Mario Castro

In vitro effects of a flavonoid-rich extract on LDL oxidation

Flavonoids are phenolic compounds of vegetable origin with antioxidant effects. The present study aimed to determine their properties as LDL antioxidants. LDL were incubated with increasing concentrations of flavonoids (0-16 micrograms/ml) and LDL oxidation was started by adding CuCl2 (2 microM) to the media. When flavonoids were present in the media, vitamin E consumption, the lag phase of conjugated diene formation, LDL electrophoretic mobility in agarose gels and the appearance of thiobarbituric acid reacting substances (TBARS) were delayed in a concentration-dependent manner. To determine whether flavonoids could terminate LDL oxidation once initiated, two sets of experiments were performed. In the first, LDL oxidation was initiated as described above. At 2 or 4 h of incubation, flavonoids were added (4 micrograms/ml) and their effect compared to samples where butylated hydroxytoluene or EDTA were added. At 5 h, in the LDL samples where flavonoids were added, the electrophoretic mobility and TBARS production were the same as those present in LDL samples incubated for the whole period in the absence of flavonoids. However, when either butylate hydroxytoluene or EDTA was added, as would be expected, the LDL oxidation process was completely arrested as shown by a reduction in the appearance of TBARS and a lower LDL electrophoretic mobility. In the second experiment, LDL oxidation was initiated as described above and at 0, 10 and 20 min, flavonoids were added (4 micrograms/ml). When vitamin E was still present in the LDL solution, the flavonoids were able to both increase the lag phase in the formation of conjugated dienes and to delay the consumption of vitamin E. The present results show that in vitro, flavonoids prevent LDL oxidation in a concentration-dependent manner, delaying the consumption of vitamin E, but they cannot terminate or delay LDL oxidation once vitamin E in LDL is consumed.

Optimization and validation of a method for the determination of caffeine, 8-chlorotheophylline and diphenhydramine by isocratic high-performance liquid chromatography: Stress test for stability evaluation

The optimization of a HPLC method for caffeine, 8-chlorotheophylline and diphenhydramine separation with UV detection at 229 nm is described. The conditions studied included: stationary phase, compositions of mobile phases with pH modulators. Optimal conditions were: SymmetryShield RP8 column and acetonitrile-(0.01 M H3PO4-triethylamine, pH 2.8) (22:78, v/v). Validation was performed using standards and a pharmaceutical preparation containing the compounds described above. Results from both standards and samples show suitable validation parameters. The pharmaceutical grade substances were tested by factors that could influence the chemical stability. These reaction mixtures were analyzed to evaluate the capability of the method to separate degradation products. Degradation products did not interfere with the determination of the substances tested by the assay.

Simultaneous determination of vitamins A and E in rat tissues by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method to determine vitamins A, Ap and E simultaneously was developed with direct extraction of vitamins from rat tissues with n-hexane and probe sonicating. The dry residue was redissolved in chloroform-methanol. Vitamins A and Ap were detected by UV-Vis and vitamin E by fluorescence. Vitamin K, used as internal standard, was detected both the UV-Vis and by fluorescence. Standards and samples were checked for linearity giving correlation coefficients that were higher than 0.99 in the concentration range of 3.1-9.4 for vitamin A, 8.2-24.7 for vitamin Ap and 0.6-1.7 nmol/g in the case of liver extracts and 0.5-3.0 nmol/g in the case of placenta. The inta-assay precision (R.S.D) varied between 1.48 and 7.25, whereas inter-assay precision was between 4.99 and 7.03. Recoveries ranged between 94 +/- 8 and 107 +/- 5%. Results from the application of this method to different rat tissues having wide range of vitamin content are presented.

