Considerable attention has recently been focused on the interrelationships of peroxidation processes, free radical related reactions and the development of a variety of pathological events (Orrenius et al. 1988) and aging processes (Packer 1991). Ingestion of oxidized lipids has been shown by others and by us to promote peroxidation in erythrocyte membranes (Hayam et al. 1993), liver (Corcos-Benedetti et al. 1987), kidney and heart (Yoshida and Kajimoto 1989), muscle microsomes (Monahan et al. 1994) and plasma lipoproteins (Hayam et al. 1995).

The phospholipid bilayer of membranes, which is largely composed of polyunsaturated fatty acids, is highly susceptible to peroxidation processes. Free radical reactions in lipid domains may also result in damage to membrane proteins, thereby leading to alteration and impairment of membrane dynamics and function (Wiseman 1996). However, limited information is available in the literature regarding the changes in membrane fluidity and function caused by dietary oxidized lipids. In a recent study, we demonstrated that feeding oxidized oil to rats resulted in a reduction in the degree of unsaturation of the erythrocyte membrane lipids and a decrease in the membrane fluidity; these effects were accompanied by enhanced activity of (Na⁺K⁺) ATPase and of acetylcholine esterase (Hayam et al. 1993).

Microsomes are rich in a large number of membrane-bound enzymes such as those associated with lipid and protein metabolism and the mixed function oxidase (MFO)⁴ system (Saito and Yamaguchi 1993). The function of such membrane-associated proteins is highly dependent on the composition, structure and dynamics of the membrane (Shinitzky 1984).

The present investigation was undertaken to study changes in rat liver microsomal membrane dynamics, composition and function after rats were fed oxidized linoleic acid. Membrane dynamics was assessed in terms of membrane fluidity and the activity of the membrane intrinsic enzymes glucose-6-phosphatase (G6Pase) and aldehyde dehydrogenase (ALDH), cytochrome C reductase was used to evaluate membrane function.

MATERIALS AND METHODS

Table 1. Composition of the basal diet1 [View Table]

The fatty acid-free diets were stored at 4°C. A mixture of each of the fatty acid preparations and an appropriate amount of starch was stored at 18°C, added to the designated diet daily and given to the animals; the residue of the previous ration was discarded. These steps were undertaken to ensure minimal additional oxidation of dietary components.

At the end of the feeding period, food was withheld overnight, and rats were killed by carbon dioxide asphyxiation; portal vein blood was collected in tubes containing EDTA. Livers were removed after perfusion with saline and kept on ice or frozen at 70°C for further analysis according to the requirements of the various analytical procedures.

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For fatty acid analysis of membrane phospholipids, liver microsome samples were subjected to saponification and methylation with boron trifluoride followed by extraction with hexane/diethyl ether (1:1) according to Miller (1984). The phospholipid fatty acyl profiles were determined using a 5890 Hewlett-Packard gas chromatograph (Avondale, PA) equipped with a flame ionization detector. The methyl esters were resolved on a wide-bore fused silica column, Supelcowax 10 (Supelco, Bellefonte, PA). The flow rate of the nitrogen carrier gas was 18 mL/min. Initial oven temperature of 160°C was maintained for 12 min and then raised to 180°C at a rate of 5°C/min, kept at this temperature for another 10 min and raised to 200°C at the same rate. The injector and detector port temperature were 230 and 250°C, respectively.

RESULTS AND DISCUSSION

Table 2. Oxidative state and composition of the dietary fatty acid preparations1 [View Table]

Liver microsomal lipids from rats fed the oxidized linoleic acid-rich preparation were shown to be in a high state of peroxidation compared with the respective lipids of the control animals, as reflected by the levels of TBARS and water-soluble fluorescent peroxidation products (Table 3). The TBARS level of 59.7 ± 2.8 nmol MDA/mg protein observed for the liver microsomal lipids of the pair-fed rats was similar to that found for the control group, suggesting that the reduced food intake did not enhance hepatic peroxidation processes in these animals.

Table 3. Peroxidative state and fluorescence anisotropy parameter of 1,6-diphenyl-1,3,5-hexatriene (DPH) of liver microsomes derived from rats fed oxidized (LAOx) and untreated (LA) linoleic acid-rich preparations1 [View Table]

Feeding oxidized linoleic acid resulted in significantly greater microsomal membrane fluidity, expressed as the fluorescence anisotropy parameter, [(r₀/r)1]¹, of DPH. The fluorescence anisotropy parameter, which is inversely related to the fluidity, was studied over a temperature range of 25-37°C (Fig. 2, Table 3). No break point was found in the Arrhenius plot, which describes the temperature dependence of the fluorescence anisotropy parameter, suggesting the absence of a lipid thermotropic phase transition in this temperature range in the microsomal membranes derived from the control and the experimental groups.

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Studies reporting changes in membrane fluidity after an in vivo oxidation stress are scarce. Membrane fluidity of erythrocyte membranes from experimental animals subjected to oxidative stress was found to decrease in a few studies. Thus, a lower erythrocyte membrane fluidity was observed in rats fed oxidized oil (Hayam et al. 1993), in rats fed riboflavin-deficient diets (Levin et al. 1990), as well as in diabetic subjects (Bryszewska et al. 1986). It was postulated (Levin et al. 1990) that oxidation of membrane lipids is likely to result in the formation of peroxidation degradation products such as the highly reactive bifunctional compound malondialdehyde (MDA), leading to cross-linking reactions of the lipid-lipid and lipid-protein types, and thereby rigidifying the membrane and decreasing the fluidity. It is conceivable that such reactions took place also in the hepatic microsomal membranes of the rats fed LAOx, acting to reduce the fluidity. Nonetheless, other factors counteracting such possible rigidifying effects might have prevailed.

