Carotenoids are Effective Inhibitors of in vitro Hemolysis of Human Erythrocytes, as Determined by a Practical and Optimized Cellular Antioxidant Assay

beta-Carotene, zeaxanthin, lutein, beta-cryptoxanthin, and lycopene are liposoluble pigments widely distributed in vegetables and fruits and, after ingestion, these compounds are usually detected in human blood plasma. In this study, we evaluated their potential to inhibit hemolysis of human erythrocytes, as mediated by the toxicity of peroxyl radicals (ROO center dot). Thus, 2,2' -azobis (2-methylpropionamidine) dihydrochloride (AAPH) was used as ROO center dot generator and the hemolysis assay was carried out in experimental conditions optimized by response surface methodology, and successfully adapted to microplate assay. The optimized conditions were verified at 30 x 10(6) cells/mL, 17 mM of AAPH for 3 h, at which 48 +/- 5% of hemolysis was achieved in freshly isolated erythrocytes. Among the tested carotenoids, lycopene (IC50 = 0.24 +/- 0.05 mu M) was the most efficient to prevent the hemolysis, followed by beta-carotene (0.32 +/- 0.02 mu M), lutein (0.38 +/- 0.02 mu M), and zeaxanthin (0.43 +/- 0.02 mu M). These carotenoids were at least 5 times more effective than quercetin, trolox, and ascorbic acid (positive controls). beta-Cryptoxanthin did not present any erythroprotective effect, but rather induced a hemolytic effect at the highest tested concentration (3 mu M). These results suggest that selected carotenoids may have potential to act as important erythroprotective agents by preventing ROO center dot-induced toxicity in human erythrocytes.