Changes in chemical composition and physico-chemical properties of chick low- and high-density lipoproteins induced by supplementation of coconut oil to the diet.

Supplementation of coconut oil to the diet for 1-2 weeks produced a significant hypercholesterolemia in 14-day-old chicks. Changes in plasma fatty acid composition correlated positively with those of diets. In this study, we have shown a different response of low- and high-density lipoprotein (LDL and HDL) fractions to dietary saturated fat (coconut oil) rich in lauric and myristic acids. Although all the components of these particles seemed to increase, the percentages of increases found in total (TC), free (FC) and esterified cholesterol (EC) were higher in LDL than in HDL. TC/phospholipid (PL) ratio, considered as an inverse index of membrane fluidity, also increased with the dietary regimen in LDL, while no significant differences were found in HDL. These results suggest that supplementation of coconut oil to the diet decreased the fluidity of LDL. The EC/triglycerides (TG) ratio was also significantly increased in LDL, corroborating the main atherogenic function of this lipoprotein fraction in response to lauric and myristic acids. We have also estimated the lipidic order parameter, S, from the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled low- and high-density lipoproteins. In LDL, temperature dependence of S shows two different behaviour zones at about 20 degrees C. In HDL, the plot of S values versus T is linear. DPH anisotropy and S increased in both LDL and HDL from treated chicks. This increase becomes more evident as temperature rises and also with dietary treatment.

Coconut oil affects lipoprotein composition and structure of neonatal chicks.

Supplementation of 10 or 20% coconut oil in the diet for 1-2 weeks produced a significant hypercholesterolemia in neonatal chicks. Plasma triacylglycerol concentration significantly increased after the addition of 20% coconut oil for 2 weeks. These results show that newborn chicks are more sensitive to saturated fatty acids from coconut oil than adult animals. The effects of this saturated fat on lipoprotein composition were studied for the first 1-2 weeks of neonatal chick life. Coconut oil supplementation in the diet (20%) for 2 weeks increased cholesterol concentration in all the lipoprotein fractions, while 10% coconut oil only increased cholesterol in low-density and very-low-density lipoproteins, an increase that was significant after 1 week of treatment. Similar results were obtained for triacylglycerol concentration after 2 weeks of treatment. Changes in phospholipid and total protein levels were less profound. Coconut oil decreased low-density and very-low-density lipoprotein fluidity, measured as total cholesterol/phospholipid ratio. Changes in esterified cholesterol/phospholipid and triacylglycerol/phospholipid ratios suggest that coconut oil affects the distribution of lipid components in the core of very-low-density particles. Likewise, the esterified cholesterol/triacylglycerol ratio was clearly increased in the low-density, and especially in the very-low-density, fraction after the first week of coconut oil feeding. Our results show that neonatal chick provides a suitable model in which to study the role of very-low-density lipoproteins in atherogenesis and the rapid response to saturated fatty acids with 12-14 carbons.

Inhibition of chick brain cholesterogenic enzymes by phenyl and phenolic derivatives of phenylalanine.

Phenylalanine and its phenyl metabolites produced a clear inhibition of chick brain mevalonate 5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives produced a similar or higher inhibition than that found in the presence of phenyl metabolites. The inhibition was progressive with increasing concentrations of inhibitors (1.25-5.00 mM). Phenylpyruvate and p-hydroxyphenyl-lactate were the most potent inhibitors of decarboxylase activity. Simultaneous supplementation of each metabolite at 0.25 mM concentration produced a considerable inhibition of brain decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. At our knowledge this is the first report on the in vitro inhibition of both brain regulatory enzymes of cholesterogenesis in phenylketonuric-like conditions.

Induction of experimental phenylketonuria-like conditions in chick embryo. Effect on amino acid concentration in brain, liver and plasma.

