Grape extract inhibits lipid peroxidation of human low density lipoprotein.

Some epidemiological data have linked dietary polyphenols with lower risk of coronary heart disease. Polyphenols might impair lipoprotein oxidation which is believed to be an important step in initiating atherogenesis. The purpose of this study was to determine if grape extract known to contain polyphenolic substances can block copper-induced oxidative modification of human low density lipoprotein (LDL).LDL oxidation was monitored spectrophotometrically by measurement of change in absorbance at 234 nm. Incubation of LDL (0.05 mg protein/ml) with 1.66 microM cupric chloride produced a lag phase of 130 min before onset of the propagation phase where polyunsaturated fatty acids undergo conversion to conjugated lipid hydroperoxides. However, in the presence of grape extract at a final concentration equal to an 8000-fold dilution, the lag phase was extended to 185 min. A 4000-fold and 2000-fold dilution of grape extract produced lag phases of 250 and 465 min, respectively. LDL oxidation was essentially blocked for at least 10 h with a 1000-fold dilution of grape extract. In other experiments, incubation of LDL (0.2 mg protein/ml) with 5 microM cupric chloride for 1-4 h increased both thiobarbituric acid-reactive substances and electrophoretic mobility of LDL on agarose gel. In addition, there was loss of immunoreactivity of LDL with a murine monoclonal antibody against human apolipoprotein B-100. However, these oxidative changes to LDL by copper were prevented when diluted grape extract was present during incubation. It is concluded that grape extract contains antioxidants in the form of polyphenols with the capacity to inhibit oxidative modification of LDL.

Hypolipidemic agents alter hepatic mitochondrial respiration in vitro.

The direct effects of three different classes of structurally diverse hypolipidemic agents on respiration were studied in mitochondria isolated from donor Sprague-Dawley rats. Two classes of peroxisome proliferators (i.e. plasticizers and hypolipidemic hormones and drugs) and one class of peroxisome inhibitors (i.e. anti-psychotic drugs) were studied. The phthalate ester plasticizers dibutylphthalate, ethylhexanoic acid and di(2-ethylhexyl) adipate, the hypolipidemic hormones or drugs dehydro-epiandrosterone (DHEA), thyroxine (T4), triiodothyronine (T3), gemfibrozil, clofibrate and naphthoflavone, and the anti-psychotic drugs chlorpromazine, thioridazine and fluphenazine were studied. As the dose of the plasticizer dibutylphthalate increased from 8 to 200 mumol/l, there was a decrease (P < 0.05) in state 3 (+ADP) respiration and in the respiratory control ratio for both substrates tested. The anti-psychotic drug chlorpromazine decreased state 3 malate + pyruvate-supported respiration and increased state 3 succinate-supported respiration. As the concentration of all three anti-psychotic drugs increased, there was a linear increase in state 4 respiration (-ADP) and a decrease in the respiratory control ratio for both substrates tested. As the dose of the hypolipidemic agents DHEA, gemfibrozil and T4 increased, there was a linear reduction in state 3 malate + pyruvate-supported respiration. However, when succinate was used as the substrate to support respiration, only the thyroid hormones significantly decreased state 3 respiration. Gemfibrozil, T4 and T3 increased state 4 respiration, regardless of the substrate used. As the dose of clofibrate, gemfibrozil, and the thyroid hormones increased, there was a linear reduction in the respiratory control ratio for both substrates tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Inverse relationship between peroxisomal and mitochondrial beta-oxidation in HepG2 cells treated with dehydroepiandrosterone and clofibric acid.

A transformed human hepatoma cell line was examined to determine if it was an appropriate model system for studying the mechanism of action of two peroxisome proliferators that lower blood lipids. Cultures of HepG2 cells were exposed to four different concentrations of either the hypolipidemic drug, clofibric acid (CLO), or the adrenal steroid, dehydroepiandrosterone (DHEA). Activities of two peroxisomal enzymes, palmitoyl-CoA oxidase and catalase, and two mitochondrial enzymes, carnitine palmitoyl-CoA transferase and succinate-INT-reductase, were measured in CLO- and DHEA-treated cells. In general, as the concentration of these hypolipidemic agents increased from 0 to 1000 microM, the specific activities of peroxisomal palmitoyl-CoA oxidase and catalase increased, and mitochondrial carnitine palmitoyl-CoA transferase and succinate-INT-reductase decreased. The activity of lactate dehydrogenase was significantly higher in the medium of cultures exposed to the 500 and 1000 microM concentration of DHEA compared with the control cultures, indicating the cytotoxic effects of this steroid at millimolar levels in vitro. In summary, the peroxisomal proliferators, DHEA and CLO, inversely altered peroxisomal and mitochondrial beta-oxidation in HepG2 cultures, but not to the extent reported for rat hepatocytes in vitro. In vitro concentrations of DHEA greater than 500 microM adversely affected the viability of HepG2 cells. The results of this study suggest that beta-oxidation in this human hepatoma cell line may not be as sensitive to hypolipidemic agents as are primary cultures of rat hepatocytes.

Rates of beta-oxidation of fatty acids of various chain lengths and degrees of unsaturation in highly purified peroxisomes isolated from rat liver.

Highly purified peroxisomes were obtained from the liver of untreated rats, and rates of peroxisomal beta-oxidation were measured using fatty acyl-CoAs differing in chain length and degree of unsaturation. A 20-24-fold purification of peroxisomes, indicated by the specific activities of the marker enzymes catalase and urate oxidase, respectively, was obtained from crude liver homogenate using differential centrifugation techniques followed by a 30% Nycodenz gradient separation. The use of a 30% Nycodenz gradient in the final step of purification was extremely effective (e.g. 5.5-fold reduction) in removing lysosomal contamination. The rate of peroxisomal beta-oxidation with lauroyl-CoA (C12:0) as substrate was the highest of all fatty acyl-CoAs tested. Butyryl-CoA (C4:0) was not oxidized by purified peroxisomes. In general, as chain length of the fatty acyl-CoAs increased above 12 carbons, the rates of beta-oxidation decreased.