Effects of beta-carotene and alpha-tocopherol on radical-initiated peroxidation of microsomes

Rat liver microsomal membranes were exposed to either beta-nicotinamide adenine dinucleotide phosphate (NADPH), adenosine 5'-diphosphate (ADP), and Fe+3 or to azocompounds, and the antioxidant activities of beta-carotene and alpha-tocopherol were studied. Lipid peroxidation was monitored either by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm or by hydroperoxide formation at 234 nm, after high-pressure liquid chromatography (HPLC) separation of phospholipid hydroperoxides. The radical initiators, water-soluble 2,2'-azobis(2-amidinopropane) (AAPH) and lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile (AMVN), when thermally decomposed at 37 degrees C under air, produced a constant rate of lipid peroxidation in microsomes and lag times inversely related to their concentrations. Using 25 mM AAPH, beta-carotene suppressed lipid peroxidation at a concentration of 50 nmol/mg protein; using 24 mM AMVN, an inhibition of MDA formation was observed at a concentration of only 5 nmol/mg protein. Inhibition by beta-carotene did not produce a clearly defined lag phase. During AAPH-induced lipid peroxidation, beta-carotene was consumed linearly, and high levels of the antioxidant were still present at the end of 45 min of incubation. Using NADPH/ADP/Fe+3, protection by beta-carotene was observed at 10 nmol/mg protein. alpha-Tocopherol effectively suppressed both MDA and hydroperoxide formation in a dose-dependent manner when either NADPH/ADP/Fe+3 or azocompounds were used. These effects were observed at very low concentrations of the added alpha-tocopherol, ranging from 2 to 3 nmol/mg protein. When the lag times were measurable (AAPH and AMVN), they were directly proportional to the concentration of alpha-tocopherol and revealed the presence of endogenous antioxidants in the microsomal membranes. Different temporal relationships between the loss of alpha-tocopherol and lipid peroxidation were observed in relation to the prooxidant used. A substantial depletion of about 70% of endogenous alpha-tocopherol preceded the propagation phase when induced by the azocompounds, while only 20% of antioxidant disappeared at the beginning of the peroxidation when induced by NADPH/ADP/Fe+3. Although our results show that both beta-carotene and alpha-tocopherol suppress the peroxidation of microsomal membranes, their antioxidant efficacy is influenced by several factors, including the type of radical initiator involved and the site and rate of radical production.