Simplified method for vitamin E determination in rat adipose tissue and mammary glands by high-performance liquid chromatography

A method for vitamin E (alpha-tocopherol) measurement in rat adipose tissue and mammary gland has been developed and validated. Tissues were homogenized in ethanol-water (1:1) and extracted with n-hexane. Vitamin K1 was used as internal standard. Separation was performed by HPLC with methanol-water (96.5:3.5) as eluent in a Nucleosil C18 column (15 x 0.46 cm) at 40 degrees C. Detection was by fluorescence with excitation at 295 nm and emission at 350 nm for vitamin E and at 330 and 440 nm for vitamin K1. Standards and tissue extracts were checked for linearity giving correlation coefficients over 0.99 in a range of concentrations from 0.56 to 4.51 nmol/g in adipose tissue and from 2.18 to 17.4 nmol/g in mammary gland tissue. Intra-assay precision (R.S.D.) varied between 3 and 4%, whereas inter-assay precision was between 8 and 9%. Recoveries ranged between 95 +/- 3% and 98 +/- 11% for the two tissues, respectively. Vitamin E was measured in rats that had previously received one oral dose of this vitamin. Whereas vitamin E content in adipose tissue did not differ between late-pregnant and virgin rats, it was significantly higher in mammary gland of pregnant rats, and this difference could be related to the enhanced lipoprotein lipase activity in this group.

Determination of α-tocopherol and α-tocopheryl acetate in diets of experimental animals: Study of stability in the diets

A simple method is described which permits, avoiding saponification, α-tocopherol and α-tocopheryl acetate measurement in semi-synthetic diets for experimental animals by HPLC, with both UV and fluorescence detection. Phenyldodecane was chosen as internal standard with remarkable performances, and EDTA and BHT were added to prevent oxidation in aqueous and non-aqueous phases respectively. The mobile phase was methanol–water (94:6, v/v) at a flow-rate of 2 ml/min. Samples were homogenized and extracted twice with n-hexane by probe sonication. Extracts were evaporated to dryness and redissolved with chloroform–methanol (1:1, v/v). Validation parameters were studied between 25 ng and 6 μg for α-tocopherol and between 3 and 24.2 μg for α-tocopheryl acetate, which corresponds to the range of values in the existing diets. Results had correlation coefficients >0.99; recoveries >85%; R.S.D.<6%, so the method is adequate to control vitamin E intake in animals as well as vitamin E stability in food during storage.

Alpha-Tocopherol Concentration in Fetal and Maternal Tissues of Pregnant Rats Supplemented with Alpha-Tocopherol

We wanted to determine whether alpha-tocopherol supplementation to pregnant rats could increase the concentration of alpha-tocopherol in maternal and fetal plasma and tissues. Pregnant rats were treated with alpha-tocopherol on days 18 and 19 gestation and studied at day 20. A control group was studied in parallel. Treatment of pregnant rats with alpha-tocopherol increased its concentration in maternal and fetal plasma, in all maternal plasma lipoprotein fractions, in maternal and fetal liver and in the placenta. The fetal and maternal concentration of alpha-tocopherol were positively correlated.

Studies with etofibrate in the rat. Part II: A comparison of the effects of prolonged and acute administration on plasma lipids, liver enzymes and adipose tissue lipolysis.

To contribute to the understanding of the hypolipidemic action of etofibrate, which is the 1,2-ethandiol ester of clofibric acid and nicotinic acid, 300 mg of this drug/kg body weight or of the medium were administered daily by a stomach tube to normolipidemic rats. Some animals were decapitated at the 10th day of daily treatment (prolonged treatment), whereas others were studied at different times after one single administration (acute treatment). In animals on prolonged treatment etofibrate decreased plasma levels of cholesterol, triacylglycerols, free fatty acids (FFA) and glycerol, as well as the total and unesterified cholesterol concentrations, in liver microsomes. In these rats, etofibrate increased the activity of liver cytosolic glycerol-3-P dehydrogenase, whereas it decreased the activity of both microsomal HMG-CoA reductase and cholesterol 7 alpha-hydroxylase and did not affect acyl-CoA: cholesterol acyltransferase (ACAT). At 3, 5 and 7 h after acute treatment, etofibrate decreased plasma levels of triacylglycerols, glycerol and FFA, and this effect disappeared at 24 h, whereas plasma cholesterol did not change 3 h after etofibrate but decreased at 5 and 7 h and remained low after 24 h, and a similar change was found in the liver microsomes free cholesterol concentration. However, with the exception of a significant reduction in cytosolic glycerol-3-P dehydrogenase at 7 h and in ACAT at 5 h, acute etofibrate treatment did not affect the activity of the liver enzymes studied. At low concentrations (10(-5) M) in the incubation medium, etofibrate decreased the release of both FFA and glycerol by epididymal fat pad pieces incubated in vitro. These findings together with those previously reported by us in rats using a similar etofibrate treatment protocol [6] indicate that etofibrate decreases the availability of lipolytic products in the liver by acting on their release from adipose tissue and on their intrinsic hepatic metabolism. Consequently, this drug would decrease liver VLDL triacylglycerol synthesis and secretion, which together with facilitating the clearance of circulating triacylglycerols causes its hypotriglyceridemic effect. The hypocholesterolemic effect of etofibrate after acute treatment may be a secondary consequence of the reduced liver VLDL production caused by decreased adipose tissue lipolysis, but after prolonged treatment, this effect also seems to be influenced by the inhibition of HMG-CoA reductase activity which would reduce cholesterol synthesis.

