The effect of antioxidant nutrients in rat kidney homogenates was studied by measuring the formation of oxidized heme proteins (OHP) and thiobarbituric acid reactive substances (TBARS) during spontaneous oxidation at 37 degrees. OHP were analyzed using a modified spreadsheet protocol; the Heme Protein Spectra Analysis Program (HPSAP). Male Sprague-Dawley (SD) rats were fed a basal diet fortified with vitamin E, selenium, or beta-carotene, or a combination of all three antioxidants. A second group of male SD rats received a basal diet fortified with Trolox, ascorbic acid palmitate, acetylcysteine, beta-carotene, coenzyme Q10, coenzyme Q0, and (+)-catechin. A control group of rats was given a vitamin E- and selenium-deficient basal diet. The amount of TBARS production during a 1 h reaction decreased as the relative antioxidant effectiveness of the dietary treatments increased. Dietary treatments providing nine antioxidants significantly reduced the formation of OHP and methemoglobin during the 1 h reaction compared to the dietary treatment providing only two antioxidant nutrients. These data suggest that increasing the diversity and quantity of antioxidants in the diet provides significantly more protection for heme proteins and lipids in kidney tissue than individual antioxidants or a combination of vitamin E and selenium.