谷胱甘肽的化学与医疗作用

谷胱甘肽 （glutathione,GSH）在体内以氧化型 （GSSG）和还原型 （GSH）2种形式存在。GSH分子中的巯基容易失氢氧化,因而可以消除体内的活性氧,具有保护酶和蛋白质免受氧化,保护脏器免受损伤的功效。人工合成的GSH已广泛用于临床。     

氧化型谷胱甘肽对还原型谷胱甘肽清除自由基的协同作用

利用分光光度法和基质辅助飞行质谱法研究了谷胱甘肽对1,1-二苯基-2-苦肼基(DPPH)自由基的清除作用.通过比较不同浓度和不同配比的还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)以及Na2SeO3混合溶液的自由基清除率,发现GSH/GSSG的配比对自由基清除率有明显影响.当GSH/GSSG的配比大于50∶ 1时,自由基清除率比同浓度的GSH大,且自由基清除率随GSH和GSSG的绝对浓度的增加而明显增加,说明适量的GSSG可协同催化GSH清除自由基过程.质谱测定结果表明: 此协同作用与GSSG 参与自由基清除过程中的自由基反应有关.Na2SeO3对GSH的清除自由基的影响主要是通过与GSH反应生成GSSG来调控GSH/GSSG配比的结果.通过测定和分析一定配比的GSH+GSSG混合溶液与DPPH作用前后的质谱图,提出了少量的GSSG共存下,GSH催化清除DPPH自由基的作用机理.     

谷胱甘肽键合柱对变性核糖核酸酶的复性研究

将氧化还原型谷胱甘肽(GSH/GSSG)共价键合到色谱固定相上, 实现了对变性核糖核酸酶(RNase)的复性. 实验发现, 谷胱甘肽键合柱具有典型的弱阳离子交换性质, 在离子交换(IEC)模式下能够对4种标准蛋白进行基线分离, 且具有较高的柱效. 当蛋白浓度为5 mg/mL, 流速为0.2 mL/min时, 在流动相中不加GSH/GSSG的条件下, GSH/GSSG柱对变性核糖核酸酶的活性回收率可达(39.5±3.8)%, 而普通IEC柱对变性核糖核酸酶的活性回收率几乎为0, 说明其对变性蛋白二硫键的正确对接具有明显的促进作用; 在收集液中加入GSH/GSSG后, 其活性回收率可达到(81.5±4.3)%. 本文结果对蛋白折叠液相色谱法的发展及降低蛋白复性成本具有一定的应用价值.     

注射用还原型谷胱甘肽钠的毛细管区带电泳分离及其光学纯度分析

运用毛细管区带电泳(CZE),以pH=6.59的60 mmol/L Na2 HPO4-NaH2 PO4作为电泳缓冲溶液,测定波长为200 nm,还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)于5 min内达到基线分离.GSH和GSSG的线性范围分别为4～1 000 μmol/L和2～1 000μmol/L,相关系...     

聚乳酸-聚半胱氨酸混纺纤维毡用于谷胱甘肽S-转移酶的捕获和洗脱

本文研究一种分离纯化GST（谷胱甘肽S-转移酶）的方法：将嵌段聚合物聚左旋乳酸-聚半胱氨酸（PLLA—PCys）电纺丝制成超细纤维,将还原型谷胱甘肽（GSH）通过二硫键偶联到纤维上,再将纤维填充玻璃管,制成吸附柱;含有GST的混合溶液通过吸附柱时,GST被纤维捕获;再用含GSH的溶液流过吸附柱,GST被洗脱下来,经冷冻干燥获得GST纯品．用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳进行检测．结果表明,键合了GSH的PLLA—PCys混纺纤维毡能够从蛋白混合溶液中专一性地捕获GST,被捕获的GST可以有效地回收．所以,此种键合了GSH的电纺丝纤维可望用于GST或以GST为标签的蛋白的分离、纯化以及检测．     

