An optimized method for determination of intracellular glutathione in mouse macrophage cultures by fluorimetric high-performance liquid chromatography

We have optimized a method for the determination of intracellular glutathione by high-performance liquid chromatography, using fluorimetric detection. To minimize artifacts and provide an accurate determination of intracellular glutathione, cell extracts were prepared using extraction conditions specifically designed to inhibit autoxidation and enzymatic degradation of glutathione. The sensitivity of the method was enhanced by adjusting the dansyl chloride derivatization reaction with regard to parameters such as pH, reaction time and dansyl chloride concentration. Both oxidized and reduced forms of glutathione were quantified using the refined method in extracts of oxidatively stressed J774A.1 mouse macrophage cells and reflected an expected shift in cellular redox status.     

Exhaled breath condensate: Determination of non-volatile compounds and their potential for clinical diagnosis and monitoring. A review

Exhaled breath condensate is a promising, non-invasive, diagnostic sample obtained by condensation of exhaled breath. Starting from a historical perspective of early attempts of breath testing towards the contemporary state-of-the-art breath analysis, this review article focuses mainly on the progress in determination of non-volatile compounds in exhaled breath condensate. The mechanisms by which the aerosols/droplets of non-volatile compounds are formed in the airways are discussed with methodological consequences for sampling. Dilution of respiratory droplets is a major problem for correct clinical interpretation of the measured data and there is an urgent need for standardization of EBC. This applies also for collection instrumentation and therefore various commercial and in-house built devices are described and compared with regard to their design, function and collection parameters. The analytical techniques and methods for determination of non-volatile compounds as potential markers of oxidative stress and lung inflammation are scrutinized with an emphasis on method suitability, sensitivity and appropriateness. The relevance of clinical findings for each group of possible non-volatile markers of selected pulmonary diseases and methodological recommendations with emphasis on interdisciplinary collaboration that is essential for future development into a fully validated clinical diagnostic tool are given.     

稀土氧化谷胱甘肽配合物的XPS研究

谷胱甘肽是人体内重要低肽,具有许多生物功能。正常状态下,体内主要以还原型(GSH,-O_2CCH(NH_3~(+))CH_2CH_2C∶ONHCH(CH_2SH)C∶ONHCH_2CO_2H)存在,氧化型(GSSG、(-O_2CCH(NH_3~(+))CH_2CH_2C∶ONHCH(CH_2S-)C∶ONHCH_2CO_2H)_2)只占还原型浓度的～1%,并由谷胱甘肽氧化还原酶维持二者的平衡。近年来发现,某些金属离子可以催化氧     

基于氧化型谷胱甘肽保护的荧光金纳米团簇的制备及其对Fe~(3+)的检测

利用氧化型谷胱甘肽作为还原剂和稳定剂,通过绿色合成法制备了具有良好生物相容性的金纳米团簇(GSSG-Au NCs),其平均粒径大小为2.9 nm。以其为荧光探针实现了对Fe~(3+)的高灵敏检测,且对干扰离子具有高度选择性,线性范围宽(0.1~30μmol/L),检出限为0.03μmol/L。该方法用于实际水样(自来水、湖水和河水)中Fe~(3+)的测定,结果满意。     

Validation of a simplified procedure for convenient and rapid quantification of reduced and oxidized glutathione in human plasma by liquid chromatography tandem mass spectrometry analysis

Endogenous glutathione (GSH) and glutathione disulfide (GSSG) status is highly sensitive to oxidative conditions and have broad application as a surrogate indicator of redox status in vivo. Established methods for GSH and GSSG quantification in whole blood display limited utility in human plasma, where GSH and GSSG levels are ~3–4 orders of magnitude below those observed in whole blood. This study presents simplified sample processing and analytical LC–MS/MS approaches exhibiting the sensitivity and accuracy required to measure GSH and GSSG concentrations in human plasma samples, which after 5-fold dilution to suppress matrix interferences range from 200 to 500 nm (GSH) and 5–30 nm (GSSG). The utility of the methods reported herein is demonstrated by assay performance and validation parameters which indicate good sensitivity [lower limits of quantitation of 4.99 nm (GSH) and 3.65 nm (GSSG), and high assay precision (intra-assay CVs 3.6 and 1.9%, and inter-assay CVs of 7.0 and 2.8% for GSH and GSSG, respectively). These methods also exhibited exceptional recovery of analyte-spiked plasma samples (98.0 ± 7.64% for GSH and 98.5 ± 12.7% for GSSG). Good sample stability at −80°C was evident for GSH for up to 55 weeks and GSSG for up to 46 weeks, with average CVs <15 and <10%, respectively.     

血浆中游离还原型及氧化型谷胱甘肽的荧光测定法

文章建立了快速、可同时检测血浆中游离的还原型和氧化型谷胱甘肽(即GSH和GSSG)的方法, 并评价了血浆中GSH/GSSG的氧化还原状态。利用二硫苏糖醇将GSSG还原成两分子的GSH,使GSH在pH 8.0时与邻苯二甲醛(OPA)结合生成荧光产物GSH-OPA,用荧光分光光度仪测定其荧光值,从而对GSH和GSSG进行定量分析。并运用此方法测量青年健康志愿者静脉血中的游离GSH和GSSG。该法最低可检测出测定管中11.4 pmol·L-1GSH以及5.7 pmol·L-1GSSG。测量GSH批内和批间变异系数分别4.6%和3.9%,GSSG批内和批间变异系数分别为3.5%和4.1%,GSH,GSSG加标准品后的回收率分别为(99.77±5.70)%和(99.28±4.73)%。测定健康青年志愿者血浆中游离GSH,GSSG的含量分别为(16.5±2.4) nmol·mL-1和(1.7±0.35) nmol·mL-1。     

Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol

A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r(2) > 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 microL injection volume.     

Derivatization of GSSG by pHMB in alkaline media. Determination of oxidized glutathione in blood

Chromatographic determination of glutathione disulfide (GSSG) without any preliminary reduction has been presented using GSSG derivatization by p-hydroxymercuribenzoate (pHMB) in strong alkaline medium followed by the determination of GS-pHMB complex by reversed phase chromatography coupled to chemical vapour generation and atomic fluorescence detector (RPC-CVGAFS). A detection limit of 35 nM for GSSG (corresponding to 1.8 pmol) detected as GS-pHMB species was achieved based on a signal-to-noise ratio of 3 in buffer and in blood. The proposed method was applied to the determination of GSSG in whole blood and validated by the classical determination of GSSG by derivatization after reduction with dithiothreitol (DTT).