In vitro effects of aged garlic extract and other nutritional supplements on sickle erythrocytes

In the circulation of sickle cell anemia patients, a certain population of erythrocytes has an elevated density. These abnormally dense cells are believed to be at the root of the painful crisis and anemia of the patients. We have developed an in vitro method for the preparation of these heavier erythrocytes by a repeated deoxy-oxy cycling of erythrocytes from sickle cell anemia patients. By using this method, we studied whether certain nutritional supplements would inhibit the formation of dense cells in vitro. It was found that aged garlic extract (AGE) as well as its components with antioxidant activity, i.e., S-allylcysteine and Nalpha-(1-deoxy-D-fructos-1-yl)-L-arginine (fructosyl arginine), inhibited the formation of dense cells in vitro. Vitamin C, vitamin E and the spin-trapping agents, 5-diethoxyphophoryl-5-methyl-1-pyrroline-N-oxide and alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone were all found to inhibit the formation of dense cells in vitro. These results suggest that, when extremely stretched sickle-shaped cells are formed by the repeated deoxy-oxy cycling, the erythrocyte membrane becomes susceptible to oxidative injury by reactive oxygen species. The protection of the erythrocyte membrane from such an oxidative injury would prevent the membranes from becoming leaky to the calcium ion, thus inhibiting the activation of the calcium-activated potassium efflux channel and the formation of dense cells. We also developed a new ex vivo method of studying the possible efficacy of antioxidants taken orally on the dense cell formation in sickle cell patients. It involved the use of blood plasma taken from a healthy donor (with normal hemoglobin) of AB blood type who had consumed different types of antioxidants orally. By suspending sickle erythrocytes in such plasma and exposing them to the deoxy-oxy cycling, the degree of dense cell formation was determined. The degree of inhibition in vitro by antioxidants taken orally may be related to their efficacy in inhibiting dense cell formation in the patients. On the basis of these in vivo and ex vivo studies, we propose that a cocktail of antioxidants would have beneficial effects in lessening the incidence and severity of crisis and reducing anemia in sickle cell disease.