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.

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.

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.

Effects of dietary fish oil on the fatty acid composition of the main lipid classes of chick plasma lipoproteins.

We have studied the effects of diet supplementation with 10% fish oil on fatty acid composition of the main lipid classes of chick plasma lipoproteins bearing in mind the relationship between platelet aggregation and eicosanoid production from arachidonic acid. Fish oil drastically increased the percentages of 20:5 n-3 and 22:6 n-3 acids in the high density lipoprotein lipids. The 20:5/22:6 ratio increased in triacylglycerol fraction whereas in phospholipids and cholesterol esters both 20:5 and 22:6 acids increased in a similar proportion. The percentage of arachidonic acid was higher in phospholipids than in the other lipid classes from this lipoprotein fraction and was significantly reduced by fish oil feeding. Linoleic acid, which was the most abundant fatty acid in cholesterol esters, strongly decreased after fish oil consumption. Changes induced in low- and very low density lipoproteins were similar to that observed in the high density lipoproteins. However, in the very low density lipoproteins, the 20:5/22:6 ratio was not increased in triacylglycerols, in contrast to that found in the high- and low density fractions. Our results suggest that decreases observed by fish oil feeding in the percentages of arachidonic acid in phospholipids and linoleic acid in cholesterol esters in the three lipoprotein fractions may be of importance to explain some pharmacological effects of n-3 PUFA with regard to vascular diseases.

An improved assay of 3-hydroxy-3-methylglutaryl-CoA reductase activity in Reuber H35 hepatoma cells without microsomes isolation.

The optimal conditions for measuring 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity in Reuber H35 hepatoma cells are described in this paper. Cells in the exponential phase of growth were lysed by incubation with Brij 97 detergent for 30 min. We used imidazole buffer supplemented with EDTA and leupeptine, two inhibitors of proteases. Disrupted cells were then centrifuged at 12,000 g. Although microsomes are usually reported as enzyme preparations for measuring HMG-CoA reductase, our data showed that hepatoma cells may be used without previous isolation of microsomes. The 12,000 g supernatant showed similar levels of total and specific activities to those found in the microsomal fraction obtained after 105,000 g centrifugation. The soluble fraction showed less than 10% of reductase activity. Reductase activity from Reuber H35 hepatoma cells increased proportionally to the reaction time from 30 to 90 min and to the amount of protein added in a range of 50-500 micrograms. Our modified method was very sensitive and reproducible, because very low specific activity (about 15-100 pmol min-1 per mg protein) could be quantified in different assay conditions obtaining similar values.

Hypocholesterolemic activity of dipyridamole: effects on chick plasma and lipoprotein composition and arachidonic acid levels.

We have studied the effects of dipyridamole treatment on chick plasma and lipoprotein composition in postprandial and fasting (12 h) conditions. Plasma cholesterol levels were higher in fasted than in fed chicks, whereas triglycerides declined during starvation. Dipyridamole treatment reduced plasma cholesterol content, mainly of the free cholesterol fraction. In postprandial conditions, total cholesterol content of high and low density lipoproteins decreased in a similar proportion to that observed in plasma. However, cholesterol and other chemical constituents of intermediate and very low density lipoproteins were more drastically reduced by dipyridamole than in plasma. Total amounts of these lipoprotein fractions were also reduced about 50%. The effects of dipyridamole in fasted animals were not significant. To our knowledge, this is one of the first reports about the response of lipoprotein cholesterol to dipyridamole treatment. A strong decrease was also found in the arachidonic acid content of plasma phospholipids and cholesterol esters fractions.

Fish oil reduces cholesterol and arachidonic acid levels in plasma and lipoproteins from hypercholesterolemic chicks.

The value of fish oil for prevention and/or treatment of human atherosclerosis has not been fully established. This study shows that replacement of saturated fat in young chick diet with menhaden oil produced a significant reversion of the hypercholesterolemia previously induced by coconut oil feeding. Fish oil also produced a clear decrease of plasma triacylglycerol levels. Coconut oil increased the percentages of 12:0 and 14:0 fatty acids, while menhaden oil increased those of 20:5 n-3 and 22:6 n-3. Percentages of 20:4 n-6, 18:2 n-6 and 18:1 n-9 significantly decreased by fish oil addition to the diet. Total cholesterol, phospholipid and protein contents of high and low density lipoproteins increased by coconut oil feeding. When coconut oil was replaced by menhaden oil, total cholesterol was significantly reduced in high, low and very low density lipoproteins. All chemical components of VLDL were decreased by menhaden oil feeding. Our results show a strong hypocholesterolemic effect of menhaden oil when this fat was supplemented to hypercholesterolemic chicks. The clear decrease found in arachidonic acid content of chick plasma and lipoproteins may contribute to the beneficial effects of fish oil consumption by lowering the production of its derived eicosanoids.

Metabolism of palmitic and docosahexaenoic acids in Reuber H35 hepatoma cells.

In this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (palmitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that palmitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.

Dietary fish oil reduces cholesterol and arachidonic acid levels in chick plasma and very low density lipoprotein.