Experimental hyperphenylalaninemia has been induced in chick embryos between 11-20 days of incubation by daily injection of ?-methylphenylalanine and phenylalanine. Brain and liver weight decreased after 8 days of treatment. An increase of nearly 14-fold in the brain phenylalanine/tyrosine ratio was observed after 9 days of treatment. Similar results were obtained in liver, although the increase found in this case was smaller than in brain. Chronic hyperphenylalaninemia induced a clear rise in the levels of plasma and liver valine, leucine and isoleucine, while in brain these levels did not change significantly. Plasma and brain glycine content was also enhanced by this treatment. Brain tyrosine concentration was clearly decreased in these conditions, in contrast to the enhancement reported after this and other treatments in various animal species. Thus, the higher value of the brain phenylalanine/tyrosine ratio obtained by ?-methylphenylalanine plus phenylalanine administration was due to both an increase in the phenylalanine and a decrease in the tyrosine levels, conditions that have been also found in human phenylketonurics. Therefore, the treatment here reported was an excellent method for imitating the conditions of phenylketonuria during the period of rapid myelination in the chick, one of the most dramatic in nervous system development.

Coconut oil induces short-term changes in lipid composition and enzyme activity of chick hepatic mitochondria.

We studied the short-term effects of a 20% coconut oil supplementation to the chick diet on lipid composition of liver and hepatic mitochondria, and changes that occurred in mitochondrial-associated enzymes as a result of this diet. No significant differences were observed in the lipid contents of liver when young chicks were fed the experimental diet, whereas hepatic mitochondria rapidly changed in response to this diet. Total cholesterol significantly increased in mitochondria at 24 hours of coconut oil diet feeding and decreased when dietary treatment was prolonged for 5 to 14 days. Changes in total mitochondrial phospholipids showed an inverse profile. A significant decrease in phosphatidylethanolamine and an increase in sphingomyelin were found at 24 hours. The cholesterol/phospholipid molar ratio significantly and rapidly (24 hours) increased in mitochondria from treated animals. Cytochrome oxidase activity drastically increased after 24 hours of experimental diet feeding and lowered to the control values when dietary manipulation was prolonged for 5 to 14 days. ATPase activity showed an inverse profile. Changes in cytochrome oxidase activity were parallel to changes in the cholesterol/phospholipid molar ratio, whereas changes in ATPase activity showed an inverse correlation with changes in this molar ratio. To our knowledge, this is one of the first reports on the very rapid response (24 hours) of mitochondrial lipid composition and function to saturated fat feeding.

Hypolipidemic activity of dipyridamole: effects on the main regulatory enzyme of cholesterogenesis.

The in vivo dipyridamole treatment for 16 days produced a significant decrease in chick plasma cholesterol, mainly due to the esterified form. This effect was especially patent in the VLDL + LDL fraction. Similar results were observed in triglyceride content. To our knowledge, this is the first report on this hypolipidemic effects of dipyridamole. Total and esterified cholesterol increased after the same treatment in chick liver, while brain cholesterol content was not affected. Hepatic 3-hydroxy-3- methylglutaryl-CoA reductase activity was drastically reduced, while other secondary regulatory enzymes such as mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase did not change significantly. No significant differences were found in cholesterol and lipidic phosphorus from liver microsomes, so that the effect of dipyridamole on reductase activity cannot be due to modifications in cholesterol/lipidic phosphorus molar ratio. Neither of these enzyme activities was affected in vitro by dipyridamole.

Effects of clofibrate on the main regulatory enzymes of cholesterogenesis.

The in vivo effect of clofibrate on the main regulatory enzymes of cholesterogenesis has been comparatively studied for the first time in chick liver and brain. 3-Hydroxy-3-methylglutaryl-CoA reductase and mevalonate 5-pyrophosphate decarboxylase from chick liver were significantly inhibited by this hypocholesterolenic drug, while mevalonate kinase and mevalonate 5-phosphate kinase were not affected. No enzyme from chick brain was significantly inhibited by the in vivo treatment. However, both liver and brain reductase activity was inhibited in vitro by clofibrate, inhibition that was progressive with increasing concentrations (1.25-5.00 mM) of drug.

Differential changes in the fatty acid composition of the main lipid classes of chick plasma induced by dietary coconut oil.

