背景:硫化氢(H2S),一种气体信号分子,影响包括衰老在内的多种生理过程,是通过选择哺乳动物酶和肠硫还原细菌产生的。H2S研究受到缺乏在生物基质中定量的准确内标测定的限制。
方法:合成[34S]H2S并制定样品制备方案,通过添加含硫醇的标准品或还原剂来避免硫化物污染,我们开发了一种稳定的同位素稀释高效液相色谱串联质谱(LC-MS/MS)方法,用于同时定量总H2S和其他丰富的硫醇(半胱氨酸,同型半胱氨酸,谷胱甘肽,谷氨酰半胱氨酸,半胱氨酰甘氨酸)在生物基质中,进行了为期20天的分析验证/正常范围研究,然后分析了400名受试者血浆中循环的总H2S和硫醇,在抗生素诱导的肠道微生物群抑制之前和之后的20名志愿者中。
结果:使用新的检测方法,所有分析物显示最小的干扰,没有结转,以及优异的日内和日间再现性(≤7.6%,≤12.7%,分别),线性度(r2>0.997),回收率(90.9%-110%)和稳定性(90.0%-100.5%)。仅循环总H2S水平在男性和女性中均显示出与年龄相关的显着降低(p<0.001),肠道微生物群抑制后显著减少(平均33.8±17.7%,p<0.001),受试者之间肠道微生物群的贡献差异很大(抗生素减少6.0-66.7%)。
结论:提出了一种稳定同位素稀释LC-MS/MS方法,用于同时定量生物基质中的总H2S和多种硫醇。然后,我们使用该测定面板来显示与年龄相关的显着下降和肠道微生物群对人体循环总H2S水平的贡献。
BACKGROUND: Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices.
METHODS: After synthesizing [34S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota.
RESULTS: Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r2 > 0.997), recovery (90.9%-110%) and stability (90.0%-100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0-66.7% reduction with antibiotics).
CONCLUSIONS: A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.