The mechanisms involved in the hypolipidemic effects of fish oil have not been clearly established. This study shows that supplementation of 10% menhaden oil to the chick diet for 7 days produced a significant hypocholesterolemia and hypotriglyceridemia. Fatty acid composition of chick plasma drastically changed by the same dietary manipulation. Percentages of 20:5 and 22:6 n-3 fatty acids strongly increased, while percentages of 20:4 n-6, 18:2 n-6, and 18:1 n-9 significantly decreased. Changes observed in the relative percentages were parallel to those obtained in the amount of each fatty acid. Ratio of n-3/n-6 clearly decreased in plasma by fish oil feeding. Total cholesterol and triacylglycerol contents decreased in high density lipoprotein (HDL) but did not change in low density lipoprotein (LDL). All chemical constituents of very low density lipoprotein (VLDL) significantly decreased after the first week of menhaden oil supplementation to the diet. Similar modifications in fatty acid composition of the three lipoprotein fractions were also found. Our results suggest that the hypocholesterolemic effects of fish oil may be mediated by the depletion in VLDL synthesis and secretion into the chick plasma. On the other hand, the strong decrease found in the arachidonic acid (AA) content of chick plasma and lipoproteins may contribute to the beneficial effects of fish oil consumption by lowering the production of its derived eicosanoids.

Differential effects of dietary fat on chick plasma and liver composition and HMG-CoA reductase activity.

The comparative effects of diet supplementation with 10% saturated fat rich in 12:0 and 14:0 fatty acids (coconut oil), without and with 1% added cholesterol, and with 10% unsaturated fat rich in n-3 polyunsaturated fatty acids (menhaden oil) on cholesterol metabolism in neonatal chicks were examined to clarify the different mechanisms of their hyper- and hypolipidemic action. Supplementation of coconut oil produced a significant hypercholesterolemia after 7 days of treatment, with a similar increase in the amount of both free and esterified cholesterol. Supplementation of coconut oil plus cholesterol produced a higher increase of plasma cholesterol levels (approximately two to three times higher than those found with standard diet). However, supplementation of menhaden oil induced a significant decrease in total cholesterol after only 2 weeks of treatment. Levels of plasma triglycerides did not change by coconut oil addition to the diet, but a significant increase was observed after coconut oil plus cholesterol feeding. Menhaden oil produced a transient decrease in plasma triglycerides. Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity did not change with coconut oil treatment. However, both coconut oil plus cholesterol and menhaden oil supplemented diets drastically decreased reductase activity after 1 week of dietary manipulation. These results show that different nutrients with the same inhibitory effect on reductase activity produced opposite effects on plasma cholesterol content, suggesting the existence of important differences in the regulatory mechanisms implied in cholesterol biosynthesis and its accumulation in plasma.

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.

Synergism between the effects of dietary cholesterol and coconut oil on plasma, liver and lipoprotein composition of neonatal chick.

The nature of the synergism between dietary factors and the development of atherosclerosis has not been fully defined. Our studies showed that simultaneous supplementation of 10% saturated fat rich in 12:0 and 14:0 fatty acids (coconut oil) plus 1% cholesterol to the diet produced a sharp increase of plasma cholesterol, indicating a synergic influence of both dietary constituents. This increase was especially patent in the VLDL fraction, modifying the distribution of other lipid components between the core and the surface of these particles. These changes are consistent with the atherogenic function of VLDL and its responsiveness to dietary manipulation.

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.

Regulation of mevalonate 5-pyrophosphate decarboxylase in HeLa cells. Inhibition of enzymatic protein synthesis by serum lipoproteins.

Mevalonate 5-pyrophosphate decarboxylase (EC 4.1.1.33) has been considered as a secondary site of regulation of cholesterogenesis. Because of this, we have studied the regulation of decarboxylase in HeLa cells by serum lipoproteins in the cell culture medium. A first group of experiments was performed with cells grown in Eagle's medium with 10% foetal calf serum. The specific activity of decarboxylase was increased when whole foetal calf serum was replaced with lipoprotein-poor serum. This increase was clearly reduced in the presence of cycloheximide. Addition of serum lipoproteins to a medium containing lipoprotein-poor serum led to a clear decrease in the decarboxylase activity. An identical decrease was observed after the addition of lipoproteins alone or in combination with cycloheximide. These results suggest for the first time that the effect of serum lipoproteins on decarboxylase activity should be a decrease in the rate of enzymatic protein synthesis, and corroborate the important role of reactions other than those catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase in the regulation of cholesterogenesis.

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.

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 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.

Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development.

Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO2 strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.

Diurnal rhythm of the in vivo acetate metabolism to CO2 and nonsaponifiable lipids by neonatal chick.

The in vivo incorporation of acetate into nonsaponifiable lipids was studied in different tissues from 14-day-old chick. Total nonsaponifiable lipids (nmol/30 min/g tissue) were mainly synthesized in testicles and liver. The in vivo CO2 production from acetate by 1-day-old chick did not exhibit diurnal variations. However, in 14-day-old chick, a maximal value was observed in the middle of the light period, while a minimal value was found 9 h after the start of the dark period. No significant diurnal differences were detected in the in vivo acetate incorporation into nonsaponifiable lipids by liver and duodenal mucosa from 1-day-old chick. Nevertheless, a clear diurnal rhythm was found in liver and duodenal mucosa from 14-day-old chick, but not in brain and kidney from animals of the same age. Distribution of radioactivity from (1-14C)acetate among the different constituents of the nonsaponifiable fraction has been also studied at 3-h intervals. Cholesterol was the major sterol formed from acetate by chick liver at any time of day. In duodenal mucosa and kidney, maximal values in the percentage of cholesterol synthesized were observed during the light period.