For a better understanding of the hyperlipidemic function of saturated fat, we have studied the comparative effects of diet supplementation with 10 and 20% coconut oil on the main lipid classes of chick plasma. Changes in fatty acid composition of free fatty acid and triglyceride fractions were parallel to that of the experimental diet. Thus, the increase in the percentages of 12:0 and 14:0 acids may contribute to the hypercholesterolemic effects of coconut oil feeding. Plasma phospholipids incorporated low levels of 12:0 and 14:0 acids whereas 18:0, the main saturated fatty acid of this fraction, also increased after coconut oil feeding. The percentage of 20:4 n-6 was higher in plasma phospholipids than in the other fractions and was significantly decreased by our dietary manipulations. Likewise, minor increases were found in the percentages of 12:0 and 14:0 acids in plasma cholesterol esters. However, the percentage of 18:2 acid significantly increased after coconut oil feeding. Our results show a relationship between fatty acid composition of diets and those of plasma free fatty acid and triglyceride fractions, whereas phospholipids and cholesterol esters are less sensitive to dietary changes.

Effect of lipid content of diet on cholesterol content and cholesterogenic enzymes of European eel liver.

The effect of dietary lipid levels on the levels of cholesterol and the activities of the major cholesterogenic enzymes of the liver has been studied in the European eel. An increase in hepatic total cholesterol was observed when the dietary lipid levels increased from 12 to 20%, while protein levels were maintained at 30%. This change paralleled an increase in mevalonate 5-pyrophosphate decarboxylase activity, while 3-hydroxy-3-methylglutaryl-CoA reductase mevalonate kinase and mevalonate 5-phosphate kinase were not affected by changes in diet composition. These results suggest that the decarboxylase may be a rate-limiting enzyme in cholesterogenesis in eel liver.

A procedure for the simultaneous determination of lipid and protein in biomembranes and other biological samples.

Very small sample sizes frequently become the limiting factor in biochemical and biomembrane studies in which routine quantification of protein and bulk lipids are required. The procedure described here allows the simultaneous determination of protein and lipid without initial, multiple aliquots. The method is based on the quantitative precipitation of proteins from a defined hexane/isopropanol mixture. The liquid phase resulting after decanting and concentrating to dryness can then be used to assay the lipid content directly. Quantitative assay of protein can be achieved after resuspension of the pelleted material by addition of sodium dodecyl sulfate (0.1%) and deoxycholate (1%). The method is also applicable to other types of lipid- and protein-containing samples with a broad range of protein/lipid ratios and lipid compositions, as they occur, for example, in serum lipoproteins.

Supplementation of coconut oil from different sources to the diet induces cellular damage and rapid changes in fatty acid composition of chick liver and hepatic mitochondria.

Supplementation of 20% coconut oil from two commercial sources pharmaceutical ("Pharmacy") and cooking ("Pastry") use, to the chick diet for 14 days produced a clear damage to the hepatic mitochondria, accompanied by an accumulation of glycogen and lipid droplets in the hepatocyte cytoplasm. These effects may be accounted for the high proportion of fat supplemented to the diets (20%). Pharmacy coconut oil induced a high percentage of cellular death when administered for 14 days. Fatty acid profiles in liver and hepatic mitochondria rapidly changed (24 hr) after both coconut oils supplementation to the diet. The accumulation of shorter chain fatty acids (12:0 and 14:0) was always higher after Pharmacy than after Pastry diet feeding. This fact may contribute, at least in part, to the cellular damage mentioned above especially after Pharmacy diet feeding. Mitochondrial ratios of saturated/unsaturated and saturated/polyunsaturated fatty acids rapidly changed in parallel to these ratios in both diets. Most of the mitochondrial parameters measured tend to recuperate the control values when diets were supplied for 5-14 days. Nevertheless, the maintenance of the mentioned ratios after 14-days Pharmacy diet feeding at significantly higher levels than those observed in control, seems to suggest the lack of the homeostatic mechanism in these membranes and could be also related with the high percentage of cellular death observed after this dietary manipulation.

Influence of fasting status on the effects of coconut oil on chick plasma and lipoprotein composition.

