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.
Palozza
P
P
Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111.
Moualla
S
S
Krinsky
N I
NI
D000578:Amidines / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001391:Azo Compounds / Q000494:pharmacology; D002338:Carotenoids / Q000494:pharmacology*; D005609:Free Radicals / Q000378:metabolism; D066298:In Vitro Techniques; D015227:Lipid Peroxidation / Q000187:drug effects*; D008315:Malondialdehyde / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D014810:Vitamin E / Q000494:pharmacology*; D019207:beta Carotene
D000578:Amidines; D000975:Antioxidants; D001391:Azo Compounds; D005609:Free Radicals; D019207:beta Carotene; D014810:Vitamin E; D002338:Carotenoids; D008315:Malondialdehyde; D009249:NADP; C046728:2,2'-azobis(2-amidinopropane)