The effect of halofenate and clofibrate on aggregation and release of serotonin by human platelets.

The hypolipidemic drugs halofenate and clofibrate inhibit the aggregation of human platelets induced by ADP, collagen and epinephrine. The effect of the drugs is primarily on the “second wave” of aggregation which is associated with the platelet release reaction. The drugs also inhibit the release of serotonin from platelets suggesting that the effect on aggregation is attributable to an inhibition of the release reaction. Halofenate is much more potent than clofibrate in inhibiting both the release of serotonin and the second wave of aggregation.     

Reversal of clofibrate-induced cholesterol oversaturation of bile with chenodeoxycholic acid.

Giving clofibrate 2 g daily to seven patients significantly increased the biliary cholesterol concentration while the proportion of bile acids fell. Five patients on established clofibrate treatment were given 750 mg of chenodeoxycholic acid (CDCA) daily for one month. Biliary lipid analysis after the CDCA treatment showed a significant fall in the proportion of cholesterol and a rise in that of bile acids. The serum lipid concentrations, which had already been reduced by diet and clofibrate, showed a further significant reduction after the introduction of CDCA. This study suggests that CDCA may be usefully combined with clofibrate to reverse the tendency towards cholesterol saturation of bile and enhance the effect of lowering serum lipid concentrations.     

Halofenate and clofibrate: mechanism of hypotriglyceridemic action in the rat.

Rats fed a fat-free diet containing no drug, 0.02% or 0.10% halofenate, or 0.25% clofibrate for 14 days were injected intravenously with equivalent amounts of either [2-3H]glycerol or [1(3)-3H]glycerol. Blood samples were collected at times up to 150 min after injection and serum triglycerides were isolated and assayed for radioactivity. Kinetic analysis of the serum appearance and clearance curves of 3H-labeled triglyceride permits estimation of serum total 3H-labeled triglyceride formation and triglyceride fractional turnover rates. The total amounts of 3H-labeled triglyceride formed from [2-3H] or from [1(3)-3H] glycerol in control-fed animals were very similar. Over 95% of the serum 3H-labeled triglyceride formed from either substrate circulated in a rapidly turning-over triglyceride pool (t1/2 = 8 min). Treatment with 0.10% halofenate or 0.25% clofibrate decreased labeling of serum triglycerides by 75-80% without increasing serum 3H-labeled triglyceride fractional turnover rates. Furthermore, both drugs decreased incorporation in vivo of 14C from [U-14C]glycerol into hepatic but not intestinal triglycerides without significantly decreasing incorporation of 14C into total phospholipids of either tissue. From these observations we suggest that, in the intact normal rat, sustained reduction of serum triglyceride levels produced by treatment with halofenate or clofibrate is due to inhibition of hepatic triglyceride formation.     

Evidence for permanent enhancement of residual ADH induced by antidiuretic agents (chlorpropamide, carbamazepine, clofibrate) in patients with pituitary diabetes insipidus.

Excretion of free water was studied in 7 patients with pituitary diabetes insipidus before treatment and after withdrawal of antidiuretic drugs (chlorpropamide, carbamazepine, clofibrate) used on a long-term basis. A statistically significant decrease in free water clearance was found after 2-4 weeks of withdrawal of antidiuretic agents. This was considered as a clinical evidence for the permanently enhanced release of residual ADH induced by chlorpropamide, carbamazepine and clofibrate in patients with partial defect in ADH secretion.     

Combined Use of Clofibrate and Cholestyramine or DEAE Sephadex in Hypercholesterolaemia

A comparison was made of the effect of DEAE (diethylaminoethyl) Sephadex (an anion exchange resin) and cholestyramine (Questran) with and without the addition of clofibrate in normal and hypercholesterolaemic patients. DEAE Sephadex (12-15 g/day) alone appeared to be as effective as cholestyramine in lowering the plasma cholesterol by 12-15%. Clofibrate acted synergistically with DEAE Sephadex and increased the activity of the latter by over twofold. This combination proved superior to that of clofibrate and cholestyramine and has the greatest potential use in the treatment of type II pattern hyperlipoproteinaemia.     

Pharmacologic effects of 4-chlorophenol in rats: comparison to clofibrate

4-Chlorophenol (4-CP) is an identified trace contaminant in commercial clofibrate preparations and the pharmacologic effects of 4-CP have not yet been widely established. We have examined the dose-dependent effects of oral 4-CP and clofibrate administration on selected hepatic parameters and on serum glucose, cholesterol, and triglyceride concentrations in male rats. 4-CP treatment (0.00125-0.08 mmol/kg, twice a day) of rats for 2 weeks increased hepatic microsomal protein (20-30%) and cytochrome P-450 (20-190%) contents without changing liver/body weight ratios. Both 4-CP (0.0025 mmol/kg body wt, twice a day) and CPIB (0.4 mmol/kg body wt, twice a day) treatment to rats for 2 weeks caused significant elevations in microsomal cytochrome P-450 content and in the maximal activities of ethylmorphine, aminopyrine, and benzphetamine N-demethylase, but not in the activity of zoxazolamine 6-hydroxylase. With the same dose of 4-CP, time-dependent increases in hepatic microsomal protein, cytochrome P-450, and the activity of benzphetamine N-demethylase were observed for a 4-week period, and the induction of hepatic microsomal benzphetamine N-demethylase activity by 4-CP was associated with an increased enzyme synthesis. 4-CP treatment produced a marked morphologic change in liver cell ultrastructure, including a proliferation of mitochondria and endoplasmic reticulum at lower 4-CP doses. A clustering of intracellular organelles (mitochondria and endoplasmic reticulum) and a foamy cytoplasm were seen at doses greater than 0.01 mmol/kg, twice a day for 2 weeks, and at 0.0025 mmol/kg, twice a day for greater than 4 weeks. The effects of 4-CP and clofibrate on fasting blood glucose and fasting serum lipid levels were also monitored throughout an 8-week period. Both 4-CP (0.005 mmol/kg body wt, twice a day) and clofibrate (0.2 mmol/kg body wt, twice a day) produced significant elevations in fasting serum glucose levels, but this dosage of 4-CP did not alter serum lipid and lipoprotein parameters, whereas clofibrate significantly reduced serum total cholesterol and high density lipoprotein cholesterol levels. These results lead us to conclude that 4-CP does not contribute to the antilipidemic effects of clofibrate.     