毛细管区带电泳检测糖尿病肾病变患者血液内红细胞中的谷胱甘肽

采用高效毛细管区带电泳法对糖尿病肾病变患者血液内红细胞中参与氧化应激的氧化型谷胱甘肽(GSSG)和还原型谷胱甘肽(GSH)进行了测定。对影响分离的条件(缓冲液pH、缓冲液浓度、分离电压和毛细管温度)进行了优化,使用非涂层的毛细管(21 cm×75 μm i.d.)和20 mmol/L pH 6.86的磷酸缓冲液,在25 kV,30 ℃和200 nm条件下,可在3 min内同时对血红细胞中GSSG和GSH定量分析。方法具有良好的重现性(迁移时间和峰面积的相对标准偏差均小于2.0%)、较高的灵敏度(GSH:     

一种便携快速测量水中铅和镉含量的仪器-ANDalyze

快速测量污染水中重金属元素含量对于监测野外突发污染至关重要.建立了使用以生物酶（DNA酶）为原理的便捷仪器快速测定污染水中铅（Pb）和镉（Cd）元素含量的方法.使用生物酶传感器对标准溶液进行测量,根据溶液推荐和测量浓度之间的线性关系对仪器进行校准后,可测量的质量浓度范围:Pb为2~100 μg/L,Cd为0.1~1.0 mg/L.仪器可以在3~5 min内方便快速完成重金属的现场测量,使用DNA酶可以快速获得污染水中的微量金属元素含量,有利于野外重金属污染的即时测量.     

工程化蛋白质纳米孔实时监测镉离子与谷胱甘肽的单分子反应

本研究利用合成的全-6-季铵基-β-环糊精(Per-6-quaternary ammonium-β-cyclodextrin,p-QABCD)装备基因工程化的α-溶血素(α-Hemolysin,αHL)蛋白质纳米孔(M113R)7,构建全新的单分子纳米孔反应器,在单分子水平实现对谷胱甘肽(GSH)和镉离子(Cd2+)的络合反应的实时原位监测,并辨认络合反应的不同路径、反应中间产物和最终产物.结果表明,溶液的pH值显著影响GSH与Cd2+络合产物的络合比例.pH=7.4时,GSH与Cd2+络合反应的最终产物为Cd(GSH)2;pH=9.0时,最终产物为Cd(GSH)2和Cd2(GSH)2.其中,Cd2(GSH)2的形成遵循两种路径:(1)一个Cd2+首先结合两个GSH分子的巯基形成Cd(GSH)2,然后,第二个Cd2+结合去质子化的氨基形成Cd2(GSH)2;(2)两个Cd2+分别结合同一个GSH分子的巯基和去质子化的氨基形成Cd2(GSH)1,然后,第二个GSH分子的巯基和去质子化的氨基结合Cd2(GSH)1的Cd2+形成Cd2(GSH)2.本研究实现了在单分子水平无标记和无化学修饰研究金属离子和生物小分子的反应,对理解细胞内重金属的解毒机理和拓展纳米孔单分子技术的研究领域具有重要意义.     

还原型和氧化型谷胱甘肽在磁性Fe3O4纳米粒子上的吸附组装

研究了Fe3O4磁性纳米粒子在水溶液中对还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)的吸附。分别测定了两种物质在不同温度下的吸附等温线,比较了研究结果,并用Freundlich吸附等温式对数据进行拟合,根据公式计算出相关的吸附热力学函数值,研究了碳化二亚胺含量和pH对吸附结果的影响,并从吸附量和脱附实验结果探讨了其可能的吸附机理。结果表明,还原型谷胱甘肽和氧化型谷胱甘肽在Fe3O4表面的吸附是不可逆的,加入强电解质基本不能够脱附,两者间形成了部分共价键。     

还原变性核糖核酸酶在疏水性液-固界面上的复性

采用疏水相互作用色谱(HIC)对还原变性核糖核酸酶A (RNase A)在疏水性液-固界面上的复性进行了研究。详细讨论了流动相中脲的浓度、还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)的比例、流动相pH和变性蛋白质浓度对还原变性RNase A复性效率和质量回收率的影响。结果表明,在最优化的复性条件(流动相中含有2.0 mol/L脲,GSH/GSSG的浓度比为8:1,流动相pH为8.0)下,还原变性RNase A能完全复性。当变性蛋白质质量浓度为5.0 mg/mL时,还原脲变性RNase A的活性回收率和质量回收率分别为98.0%和61.9%,还原胍变性RNase A分别为100.1%和66.8%。研究表明HIC是还原变性蛋白质复性的有力工具之一,可为蛋白质复性研究提供新方法和新思路。     

