H2S analysis in biological samples using gas chromatography with sulfur chemiluminescence detection - PubMed (original) (raw)

H2S analysis in biological samples using gas chromatography with sulfur chemiluminescence detection

Victor Vitvitsky et al. Methods Enzymol. 2015.

Abstract

Hydrogen sulfide (H2S) is a metabolite and signaling molecule in biological tissues that regulates many physiological processes. Reliable and sensitive methods for H2S analysis are necessary for a better understanding of H2S biology and for the pharmacological modulation of H2S levels in vivo. In this chapter, we describe the use of gas chromatography coupled to sulfur chemiluminescence detection to measure the rates of H2S production and degradation by tissue homogenates at physiologically relevant concentrations of substrates. This method allows separation of H2S from other sulfur compounds and provides sensitivity of detection to ~15 pg (or 0.5 pmol) of H2S per injected sample.

Keywords: Biological samples; Gas chromatography; Hydrogen sulfide; Sulfur chemiluminescence detection.

© 2015 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Scheme showing analysis of sulfur compounds using GC coupled to a sulfur chemiluminescence detector (SCD). Sulfur compounds in the sample are separated on a GC column prior to entering the SCD. In the reaction furnace, the samples undergo combustion in an air and hydrogen mixture producing sulfur monoxide. In the reaction cell, sulfur monoxide reacts with ozone to produce sulfur dioxide and light emission. The latter is detected using a photomultiplier tube (PMT).

Figure 2

Figure 2

Analysis of a mixture of sulfur gases using GC–SCD. The mixture (200 μl total injection volume) contained 2.7 ppm H2S, 5 ppm carbonyl sulfide (COS), 5 ppm 3-methanethiol (CH3SH), 5 ppm ethanethiol (CH3CH2SH), and 5 ppm 5-dimethylsulfide (CH3SCH3) in N2.

Figure 3

Figure 3

Linear dependence of the H2S peak area on H2S amount. The symbols and line represent the experimental data and linear fit, respectively. Each experimental point represents the mean ± SD of 2–4 independent measurements. In most cases, the standard deviation is equal to or less than the symbol size. Samples (200 μl) containing different amounts of H2S were prepared by dilution of the stock solution (40 ppm H2S) with N2.

Figure 4

Figure 4

Scheme showing setup for sample preparation. The solid arrows depict the direction of N2 flow, the movement of the syringe plunger and movement of the three-way stopcock valve during flushing of the syringe with N2. The dashed arrow indicates the position where the sleeve stopper septum is attached during sample incubation.

Figure 5

Figure 5

A representative chromatogram of H2S production by murine liver homogenate at different cysteine concentrations. Peaks 1–3 were obtained after anaerobic incubation for 20 min of the homogenate (pH 7.4, 37 °C) with 0.1, 0.2, and 0.5 m_M_ cysteine, respectively. The arrow indicates the control sample lacking cysteine.

Figure 6

Figure 6

A representative chromatogram showing the kinetics of aerobic H2S degradation by murine liver homogenate. The numbers correspond to samples (200 μl) removed at 1, 2, 3, 5, 8, 10, and 15 min following incubation of liver homogenate with H2S at 25 °C. The calibration peak (12.2 ng H2S) is denoted by Std. The inset shows the kinetics of H2S disappearance. The amount of H2S at t = 0, shows the amount of H2S added to the reaction mixture.

References

    1. Almgren T, Dyrssen D, Elgquist B, Johansson O. Dissociation of hydrogen sulphide in seawater and comparison of pH scales. Marine Chemistry. 1976;4:289–297.
    1. Furne J, Saeed A, Levitt MD. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2008;295(5):R1479–R1485. - PubMed
    1. Hershey JP, Plese T, Millero FJ. The pK1 for the dissociation of H2S in various ionic media. Geochimica et Cosmochimica Acta. 1988;52:2047–2051.
    1. Jurkowska H, Roman HB, Hirschberger LL, Sasakura K, Nagano T, Hanaoka K, et al. Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate. Amino Acids. 2014;46(5):1353–1365. - PMC - PubMed
    1. Kabil O, Banerjee R. The redox biochemistry of hydrogen sulfide. The Journal of Biological Chemistry. 2010;285:21903–21907. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources