Abstract
Plasma glutathione peroxidase (GSHPx) has been suggested to reduce submicromolar levels of free fatty acid hydroperoxides and phosphatidylcholine hydroperoxides (PC-OOH), and therefore these hydroperoxides are undetectable in human blood plasma. The capacity for the reduction should be about 2.5 μm as the level of glutathione in human plasma is about 5 μm. However, 2 h of aerobic incubation of 58 μm PC-OOH in human plasma at 37°C resulted in the formation of 36 μm phosphatidylcholine hydroxide (PC-OH). The presence of PC-OOH-reducing protein other than plasma GSHPx was suggested by the results. a) The same rates of PC-OOH decay and PC-OH formation were observed in both sera from rats with selenium-deficient and selenium-supplemented diet; b) the PC-OOH-reducing activity was observed only in the high molecular weight fraction but not in the low molecular weight fraction; and c) albumin did not work as a reducing substrate of plasma GSHPx. We have isolated two hydroperoxide-reducing protein fractions from human plasma by a sequential purification scheme, comprising an ammonium sulfate precipitation followed by sequential chromatography on anion exchange, hydrophobic interaction, and heparin columns. One of the proteins was identified as apolipoprotein A-I by N-terminal amino acid sequence analysis. Moreover, the hydroperoxide-reducing activity of one of the fractions was inhibited almost completely by the addition of anti-apolipoprotein A-I antibody.▪ These findings demonstrate that apolipoprotein A-I in high density lipoprotein can reduce PC-OOH to PC-OH.—Mashima, R., Y. Yamamoto, and S. Yoshimura. Reduction of phosphatidylcholine hydroperoxide by apolipoprotein A-I: purification of th hydroperoxide-reducing proteins from human blood plasma. J. Lipid Res. 1998. 39: 1133–1140.
Highlights
Plasma glutathione peroxidase (GSHPx) has been suggested to reduce submicromolar levels of free fatty acid hydroperoxides and phosphatidylcholine hydroperoxides (PC-OOH), and these hydroperoxides are undetectable in human blood plasma
We have shown that PCOOH in high density lipoprotein (HDL) can be converted to CE-OOH by the action of lecithin:cholesterol acyltransferase (LCAT) [13]
We report the inability of albumin to act as a reducing substrate for plasma GSHPx, a lack of difference in the reduction of 50 m PC-OOH between sera from selenium-deficient and selenium-supplemented rats, and the isolation of two protein fractions that can reduce PC-OOH
Summary
Soybean phosphatidylcholine (PC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), rat serum albumin, tert-butyl hydroperoxide (BOOH), cumene hydroperoxide, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma (Tokyo). 2,2Ј-Azobis(2,4-dimethylvarelonitrile) (AMVN) and choline chloride were obtained from Wako Pure Chemical (Osaka). After exchanging solvent from hexane to methanol, PC-OOH was purified on a semipreparative octadecylsilyl column (25 cm ϫ 10 mm i.d., Japan Spectroscopic, Tokyo) using methanol containing 0.02% triethylamine as the mobile phase at a flow rate of 4 ml/ min. Plasma GSHPx was separated from rat serum as described previously [21]. 5 l of 1.2 mm PLPC-OOH in methanol was mixed with 95 l of human heparinized plasma obtained from a 26-year-old healthy male donor and incubated at 37ЊC under aerobic conditions. Incubation of PLPC-OOH in sera obtained from rats fed selenium-deficient or selenium-supplemented diets was carried out . Aliquots (5 l) of human plasma or rat serum were withdrawn during the course of incubation and stored at Ϫ80ЊC until analysis. The mobile phase used was acetonitrile–methanol–water 100:99:1 (v/v/v) containing 10 mm choline chloride and the flow rate was 2 ml/min
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