The Antioxidant Effect of Curcumin and Rutin on Oxidative Stress Biomarkers in Experimentally Induced Periodontitis in Hyperglycemic Wistar Rats

Background: There is a growing interest in the correlation between antioxidants and periodontal disease. In this study, we aimed to investigate the effect of oxidative stress and the impact of two antioxidants, curcumin and rutin, respectively, in the etiopathology of experimentally induced periodontitis in diabetic rats. Methods: Fifty Wistar albino rats were randomly divided into five groups and were induced with diabetes mellitus and periodontitis: (1) (CONTROL)—control group, (2) (DPP)—experimentally induced diabetes mellitus and periodontitis, (3) (DPC)—experimentally induced diabetes mellitus and periodontitis treated with curcumin (C), (4) (DPR)—experimentally induced diabetes mellitus and periodontitis treated with rutin (R) and (5) (DPCR)—experimentally induced diabetes mellitus and periodontitis treated with C and R. We evaluated malondialdehyde (MDA) as a biomarker of oxidative stress and reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG and catalase (CAT) as biomarkers of the antioxidant capacity in blood harvested from the animals we tested. The MDA levels and CAT activities were also evaluated in the gingival tissue. Results: The control group effect was statistically significantly different from any other groups, regardless of whether or not the treatment was applied. There was also a significant difference between the untreated group and the three treatment groups for variables MDA, GSH, GSSG, GSH/GSSG and CAT. There was no significant difference in the mean effect for the MDA, GSH, GSSG, GSH/GSSG and CAT variables in the treated groups of rats with curcumin, rutin and the combination of curcumin and rutin. Conclusions: The oral administration of curcumin and rutin, single or combined, could reduce the oxidative stress and enhance the antioxidant status in hyperglycemic periodontitis rats.     

Oxidative stress and immunologic responses following a dietary exposure to PAHs in <Emphasis Type="Italic">Mya arenaria</Emphasis>

Background  

The aim of this research was to investigate oxidative stress and immune responses following a dietary polycyclic aromatic hydrocarbon (PAH) exposure in a marine bioindicator organism, the soft shell clam, Mya arenaria. Immune parameters in hemolymph (haemocyte number, efficiency of phagocytosis and haemocyte activity) and assessment of oxidative stress using catalase (CAT) activity and levels of malondialdehyde (MDA) performed on the digestive gland were estimated as biomarkers in clams fed in mesocosm with PAH contaminated phytoplankton. MDA levels and CAT activities were also measured in situ in organisms sampled in a control site (Metis Beach, Québec, Canada) as well as organisms sampled in a site receiving domestic effluents (Pointe-au-Père, Québec, Canada), to assess effects of abiotic variables related to seasonal variations and mixed contamination on the selected parameters.     

Effects of CO2 laser pretreatment on drought stress resistance in wheat

In order to determine the role of laser in drought stress resistance of spring wheat (Triticum aestivum L.), seed embryos were exposed to CO2 laser radiation for 0min, 1min, 3min and 5min, respectively, and when the seedlings were 12 days old they were treated with 10% (w/v) PEG6000 solution for 10 days. Changes in the concentration of malondialdehyde (MDA), hydrogen peroxide (H2O2), glutathione (GSH), ascorbate (AsA), oxidized glutathione (GSSG), carotenoid, zeaxanthin, the production rate of superoxide radical (O2(-)), the activities of ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GP), glutathione-S-transferase (GST) and the growth parameters of seedlings (plant height, leaf area and dry weight) were measured to test the effects of laser pretreatment. The results showed that suitable laser pretreatment of embryos enhanced drought stress resistance in wheat seedlings by decreasing the concentration of MDA and H2O2, GSSG, the production rate of O2(-), leaf area and increasing the activities of APX, GST, GP and POD and AsA, carotenoid and zeaxanthin concentration. It is suggested that those changes in MDA, O2(-) H2O2, anti-oxidative enzymes and anti-oxidative compounds were responsible for the increase in drought stress resistance observed in the experiments. The results also showed that the laser had a long-term positive physiological effect on the growth of drought stress seedlings. This is the first investigation reporting the use of CO2 laser pretreatment to enhance drought stress resistance of spring wheat.     

Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice

Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC–MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress.     

Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) column chemistries for the UPLC-MS-MS analysis of glutathione, glutathione disulfide, and ophthalmic acid in mouse liver and human plasma

Glutathione (GSH), glutathione disulfide (GSSG), and ophthalmic acid (OA) are important biological oxidative stress biomarkers to be monitored in pathological and toxicological studies. With the advent of liquid chromatography tandem mass spectrometry (LC-MS-MS) technology, sensitive and selective analysis of these biomarkers in biological samples is now being performed routinely. Due to the hydrophilic and polar natures of GSH and its endogenous derivatives, achieving good retention, resolution, and peak shape is often a chromatographic challenge. In this study, three ultra-performance (UP) LC column chemistries (namely, BEH C18, BEH HILIC, and HSS T3 [C18]) are evaluated for the UPLC-MS-MS analysis of GSH, GSSG, and OA extracted from mouse liver and human plasma samples. The chromatographic parameters evaluated are retentivity, tailing factor, MS sensitivity, and resolution of the three analytes. Based on the optimized method for each column chemistry, our results indicate that the HSS T3 (C18) column chemistry affords the best retention and separation of these analytes when operated under the ultra high-pressure chromatographic conditions.     

Effects on feeding rate and biomarker responses of marine mussels experimentally exposed to propranolol and acetaminophen

Environmental risk assessments of human pharmaceuticals and other ‘emerging contaminants’ should integrate both population-relevant endpoints and biomarkers of potential modes of action in a range of species. Adult Mytilus galloprovincialis were exposed to the beta-adrenergic receptor blocker propranolol or to the anti-inflammatory drug acetaminophen (paracetamol), both commonly used therapeutic drugs present in aquatic ecosystems. Mussels were exposed under semi-static conditions for 10 days to either acetaminophen (CAS number 103-90-2; mean measured concentrations 23 and 403 μg/L) or propranolol hydrochloride (CAS number 318-98-9; mean measured propranolol concentrations 11 and 147 μg/L) at 15 ± 1 °C sea water. Feeding rate was assessed as an indicator of general toxicity. For propranolol, the 10-day no-observed effect concentration (^{feeding rate}NOEC) and lowest observed effect concentration (^{feeding rate}LOEC) were 11 and 147 μg/L, respectively. For acetaminophen, feeding rate was increased at both 23 and 403 μg/L, suggesting a 10-day ^{feeding rate}NOEC of 403 μg/L. Primarily, phase I carboxylesterase (CbE), phase II glutathione S-transferase (GST) and the anti-oxidant catalase activities were evaluated in digestive gland. Gill GST and acetylcholinesterase (AChE) activities were also measured. Lipid peroxidation (LPO) levels were measured in both tissues to assess oxidative stress. Some enzymatic activities in liver were also reduced after propranolol exposure whilst acetaminophen enhanced them (CbE p < 0.05). Acetaminophen exposure significantly increased hepatic LPO levels and inhibited AChE activity in gill (10-day NOEC and LOEC of 23 and 403 μg/L, respectively), whereas propranolol (11 μg/L) enhanced gill GST.     

Neuroprotective activity of lavender oil on transient focal cerebral ischemia in mice

The air-dried aerial parts of Lavandula angustifolia Mill, a traditional Uygur herbal drug, is used as resuscitation-inducing therapy to treat neurodisfunctions, such as stroke. This study was designed to assess the neuroprotective effects of lavender oil against ischemia/reperfusion (IR) injury in mice. Focal cerebral ischemia was induced by the intraluminal occlusion method with a nylon string. The neurodysfuntion was evaluated by neurological deficit and the infarct area was showed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The histopathological changes were observed by hematoxylin and eosin staining. The levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA) and carbonyl, the ratio of reduced glutathione (GSH)/glutathione disulfide (GSSG), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GSH-Px) in brain tissue were measured to estimate the oxidative stress state. Neurological deficit, infarct size, histopathology changes and oxidative stress markers were evaluated after 22 h of reperfusion. In comparison with the model group, treatment with lavender oil significantly decreased neurological deficit scores, infarct size, the levels of MDA, carbonyl and ROS, and attenuated neuronal damage, upregulated SOD, CAT, GSH-Px activities and GSH/GSSG ratio. These results suggested that the neuroprotective effects of lavender oil against cerebral ischemia/reperfusion injury may be attributed to its antioxidant effects.     

