学意义。男性较女性血tHcy明显升高是致男性较女性多发脑血管病的一个重要原因。男、女Scr水平位于70～100μmol/L区间肾脏对血tHcy代偿调节不同。     

老年轻度认知功能障碍与血清总同型半胱氨酸、血脂水平相关性分析

目的观察老年轻度认知功能障碍（MCI）与血清总同型半胱氨酸（tHcy）、血脂水平之间的关系。方法测定50例老年MCI患者（MIC组）和50例年龄相匹配的健康者（对照组）血清总胆固醇（TC）、三酰甘油（TG）、高密度脂蛋白（HDL）、低密度脂蛋白（LDI。）及血清tHcy水平。结果MCI组患者血清Tc、TG、HDL、LDL的水平分别为（5．72±1．25）mmol／L、（1．90±O．31）mmol／L、（1．034-0．12）mmol／L、（3．97±1．02）mmol／L;对照组健康者相应指标分别为（4．27土1．04）mmol／L、（1．21±0．17）mmo／I。（1．34士0．25）mmol／I．、（3．03±1_00）mmol／I。。MCI组TC、LDI,、TG均明显高于对照组,差异有统计学意义（P〈0．05）。MCI组血清tHcy水平明显高于对照组（P〈0．05）。血清Hcy、血脂对预测MCI具有价值（P〈0．05）。Logistic多因素回归分析提示血浆Hcy、血脂是MCI的影响因素。结论MCI患者存在明显的脂质代谢紊乱及高同型半胱氨酸血症,Hey、TC、LDL、TG可能对MCI的发病有一定的影响,而HDI。与MCI发病可能无关联。,