For a better understanding of the hyperlipidemic function of saturated fat, we have studied the effects of diet supplementation with 10-20% coconut oil on the chick plasma and lipoprotein composition under postprandial and starvation conditions. A significant hypercholesterolemia was found in chicks fed the standard diet after 12 h of food deprivation. In these conditions, LDL-cholesterol also increased, whereas triglyceride levels were reduced in HDL, VLDL and chylomicron fractions. Coconut oil induced a significant hypercholesterolemia under both conditions, also increasing the plasma triglyceride content under postprandial conditions, but not after starvation. Coconut oil feeding increased all the chemical components of HDL, especially under postprandial conditions, but did not affect the HDL-triglycerides under food-deprivation conditions. Total cholesterol and triglyceride levels in LDL increased after coconut oil supplementation to the diet. Differences were more pronounced under postprandial conditions. Changes in VLDL and chylomicron composition were less evident.

Changes in plasma lipid composition induced by coconut oil. Effects of dipyridamole.

The comparative effects of 10-20% coconut oil feeding on fatty acid composition of the main lipid classes of chick plasma have been studied with and without simultaneous treatment with dipyridamole in order to clarify the hypolipidemic role of this drug. Coconut oil drastically increased the percentages of lauric and myristic acids in free fatty acid and triacylglycerol fractions, whereas these changes were less pronounced in phospholipids and cholesterol esters. The percentage of arachidonic acid was higher in plasma phospholipids than in the other fractions and was significantly decreased by coconut oil feeding. Linoleic acid, the main fatty acid of cholesterol esters, was drastically increased by coconut oil feeding. Changes induced by the simultaneous administration of dipyridamole were more pronounced in the phospholipids and cholesterol esters than in the other fractions. The fall observed in linoleic acid levels after dipyridamole treatment may be of interest for a lower production of its derived eicosanoids, especially in plasma phospholipids and cholesterol esters.

Effect of experimental hyperphenylalaninemia induced by dietary phenylalanine plus alpha-methylphenylalanine administration on amino acid concentration in neonatal chick brain, plasma, and liver.

Supplementation of 5% phenylalanine plus 0.4% alpha-methylphenylalanine to the standard diet or 1% phenylalanine plus 0.08% alpha-methylphenylalanine to the drinking water produced phenylketonuria-like conditions in 5-day-old chicks. An increase of 10 to 15-fold in the phenylalanine content was observed in plasma or brain of animals after 9 days of both types of treatment. A smaller but significant increase was also observed in liver. However, practically no changes were found in the levels of tyrosine in the same conditions. Thus, the high values of plasma and brain phenylalanine/tyrosine ratio obtained by these treatments were mainly due to an increase in the phenylalanine levels, without increasing those of tyrosine. Chronic hyperphenylalaninemia induced a nonsignificant decrease in the most of amino acid contents in brain, especially after 9 days of treatment, although the levels of glycine and serine were significantly increased. A similar decrease was found in the plasma and liver concentration of various amino acids, although the variations observed in the liver were smaller than those found in plasma and brain.

Inhibition of brain and liver 3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphate decarboxylase in experimental hyperphenylalaninemia.

Experimental hyperphenylalaninemia has been induced in 5-day-old chicks by dietary treatments with phenylalanine and alpha-methylphenylalanine. An increase of nearly 8-fold in plasma Phe/Tyr ratio was found after 4 days of supplementation the standard diet with 5% phenylalanine plus 0.4% alpha-methylphenylalanine. The increase in this ratio was about 13-fold after 9 days of the same treatment. Similar results were observed in brain and liver, although the increases were smaller than those found in plasma. Total body, brain and liver weight decreased after 9 days of treatment. Phenylalanine plus alpha-methylphenylalanine administration to 5-day-old chicks produced a significant decrease in the 3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphate decarboxylase specific activities from both brain and liver. These results demonstrated for the first time that experimental hyperphenylalaninemia inhibited different enzyme activities directly implicated in the regulation of cholesterogenesis. Therefore, a reduced cholesterol synthesis in brain may evidenciate the theory of an impaired myelination leading to mental retardation in phenylketonuria patients.

Effect of phenylpyruvate on mevalonate-activating enzymes from chick brain and liver.

Mevalonate-activating enzymes from chick brain and liver were stable when 105,000 x g supernatants were stored at -4 degrees C for 168 h. Mevalonate kinase and mevalonate 5-phosphate kinase retained their activities for 72 h at 4 degrees C while mevalonate 5-pyrophosphate decarboxylase activity significantly decreased after 24-48 h of storage at 4 degrees C. Direct addition of 2.5 mM phenylpyruvate to the reaction mixture produced a significant inhibition of decarboxylase activity in brain and liver. When enzyme preparations were preincubated with 2.5 mM phenylpyruvate for 20 min before the addition of substrate, an increased inhibition was observed. Mevalonate kinase and mevalonate 5-phosphate kinase from both tissues were not affected in the same conditions. The inhibition of brain and liver decarboxylase was progressive with increasing concentrations (2.5-10.0 mM) of phenylpyruvate. No significant difference was observed in the inhibition of decarboxylase after 10 or 20 min of preincubation.

Development of lipidic composition of neonatal chick liver and intestine microsomes: changes induced by cholesterol feeding.

Changes in cholesterol and phospholipid content of chick liver and intestine microsomes were studied throughout the two first weeks of life. Differences observed throughout postnatal development were mainly due to the free cholesterol. Cholesterol feeding resulted in a clear increase of the amounts of both free and esterified cholesterol. Phospholipid content of chick liver and intestine microsomes did not change significantly after hatching. Phosphatidylcholine and phosphatidylethanolamine were found to be the major phospholipids. Although the amount of each phospholipid could be affected by cholesterol feeding, its relative percentage did not change by this treatment.

Influence of protein/lipid ratio of diet on cholesterol synthesis and esterification in eel liver.

The effect of protein/lipid ratio of diets on hepatic cholesterol has been studied in European eel and correlated with changes in the main enzymes responsible for cholesterol metabolism. The growth rates of animals were similar when dietary lipid level was 12%. However, a 25% protein/20% fat (25/20) diet produced a decrease in the weight gain when compared with that observed after feeding a 30/20 diet. At low fat level (12%), the decrease in dietary protein produced a little but significant increase in total cholesterol, mainly due to the esterified form. On the contrary, a 25/20 diet produced a lower cholesterol accumulation than that a 30/20 diet. These results suggest that a minimal protein level was required for an optimal utilization of dietary fat for cholesterol deposition in liver. No significant differences were found in 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase when compared the effect of 40/12 and 30/12 diets as well as that of 30/20 and 25/20 diets, suggesting that differences in hepatic cholesterol content were not due to differences in cholesterol synthesis but in the transport to the liver. Changes in the esterified cholesterol were parallel to those found in acyl-CoA: cholesterol acyltransferase, corroborating the main role of this enzyme in the regulation of hepatic cholesterol esterification.

Effect of dietary coconut oil on lipoprotein composition of young chick (Gallus domesticus).

1. The composition of HDL, the major lipoprotein fraction from chick serum, drastically changed after 2 weeks of coconut oil feeding. Total cholesterol and triacylglycerols significantly increased following dietary 10 or 20% coconut oil supplementation. 2. Changes in LDL composition were less profound, cholesterol being the only component that increased by coconut oil supplementation (10 or 20%). 3. IDL proteins were the only components that increased following the same dietary treatment (20%). 4. VLDL cholesterol and proteins also increased after 1-2 weeks of 20% coconut oil supplementation to the diet. 5. Of total lipoproteins, the cholesterol content strongly increased after dietary treatment, while triacylglycerols did not change significantly.

Effect of clofibrate on brain mevalonate-5-pyrophosphate decarboxylase.

The effect of clofibrate on the activity of the three mevalonate-activating enzymes has been studied for the first time in brain by reactions carried out using [2-14C] mevalonic acid as substrate and 105,000 g supernatants from 14-day-old chick brain. Mevalonate-5-pyrophosphate decarboxylase was clearly inhibited, while mevalonate kinase and mevalonate-5-phosphate kinase were not significantly affected. The effect of clofibrate on decarboxylase activity was progressive with increasing concentrations (1.25-5.00 mM) of the inhibitor. A transient inhibition and a subsequent activation as a function of clofibrate concentration seemed to occur for mevalonate kinase. Direct measurements of decarboxylase activity utilizing [2-14C] pyrophosphomevalonate as the specific substrate of this enzyme corroborated these results. Kinetic studies showed that clofibrate competes with the substrate ATP.