Table 4. Phospholipid fatty acyl groups and phospholipids and cholesterol levels of microsomes derived from rats fed oxidized (LAOx) and untreated (LA) linoleic acid-rich preparation1 [View Table]

The changes in microsome lipid composition in the experimental group, i.e., the reduction in cholesterol to phospholipid molar ratio and the elevation in arachidonic acid content, though at the expense of decreased linoleic acid, should contribute to a higher membrane fluidity. Because these profound alterations in membrane lipid composition overwhelmed those of the possible cross-linking reactions, which should have acted to lower the fluidity, a higher membrane fluidity resulted for the microsomes of the experimental group than that observed for the respective control rats. Elevated levels of arachidonic acid in hepatic microsomes of animals fed oxidized lipids were observed by other investigators and were suggested to be a protective mechanism against oxidative stress (Buttriss and Diplock 1988). Furthermore, Osada et al. (1996) recently showed that enhanced activity of 6-desaturase, the rate-limiting enzyme in the biosynthesis of arachidonic acid from linoleic acid, was associated with the consumption of oxidized cholesterol in diets containing casein as a protein source. Indeed, preliminary gaschromatography-mass spectroscopy results from our laboratory suggest the presence of oxidized cholesterol in liver lipid extracts of LAOx-fed rats.

The lipid composition of the livers of the two groups is presented in Table 5. Low cholesterol and high phospholipid levels were found in the livers of rats fed LAOx compared with those fed the fresh preparation. In addition, a higher ratio of free to esterified cholesterol was observed for the experimental rats. It should be noted that the pair-fed rats exhibited a hepatic lipid composition similar to that observed for the control rats (data not shown), suggesting that reduced food consumption did not alter cholesterol and phospholipid metabolism.

Table 5. Liver phospholipid and cholesterol and plasma and fecal cholesterol levels of rats fed oxidized (LAOx) and untreated (LA) linoleic acid-rich preparations1 [View Table]

The significantly higher plasma cholesterol level along with the low hepatic cholesterol level (Table 5) observed for the rats fed LAOx suggests the occurrence of impaired liver uptake of cholesterol in these animals. Staprans et al. (1993) demonstrated a low hepatic uptake of a chylomicron preparation isolated from the mesenteric duct of rats fed corn oil of a high peroxide value.

A low liver cholesterol level may induce enhanced hepatic cholesterol biosynthesis as a result of the feedback mechanism characteristic of the regulation of cholesterol synthesis. Rudney and Sexton (1986) suggested that hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity is mediated by the microsomal membrane fluidity, implying the occurrence of a positive correlation between the activity of this membrane-bound enzyme and the membrane fluidity. Thus, in the present study, the elevated membrane fluidity observed for the LAOx-fed rats may be indicative of enhanced HMG-CoA reductase activity. In addition, the apparently coupled activities of cholesterol-7-hydroxylase and HMG-CoA reductase (Sudjana-Sugiaman et al. 1994) may indicate the occurrence of an enhanced hepatic bile acid secretion, needed to enable the high rate of cholesterol secretion, in the rats subjected to the oxidative stress.

The high ratio of free to esterfied cholesterol found in the livers of the experimental rats (Table 5) is indicative of low hepatic acyl-CoA:cholesterol acyl transferase (ACAT) activity in these animals. Reduced hepatic ACAT activity was shown to be associated with a low liver cholesterol influx (Fernandez and McNamara 1994), which appears to agree with the apparently impaired hepatic cholesterol uptake suggested to occur with the LAOx-fed rats. In addition, it should be noted that reduced hepatic ACAT activity is associated with enhanced biliary cholesterol secretion (Suckling and Stange 1985).

It is therefore likely that under the oxidative stress caused by the dietary lipids, the lower hepatic and microsomal cholesterol levels as well as the enhanced fecal cholesterol excretion are associated with elevated cholesterol biosynthesis and secretion, and with impaired liver cholesterol uptake.

Table 6. Activity of hepatic microsomal enzymes derived from rats fed untreated (LA) and oxidized (LAOx) linoleic acid-rich preparations1 [View Table]

No difference in the activity of G6Pase was observed between the two dietary groups. Rats fed LAOx exhibited a elevated activities of microsomal ALDH and NADPH- cytochrome C reductase, compared with those fed the control LA. The absence of an effect of oxidized lipids on the activity of hepatic G6Pase was also reported by Kanazawa et al. (1989). It has been suggested that this enzyme serves as a marker of toxicity for orally administered secondary oxidation products of linoleic acid (Kanazawa et al. 1989).

Microsomal ALDH is known to play a role in the detoxification of aldehydes formed from peroxidized dietary lipids (Mitchell and Petersen 1989). It is thus conceivable that the activity of this enzyme would increase upon consumption of the LAOx preparation. An increase in the activity of cytochrome C reductase and other MFO enzymes under oxidative stress was also observed by others (Hogberg et al. 1973). It was suggested that increased microsomal membrane fluidity elevated the activity of this enzyme (Saito and Yamaguchi, 1994). However, the possibility that the activity of cytochrome C reductase was enhanced as the result of an excessive physiological need arising from the apparent oxidative stress cannot be excluded.

In conclusion, the study demonstrates that feeding oxidized linoleic acid to rats resulted in alterations in the composition of the lipids of liver microsomes, elevated fluidity of the microsomal membrane and changes in the activity of some membrane-bound enzymes.