Characterization of Cystathionine Beta‐Synthase and Cystathionine Gamma‐Lyase in the Production of Hydrogen Sulfide Biomarkers, Lanthionine and Homolanthionine, in a HepG2 Cell Culture Model

Hydrogen sulfide (H 2 S) is an essential gasotransmitter with the ability induce vasodilation and exert other cardioprotective effects in‐vivo, yet accurate quantification of H 2 S is difficult and often erroneous. Proposed biomarkers, lanthionine and homolanthionine, are produced concurrently with H 2 S by the pyridoxal 5′‐phosphate (PLP) dependent‐transsulfuration enzymes, cystathionine beta‐synthase (CBS) and cystathionine gamma‐lyase (CSE). This study sought to determine the contribution of CBS and CSE in the production of H 2 S biomarkers in a HepG2 cell culture model. Isotopic tracers, [U‐ 13 C 5 ] L‐methionine and (3, 3 D2) L‐cysteine, were used to determine metabolic fluxes through the transsulfuration pathway before and after the inhibition of CSE by 1mM propargylglycine (PAG), a CSE specific inhibitor. Cellular methionine concentrations and remethylation kinetics were not affected by PAG addition, nor was extracellular cysteine pool size. CSE inhibition caused intracellular cystathionine to accumulate (P=0.012). We evaluated the CBS‐catalyzed appearance of [D2]‐cystathionine and [ 13 C 4 ]‐cystathionine from homocysteine condensation with cysteine or serine, respectively. The labeling pattern of cellular cystathionine showed that only 4% of the cystathionine pool was derived from the CBS‐catalyzed condensation of homocysteine with cysteine. Lanthionine concentrations were four times greater in cells with PAG compared to control cells (P<0.001) but enrichments were not affected by PAG addition. This suggests CSE does not contribute to lanthionine synthesis but CSE may catalyze some lanthionine cleavage. Homolanthionine production in control cells greatly exceeded that observed in PAG‐treated cells (P<0.001), which confirmed that CSE is essential in homolanthionine synthesis. These results support our primary human hepatocyte studies which showed that CSE is the primary source of homolanthionine production while CBS is solely responsible for lanthionine production. These data illustrate how this HepG2 model is useful for mechanistic investigation of H 2 S production. Support or Funding Information Supported by NIH DK072398.