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Analytical measurement of discrete hydrogen sulfide pools in biological specimens.

ABSTRACT: Hydrogen sulfide (H₂S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H₂S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H₂S. The complexity of analytical H₂S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H₂S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H₂S. Acid-labile H₂S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H₂S. Volatilized H₂S is then trapped in 100 mM Tris-HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H₂S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H₂S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H₂S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens.

SUBMITTER: Shen X

PROVIDER: S-EPMC4413934 | biostudies-other | 2012 Jun 1-15

REPOSITORIES: biostudies-other

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Project description:By electrospinning of polycaprolactone (PCL) solutions containing N-(benzoylthio)benzamide (NSHD1), a H2S donor, fibrous scaffolds with hydrogen sulfide (H2S) releasing capability (H2S-fibers) are fabricated. The resultant microfibers are capable of releasing H2S upon immersion in aqueous solution containing biological thiols under physiological conditions. The H2S release peaks of H2S-fibers appeared at 2-4h, while the peak of donor alone showed at 45 min. H2S release half-lives of H2S-fibers were 10-20 times longer than that of donor alone. Furthermore, H2S-fibers can protect cells from H2O2 induced oxidative damage by significantly decreasing the production of intracellular reactive oxygen species (ROS). Finally, we investigated the H2S-fibers application as a wound dressing in vitro. Given that H2S has a broad range of physiological functions, H2S-fibers hold great potential for various biomedical applications.Hydrogen sulfide, as a gaseous messenger, plays a crucial role in many physiological and pathological conditions. Recent studies about functions of H2S suggests H2S-based therapy could be promising therapeutic strategy for many diseases, such as cardiovascular disease, arthritis, and inflammatory bowel disease. Although many H2S donors have been developed and applied for biomedical studies, most of H2S donors have the shortage that the H2S release is either too fast or uncontrollable, which poorly mimic the biological generation of H2S. By simply combining electrospinning technique with our designed biological thiols activated H2S donor, NSHD1, we fabricated H2S releasing microfibers (H2S-fibers). This H2S-fibers significantly prolonged the releasing time compared to H2S donor alone. By adjusting the electrospinning parameters, tunable releasing profiles can be achieved. Moreover, the H2S fibers can protect cardiac myoblasts H9c2 and fibroblast NIH 3T3 from oxidative damage and support their proliferation as cellular scaffolds. To our knowledge, this is the first report of electrospun fibers with H2S releasing capacity. We anticipate this H2S-releasing scaffold will have great potential for biomedical applications.

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Analytical measurement of discrete hydrogen sulfide pools in biological specimens.

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