Background

Hydrogen sulfide (H₂S) gas is produced endogenously by at least three different enzymes in mammals (CGL, CBS, 3-MST) with a range of tissue and cell-type distributions. H₂S functions as a gasotransmitter and effector molecule (Wang, 2012) in a wide range of biological functions related to metabolism (Módis et al., 2013), stress resistance (Hine et al., 2015), and redox biology (Dickhout et al., 2012). Reduced H₂S is linked to cardiovascular problems including hypertension in rodents (Yang et al., 2008) and cardiac hypertrophy in man (Polhemus et al., 2014). Increased H₂S can also cause pathology, for example in rodent pancreatitis (Bhatia et al., 2005). Thus, accurate and quantitative detection of H₂S from biological sources could facilitate a better understanding of its biological effects as well as its potential use as a clinical biomarker.

Techniques to measure absolute concentrations of H₂S present in biological samples, along with their pros and cons, have been reviewed extensively (Olson, 2012; Wang, 2012; Hartle and Pluth, 2016; Takano et al., 2016). For example, free and sulfane-bound H₂S pools can be measured in biological samples including serum or tissue homogenates ex vivo using headspace GC-MS, which is highly sensitive and selective, but requires expensive equipment. Nonetheless, due to the volatility of H₂S, its interaction with other biological macromolecules and its breakdown into different sulfur-containing compounds, quantitative detection of steady-state free H₂S levels in vivo remains challenging (Olson, 2009).

An alternate approach is to measure the capacity of a tissue homogenate or extract to produce H₂S in a reaction mixture containing optimized levels of substrate and cofactor, thus allowing for H₂S detection methods that are specific but less sensitive. An example is the methylene blue method in which H₂S in solution is trapped by lead acetate to form lead sulfide, which upon conversion to methylene blue can be easily read in a standard spectrophotometer (Stipanuk and Beck, 1982; Ikeda et al., 2017). The pros and cons that must be taken into account with each method are based on the question being asked, the biological system and tissue being studied, the relative need for sensitivity, selectivity, or speed, and the cost and resources of the investigator.

Here, we describe an inexpensive, rapid, and moderately high throughput methodology for measuring H₂S production capacity in extracts of relatively small amounts of biological material. This method is based on the reaction of H₂S present in the headspace above a biological sample with lead acetate to form the black precipitate lead sulfide, a technique used throughout the past 100 years to detect H₂S and H₂S-producing bacteria (McBride and Edwards, 1914; Kuester and Williams, 1964; Zhang and Weiner, 2014). Previously, we used this method to detect changes in H₂S production capacity as a function of diet or genetic background in a variety of biological samples including yeast, worms, flies, and rodent tissues/organs including liver (Hine et al., 2015; Mitchell et al., 2016; Nikonorova et al., 2017). Here, we present an optimized procedure to measure H₂S production capacity in mammalian liver via (B) an end-point assay using Whatman paper-embedded lead acetate, or (C) a kinetic assay using agar-embedded lead acetate. As the liver is a strong producer of H₂S in mammalian systems via the enzyme cystathionine gamma lyase (CGL) (Kabil et al., 2011), we feel this is a good starting point for researchers to understand and confidently develop this protocol for their own research questions. Furthermore, this procedure can be easily adapted to other biological samples and organisms, although the procedure may need to be optimized by the investigator in order to obtain suitable results.