Simultaneous femtomole determination of cysteine, reduced and oxidized glutathione, and phytochelatin in maize (Zea mays L.) kernels using high-performance liquid chromatography with electrochemical detection

Thiol compounds such as cysteine (Cys), reduced (GSH) and oxidized (GSSG) gluathione, and phytochelatins (PCs) play an important role in heavy metal detoxification in plants. These thiols are biological active compounds whose function is elimination of oxidative stress in plant cells. The aim of our work was to optimise sensitive and rapid method of high-performance liquid chromatography coupled with electrochemical detector (HPLC-ED) for determination of the abovementioned thiol compounds in maize (Zea mays L.) kernels. New approach for evaluation of HPLC-ED parameters is described. The most suitable isocratic mobile phase for the separation and detection of Cys, GSH, GSSG and PC2 consisted of methanol (MeOH) and trifluoroacetic acid (TFA). In addition, the influence of concentrations of TFA and ratio of MeOH:TFA on chromatographic separation and detection of the thiol compounds were studied. The mobile phase consisting from methanol and 0.05% (v/v) TFA in ratio 97:3 (%; v/v) was found the most suitable for the thiol compounds determination. Optimal flow rate of the mobile phase was 0.18 ml min(-1) and the column and detector temperature 35 degrees C. Hydrodynamic voltammograms of all studied compounds was obtained due to the selection of the most effective working electrodes potentials. Two most effective detection potentials were selected: 780 mV for the GSSG and PC2 and 680 mV for determination of Cys and GSH. The optimised HPLC-ED method was capable to determine femtomole levels of studied compounds. The detection limits (3 S/N) of the studied thiol compounds were for cysteine 112.8 fmol, GSH 63.5 fmol, GSSG 112.2 fmol and PC2 2.53 pmol per injection (5 microl). The optimised HPLC-ED method was applied to study of the influence of different cadmium concentrations (0, 10 and 100 microM Cd) on content of Cys, GSH, GSSG and PC2 in maize kernels. According to the increasing time of Cd treatment, content of GSH, GSSG and PC2 in maize kernels increased but content of Cys decreased. Decreasing Cys concentration probably relates with the increasing GSH and phytochelatins synthesis.     

A novel approach for concurrent quantitation of glutathione,glutathione disulfide,and 2‐hydroxyethylated glutathione in lungs of mice exposed to ethylene oxide,using liquid chromatography–positive electrospray tandem mass spectrometry

Glutathione (GSH), glutathione disulfide (GSSG) and 2‐hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography–tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra‐day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter‐day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4‐week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm. Copyright © 2015 John Wiley & Sons, Ltd.     

Crayfish Procambarus clarkii shows circadian variations in different parameters of the GSH cycle

The present study investigated the rhythmic changes in glutathione status in midgut gland and hemolymph as well as in glutathione reductase (GR) activity in the crayfish Procambarus clarkii. In order to determine the circadian nature of these rhythms different groups of crayfish were submitted to constant-darkness conditions for 24 or 72 h after they had spent 15 days under light-dark 12:12 cycles. The animals of the different batches were killed at 6 h intervals during a 24 h cycle. Reduced glutathione (GSH) and oxidized glutathione (GSSG) in hemolymph and midgut as well as midgut GR activity were determined in midgut gland and hemolymph by fluorometric and spectrophotometric method. Data analysis by chronogram and single Cosinor revealed circadian rhythmicity for GSH and GSSG concentration in both tissues as well as midgut GR activity. The rhythm parameters revealed oxidative stress induced by light. The possible correlation between the glutathione rhythm and other metabolic and behavioral rhythms of crayfish as well as the importance of the glutathione circadian temporal order in the adaptation of crayfish are discussed.