M. F. Zafra

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 α-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% α-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 α-methylphenylalanine administration to 5-day-old chicks produced a significant decrease in the 3-hydroxy-3-methylglutary-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 activites 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 phenylalanine derivatives on the main regulatory enzymes of hepatic cholesterogenesis.

Phenylalanine, phenylpyruvate and phenylacetate produced a considerable inhibition of chick liver mevalonate 5-pyrophosphate decarboxylase while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives of phenylalanine produced a similar inhibition of decarboxylase activity than that found in the presence of phenyl metabolites. The degree of inhibition was progressive with increasing concentrations of inhibitors (1.25–5.00 mM). Simultaneous supplementation of different metabolites in conditions similar to those in experimental phenylketonuria (0.25 mM each) produced a clear inhibition of liver decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. To our knowledge, this is the first report on the in vitro inhibition of both liver regulatory enzymes of cholesterogenesis in phenylketonuria-like conditions. Our results show a lower inhibition of decarboxylase than that of reductase but suggest an important regulatory role of decarboxylase in cholesterol synthesis.

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.

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.

Hypolipidemic activity of dipyridamole: Effects on the main regulatory enzymes 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.

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.

Different hypercholesterolemic effects of cholesterol and saturated fat on neonatal and adult chicks

Abstract Supplementation of 2% cholesterol to the diet produced a significant hypercholesterolemia in adult chicks after 3 days of treatment. The amounts of both free and esterified cholesterol were about 3-fold in cholesterol fed chicks than those found in control animals. In newborn chicks, a significant hypercholesterolemia was observed only after 15 days of the same treatment. Only esterified cholesterol was increased in these conditions. Coconut oil (10 or 20%) supplementation to the diet produced a significant increase of plasma total cholesterol levels after 7 days of treatment of newborn chicks. Both free and esterified forms resulted increased in these conditions. A significant increase of plasma triacylglycerol content was observed 14 days after 20% coconut oil feeding. Contrary to that observed after cholesterol feeding, the hypercholesterolemic effect of coconut oil was not accompanied by changes in the levels of liver cholesterol.

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 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 105,000g 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.

Induction in Gallus domesticus of experimental hypercholesterolemia by saturated fat. Effects on cholesterogenic enzyme activity

The effect of coconut oil supplementation to the diet (10 or 20%) on lipid levels in plasma and liver as well as on the cholesterogenic enzyme activity were studied in 14-day-old chicks. Treatments for 1 or 2 weeks did not interfere in the growth rate of animals nor in the liver weight. The 10% coconut oil group showed a significant increase of plasma cholesterol after 2 weeks of treatment, while after 1 week the increase was not statistically significant. The 20% coconut oil group increased plasma cholesterol from the first week. Triacylglycerol content increased after each coconut oil supplementation to the diet during the first week. Hepatic cholesterol did not change significantly after any treatment assayed. No significant difference was observed in the cholesterogenic activity, measured as hepatic 3-hydroxy-3-methylglutaryl-CoA reductase, so that this study provides a perfect model of hypercholesterolemic animals without changes in their cholesterogenic ability.