Aged Garlic Extract Enhances Endothelial Nitric Oxide Release via Increased Tetrahydrobiopterin Bioavailability

Introduction: Aged garlic extract (AGE) has been shown to enhance endothelial nitric oxide (NO) production in mice [1] and to restore endothelial dysfunction due to impaired NO bioavailability in humans with acutely elevated homocysteine (hcy) levels [2]. As reduced NO bioavailability during elevated hcy seems to be due to reduction of the NO synthase cofactor tetrahydrobiopterin (BH 4 ) leading to „uncoupling“ of the enyzme [3] we hypothesized that AGE may preserve endothelial BH 4 levels via its antioxidant and thiol-modifying properties, thereby increasing NO release. Methods: Human endothelial cells (EA.hy 926) were incubated for 24h with hypoxanthine, aminopterin, thymidine and methionine (HAT/MET) to increase cellular hcy levels, and with and without AGE (5mg/mL). Cellular levels of hcy, BH 4 , glutathione, and total thiols were measured by HPLC, and endothelial NO production using the fluorescent probe DAF-2. Results: Incubation of endothelial cells with HAT/MET resulted in a significant 2-fold increase in cellular hcy levels (from 0.17±0.03 to 0.35±0.08µmol/mg), coincubation with AGE had no significant effect on hcy in both control and HAT/MET treated cells. Elevated cellular hcy went along with significantly decreased levels of BH 4 (2.23±0.28 vs. 4.34±0.64pmol/mg). Incubation with AGE slightly increased BH 4 in control cells (5.33±0.65pmol/mg), and prevented the decline in BH 4 in HAT/MET treated cells (5.23±0.71pmol/mg). AGE increased cellular levels of total thiols and glutathione, and prevented HAT/MET induced decrease in endothelial NO release. Conclusions: AGE maintains NO bioavailability in endothelial cells even under conditions of elevated hcy levels via increasing cellular BH 4 levels, thereby maintaining normal endothelial function. This may contribute to AGE's antiatherosclerotic properties. Acknowledgement: Supported by Wakunaga of America, Mission Viejo, CA., USA. References: [1] Morihara N. et al. (2002) Life Sci 71: 509 [2] Weiss N. et al. (2006) J Nutr 136: 750S [3] Topal G et al. (2004) Free Radic Biol Med. 36: 1532.