Prolonged incubation of blood prior to plasma preparation can significantly influence the quality of the resulting data. Different markers for this pre-clinical variability have been proposed over the years but with limited success. In this study we explored the usefulness of glutathione (GSH) status, namely ratio of reduced to oxidized glutathione (GSH/GSSG), as potential marker of plasma preparation delay. For that purpose, blood from 20 healthy volunteers was collected into tubes with a cysteine quencher (N-ethylmaleimide; NEM) for GSH stabilization. Plasma preparation was delayed at room temperature for up to 3 hours and every hour, a plasma sample was prepared and the GSH/GSSG ratio measured. We report that over the course of the investigation, plasma concentrations of both GSH and GSSG increased linearly (R2 = 0.99 and 0.98, respectively). Since GSH increased at a much faster rate compared to GSSG, the GSH/GSSG ratio also increased linearly in a time dependent manner (R2 = 0.99). As GSH is an intracellular antioxidant, we speculated that this might stem from ongoing blood hemolysis, which was confirmed by the time dependent rise in lactate dehydrogenase (LDH) activity in the plasma samples. Moreover, we demonstrate that the addition of the thiol alkylating reagent NEM directly to the blood tubes does not seem to influence downstream analysis of clinical parameters. In conclusion we propose that the glutathione status could be used as an indicator of the centrifugation delay prior to plasma preparation.

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血浆谷胱甘肽状态作为分析前离心延迟的指标

在血浆制备之前长时间孵育血液会显着影响所得数据的质量。多年来，已经提出了用于临床前变异性的不同标志物，但取得的成功有限。在这项研究中，我们探索了谷胱甘肽（GSH）状态（即还原型与氧化型谷胱甘肽的比例（GSH / GSSG））作为血浆制备延迟的潜在标志的有用性。为此，将来自20名健康志愿者的血液收集到带有半胱氨酸淬灭剂（N-乙基马来酰亚胺； NEM）的试管中，以稳定GSH。血浆制备在室温下延迟最多3个小时，并且每小时制备血浆样品并测量GSH / GSSG比。我们报告说，在调查过程中，GSH和GSSG的血浆浓度均呈线性增加（R2分别为0.99和0.98）。由于GSH的增长速度比GSSG快得多，因此GSH / GSSG比率也以时间依赖性线性增长（R2 = 0.99）。由于GSH是一种细胞内抗氧化剂，我们推测这可能是由于正在进行的血液溶血引起的，血浆血浆中乳酸脱氢酶（LDH）活性随时间的升高证实了这一点。此外，我们证明了将硫醇烷基化试剂NEM直接添加到血管中似乎并不影响临床参数的下游分析。总之，我们建议谷胱甘肽状态可以用作血浆制备前离心延迟的指标。我们推测这可能源于持续的血液溶血，血浆样本中乳酸脱氢酶（LDH）活性随时间的升高证实了这一点。此外，我们证明了将硫醇烷基化试剂NEM直接添加到血管中似乎并不影响临床参数的下游分析。总之，我们提出谷胱甘肽状态可以用作血浆制备前离心延迟的指标。我们推测这可能源于持续的血液溶血，血浆样本中乳酸脱氢酶（LDH）活性随时间的升高证实了这一点。此外，我们证明了将硫醇烷基化试剂NEM直接添加到血管中似乎并不影响临床参数的下游分析。总之，我们提出谷胱甘肽状态可以用作血浆制备前离心延迟的指标。