Abstract
Vitamin E, the major lipid chain-breaking antioxidant in erythrocyte membranes, is present in low concentration, suggesting that mechanisms should exist to protect against its loss. Enzymatic pathways for the recycling of vitamin E from its tocopheroxyl radical have been observed previously in inner membranes of mitochondria and microsomes. These pathways use electron transport enzymes and their substrates to regenerate vitamin E. Erythrocyte membranes also contain significant NADH-cytochrome c reductase activity, as well as cytochrome b5, the function of which is not yet known. Using an enzymatic oxidation system composed of lipoxygenase and arachidonic acid, free radicals were produced in human erythrocyte membranes, and their reaction with chromanols was followed by ESR and high performance liquid chromatography (HPLC). Since the endogenous vitamin E content of the membranes is very low, we used a vitamin E homologue lacking the hydrocarbon chain (2,2,5,7,8-pentamethyl-6-hydroxychromane) as a probe molecule for ESR measurements. However, parallel HPLC determinations of lipid hydroperoxides and of endogenous vitamin E confirmed the results obtained by ESR. It was found that protection against the loss of vitamin E can be provided either by NADH-cytochrome b5-dependent enzymatic recycling or by a nonenzymatic pathway involving ascorbate and dihydrolipoic acid.
Highlights
Vitamin E, the major lipid chain-breaking antioxidant in erythrocyte membranes, is present in low concentration, suggesting that mechanisms should exist to protect against its loss
Using an enzymatic oxidation system composed of lipoxygenase and arachidonic acid, free radicals were produced in human erythrocyte membranes, and their reaction with chromanols was followed by ESR and high performance liquid chromatography (HPLC)
In the present study we investigated the possible role of NADH-cytochrome b5 reductase in therecycling of vitamin E inerythrocytemembranes,as well ason a nonenzymatic pathwatyhat utilizes ascorbataend dihydrolipoiaccid (DHLA)' to protect against thleoss of vitamin E
Summary
(Received for publication, January 11, 1993, and in revised form, February 9, 1993). From the Departmentof Molecular and Cell Biology, University of California, Berkeley, California94720. Using an enzymatic oxidation system composed of lipoxygenase and arachidonic acid, free radicals were produced in human erythrocyte membranes, and their reaction with chromanols was followed by ESR and high performance liquid chromatography (HPLC). In the present study we investigated the possible role of NADH-cytochrome b5 reductase in therecycling of vitamin E inerythrocytemembranes,as well ason a nonenzymatic pathwatyhat utilizes ascorbataend dihydrolipoiaccid (DHLA)' to protect against thleoss of vitamin E. determined by two different procedures: the decay in fluorescence of a polyunsaturated fatty acid, cis-parinaric acid (9,11,13,15-cis-transstrans-cis-octadecatetraenoicacid), incorporated in erythrocyte membranes as a probe molecule [13] and HPLC. The degree of lipid peroxidation was calculated from erythrocyte membrane asa probe molecule for ESR detection of chromanoxyl signals We used this homologue because in ESR measurements itproduces a high signal at low concentrations, at which the vitamin E radical is almost undetectable. HPLC Measurement of Chromano Content-Endogenous a-tocopherol was analyzed by HPLC using an in-line electrochemical detector.Tocopherols were extracted and measured as described previously [28]
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