Measurement of plasma hydrogen sulfide in vivo and in vitro.
Measurement of plasma hydrogen sulfide in vivo and in vitro.
复制标题
DOI：
10.1016/j.freeradbiomed.2011.01.025
复制
发表时间：
2011-05-01
影响因子：
7.4
通讯作者：
Kevil CG
中科院分区：
文献类型：
--
作者：
Shen X;Pattillo CB;Pardue S;Bir SC;Wang R;Kevil CG
已知气体递质硫化氢在正常和病理生理状态下调节多种细胞功能。然而，缺乏关于硫化氢参与生理和病理反应的定量信息。这主要是由于各种测量游离硫化氢的方法存在分歧。在这篇文章中，我们描述了一种非常敏感的方法来测量H2S在血浆中的存在低至纳摩尔水平，使用单溴莫烷（MBB）。目前使用亚甲基蓝的标准分析提供了错误的结果，实际上并没有测量H2S。本文提出的方法是将硫化物与过量的MBB在100 mM Tris-HCl缓冲液（pH 9.5, 0.1 mM DTPA）中衍生化30分钟，室温下在1%的氧气中。荧光产物硫化物-二bimane （SDB）采用RP-HPLC分析，色谱柱为eclipse XDB-C18 （4.6×250 mm）柱，用0.1% （v/v）三氟乙酸在乙腈中梯度洗脱。硫化物-二双烷的检出限为2 nM， SDB产品在一段时间内非常稳定，允许批量存储和分析。总之，我们的MBB方法适用于多种生物样品（如血浆、组织和细胞培养裂解物或培养基）中游离硫化氢的敏感定量测量。
The gasotransmitter hydrogen sulfide is known to regulate multiple cellular functions during normal and pathophysiological states. However, a paucity of concise information exists regarding quantitative amounts of hydrogen sulfide involved in physiological and pathological responses. This is primarily due to disagreement among various methods employed to measure free hydrogen sulfide. In this article, we describe a very sensitive method of measuring the presence of H2S in plasma down to nanomolar levels, using monobromobimane (MBB). The current standard assay using methylene blue provides erroneous results that do not actually measure H2S. The method presented herein involves derivatization of sulfide with excess MBB in 100 mM Tris–HCl buffer (pH 9.5, 0.1 mM DTPA) for 30 min in 1% oxygen at room temperature. The fluorescent product sulfide-dibimane (SDB) is analyzed by RP-HPLC using an eclipse XDB-C18 (4.6×250 mm) column with gradient elution by 0.1% (v/v) trifluoroacetic acid in acetonitrile. The limit of detection for sulfide-dibimane is 2 nM and the SDB product is very stable over time, allowing batch storage and analysis. In summary, our MBB method is suitable for sensitive quantitative measurement of free hydrogen sulfide in multiple biological samples such as plasma, tissue and cell culture lysates, or media.