Studies with etofibrate in the rat. Part I: Effects on glycerol, free fatty acid and triacylglycerol metabolism

Etofibrate is the 1,2-ethandiol diester of clofibric acid and nicotinic acid that decreases circulating levels of triacylglycerols and cholesterol. To understand the mechanism by which the drug affects plasma triacylglycerols, normolipemic rats were treated daily with 300 mg of etofibrate/kg body weight or with the medium by a stomach tube. They were decapitated on the 10th day, and showed lower levels of plasma beta-hydroxybutyrate, glycerol, free fatty acids (FFA), total triacylglycerols and cholesterol and VLDL triacylglycerols and cholesterol, whereas glucose and RIA-determined insulin levels were unmodified. Epididymal fat pad pieces from etofibrate-treated rats incubated in vitro released more glycerol but the same amount of FFA to the medium, and had greater uptake of [U-14C]glycerol for [14C]acylglycerol formation. In the presence of heparin, they also showed an enhanced release of lipoprotein lipase activity to the medium. The disappearance from plasma of intravenously administered [1-14C]palmitate was faster in the etofibrate-treated rats, and although they showed a decrease in 14C-esterified fatty acids of neutral lipids in both liver and plasma VLDL, there was an increase in liver 14C-labelled water-soluble components. After intravenous [U-14C]glycerol administration, there was a decrease in plasma VLDL [14C]acylglycerol and [14C]glucose and in liver [14C]acylglycerol, but an increase in plasma [14C]lactate. In the liver, etofibrate treatment heightened the cytosolic glycerol-3-phosphate dehydrogenase activity and the total carnitine concentration, whereas it reduced triacylglycerol and cholesterol concentrations. It is proposed that etofibrate enhances the reesterification of fatty acids and glycerol in adipose tissue, which, together with its augmented lipoprotein lipase activity, may facilitate the clearance of circulating triacylglycerols. These effects may act concomitantly with the decreased synthesis of triacylglycerols, secondary to the increased utilization of their precursors, acyl-CoA and glycerol-3-phosphate, in other pathways, causing the reduction of plasma VLDL triacylglycerols produced by etofibrate treatment.

Effect of etofibrate on bile production in the normolipidemic rat

1. The effect of etofibrate, the ethandiol-1,2 diester of nicotinic and clofibric acids on bile production was studied in male rats that received a daily dose of 300 mg of etofibrate/kg body weight by stomach tube for 10 days and were compared with control rats receiving the medium. 2. The bile duct was cannulated, animals were intravenously given 1 microCi (4-14C)-cholesterol/100 b.w. and bile was collected at different intervals for a total of 4 hr. 3. Etofibrate treatment decreased plasma cholesterol and triglyceride concentrations and increased the bile flow. The cummulative amount of both bile volume and total bile radioactivity secreted increased linearly in all the animals; the respective slopes being higher in etofibrate treated rats than in controls. 4. The main labelled component found in the bile was always bile acids rather than cholesterol and the proportion of each of these compounds was similar in both groups. Neither was any difference between the groups found in the concentration of bile acids, cholesterol and phospholipids nor in the cholesterol/(bile+phospholipid) ratio. 5. Besides other factors, the present results indicate that an increase in bile flow and biliary cholesterol excretion in its free form and after its conversion into bile acids should contribute to the hypocholesterolemic effect of etofibrate.