Further Studies on Lipid‐Peroxide Formation in Isolated Hepatocytes
Open Access
- 1 November 1975
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 59 (2), 449-455
- https://doi.org/10.1111/j.1432-1033.1975.tb02473.x
Abstract
Lipid peroxide formation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30–60 min, and the response was dose-related with iron concentrations ranging from 19 to 187 μM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The addition in vitro of N,N,N′;N′-tetramethyl-p-phenylenediamine, menadione or p-benzoquinone inhibited the iron-induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N′,N′-tetramethyl-p-phenylenediamine (100 μM). The attenuation of the iron-induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells. Cumene hydroperoxide-induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 μM to the liver cell incubate. With concentrations below 150 μM, a lag phase was present which seemed to be glutathione-dependent. It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPH via the NADPH–cytochrome P-450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron-apoferritin complex formation.Keywords
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