Garlic-induced H₂S production and GSH and GSSG levels. (A) Representative polarographic traces of garlic-induced H₂S production in 20% (vol/vol) RBCs in anoxic 10 mM PBS with 50 μM DTPA, with (black line) and without (blue line) 10 mM glucose (Glc), with sequential 1 mg/ml garlic additions at arrows, compared with the same garlic additions to 10 mM PBS and 50 μM DTPA alone in the absence of RBCs (red line) (pH 7.35) at 37°C. (B) HPLC analysis of GSH (black bars) and GSSG (expressed in GSH equivalents; white bars) in 40% (vol/vol) RBCs subject to no treatment, 10 mM glucose (Glc), 10 mM glucose and 1 mg/ml garlic, 1 mg/ml garlic alone, 10 mM IAA, and 10 mM IAM; each bar represents the mean ± SD of three to five experiments. *, GSH and GSSG levels are statistically different compared with levels in untreated RBCs by Student's t test (P ≤ 0.01). (C and D) Representative polarographic traces of H₂S production in anoxic 10 mM PBS with 50 μM DTPA in the absence of RBCs, with 2 mM each GSH, cysteine (Cys), homocysteine (Hcys), or N-acetylcysteine (NAC) (pH 7.35) at 37°C upon addition of 1 mg/ml garlic at arrow (C) and garlic-induced H₂S production in 20% (vol/vol) RBCs with 10 mM glucose previously treated with 10 mM IAA (blue line) or 10 mM IAM (red line) compared with untreated RBCs (black line) with 1 mg/ml garlic additions at arrows (D).

Single garlic-derived organic polysulfide-induced (nonenzymatic) H₂S production by GSH. (A) Representative polarographic traces of H₂S production in anoxic 10 mM PBS with 50 μM DTPA, with 100 μM each DATS (red line), DADS (green line), DAS (brown line), AMS (yellow line), and DPDS (blue line), by addition of 2 mM GSH at arrow. (B) Total H₂S yield from each compound under the same conditions as in A; each bar represents the mean ± SD of three to six experiments. *, Yields are statistically different compared with yield from DPDS by Student's t test (P ≤ 0.005).

Garlic- and single garlic-derived organic polysulfide-induced vasoactivity and H₂S production of PE-precontracted rat aorta rings at physiological O₂ levels. Representative simultaneous traces of vasoactivity (blue line) and H₂S production (red line) in a 15-ml organ bath of Krebs–Henseleit buffer (pH 7.35) at 37°C containing 5 μM indomethacin and 100 μM L-NAME, with 100 nM PE and 1 mM GSH additions at arrows with alterations of O₂ from 196 to 50 μM at the dashed line and 500 μg/ml garlic addition at the arrow (A) and alterations of O₂ from 100 to 30 μM at the dashed line and addition of 100 μM each AMS and DADS at the arrows (B). (C and D) Percentage vasorelaxation of aorta rings supported by 50, 200, and 500 μg/ml garlic (black bars) compared with 20 μM H₂S (white bar) (C; each bar represents the mean ± SD of four to six experiments) and 100 μM each DPDS, AMS, DAS, DADS, or DATS, as well as ethanol vehicle and 20 μM H₂S (D; each bar represents mean ± SD of three to nine experiments). Endothelium-intact aortic segments (black bars) are compared with endothelium-denuded segments (white bars). Levels are statistically different compared with DPDS (*, P ≤ 0.001; n = 6–9) and AMS (**, P ≤ 0.01; n = 3) by Student's t test. (E) Percent relaxation by 100 μM DPDS or DATS with or without 1 mM GSH; each bar represents mean ± SD of four to six experiments. (F) Representative polarographic traces of DADS-induced H₂S production by entire aorta (eight segments, ≈30 mg fresh weight; red line) and simultaneous O₂ concentration (blue line) in 10 mM PBS with 50 μM DTPA, with 10 mM glucose (Glc) and 100 μM DADS additions at the arrows, in the 2-ml closed multisensor respirometer chamber.

Proposed model of garlic-induced H₂S production and H₂S function in the vascular system. Garlic-derived organic polysulfides with allyl moieties and more than two sulfur atoms (see Fig. 5) react with exofacial membrane thiols and cross the cell membrane to react with GSH to produce H₂S. Glucose is the main energy source of RBCs, supporting glycolysis and pentose phosphate pathway (PPP) reduction of NADP⁺ to NADPH, a cofactor of GSH reductase (GR), which maintains the intracellular GSH pool. GSH may also participate in transmembrane electron transfer to reduce exofacial thiols (16). H₂S production then leads to vasorelaxation via vascular smooth muscle cell K_ATP-linked hyperpolarization (8).

H₂S production from organic polysulfides by thiol reactions. (A) Proposed mechanism of H₂S production by reaction of DADS and GSH via α carbon nucleophilic attack (red); H₂S is not produced by thiol/disulfide exchange (blue). (B) Nucleophilic substitution of allyl perthiol at the α carbon followed by (C) thiol/disulfide exchange. (D and E) Thiol/disulfide exchange of organic trisulfides and higher polysulfides. See text below for description.