Effect of clofibrate on fatty acid desaturation of rats treated with ethanol

The effect of clofibrate and ethanol in the rat was studied on the following aspects of lipid composition and metabolism: liver delta 5, delta 6 and delta 9 fatty acid desaturases, fatty acid synthetase and fatty acid desaturase microsomal electron transport chain activity and serum cholesterol, triacylglycerols and high (HDL), low (LDL) and very low density lipoprotein (VLDL) levels. Clofibrate administered for 9 days (0.3% W/W) did not modify the relative composition of liver phospholipids and cholesterol, but did diminish triacylglycerol levels increased by ethanol. This effect could be explained by the possible beta-adrenergic blocking properties of clofibrate or by an increased activity of peroxisomal beta-oxidation. Clofibrate also promoted a decrease in serum cholesterol and triacylglycerol levels, delta 6 desaturase activity and a suppression of the electron transport chain as measured by NADH cytochrome b5 reductase and NADH cytochrome c reductase. The drug increased delta 9 desaturase activity and fatty acid synthetase, while no effect could be found in delta 5 desaturase activity. The hypocholesterolenic effect of clofibrate can not be explained through the delta 6 desaturase inhibition, or the fatty acid synthetase enhancement. Ethanol increased the HDL and VLDL and lowered LDL serum concentrations, while clofibrate reversed these results. Considering that clofibrate could have antiatherosclerotic effect in the rat, it is difficult to explain it through these changes in lipoprotein levels, since according to Miller and Miller low HDL levels are predictive of coronary heart disease.     

Clofibrate feeding increases cytoplasmic but not mitochondrial malic enzyme activity in rat kidney cortex

Administration of clofibrate for 21 days to rats increased the malic enzyme activity in the kidney cortex by about 80 per cent. This effect seems to be specific since the drug did not alter significantly the activity either of lactate dehydrogenase, citrate synthase or total mitochondrial protein content in this organ. The increase in activity of malic enzyme in the 13,000 g supernatant (extramitochondrial) fraction in rats treated with the drug was about 80 per cent, whereas in the pellet (mitochondrial fraction) it was about 40 per cent. The specific activity of malic enzyme in the kidney cortex cytosol from clofibrate-treated rats was about twice that in controls. In contrast clofibrate treatment did not affect its specific activity in isolated mitochondria. Calculations showed that 0.57 and 0.53 mumoles min-1 g-1 wet tissue of mitochondrial malic enzyme was obtained in control and clofibrate-treated rats respectively. Thus, clofibrate feeding increases the amount of cytoplasmic but not mitochondrial malic enzyme activity.     

Small intestinal alkaline phosphatase activity and changes induced by cysteamine administration in fecal excretions of male Wistar rats

Alkaline phosphatase (ALP) activity in fecal excretions was measured in male Wistar rats. Total daily ALP activity in fecal extracts was 133.1 +/- 21.2 mumoles/min per rat weighing approximately 150 g. We found that 63.7% of the fecal ALP activity was inhibited by 30 mM L-phenylalanine (L-Phe), a specific inhibitor for intestinal ALP. As body weight increased from 150 g to 300 g, total daily ALP activity in fecal extracts decreased rapidly to 33.7 +/- 6.08 mumoles/min/rat. However, the percentage of L-Phe-sensitive ALP to total enzyme activity was less variable (40-65%) in the growing rats. Cysteamine-HCl, an ulcerogenic drug, was injected subcutaneously to adult rats (300 g b. w) at a dosage of 400 mg/kg. b. w. Daily excretion of L-Phe-sensitive ALP in fecal extracts decreased to one-third 2 days after injection. Afterwards, a steep and transient increase in the enzyme activity was detected in fecal extracts between days 4 and 7 after injection. ALP activity in fecal excretions may be a clinical indicator of duodenal mucosal damage.     

Peroxisomal proliferation induced by treatment with clofibrate in a patient with a peroxisomal disease

The induction of peroxisomal proliferation in liver parenchymal cells of rats fed a diet containing clofibrate, a hypolipidemic drug, is a well-established event. However, the available data on human hepatocytes in vivo and in vitro indicate that agents that induce peroxisomal proliferation in rats and mice have no effect on human liver cells. The authors are reporting the case of a patient with clinical and laboratory diagnosis of X-linked-adrenoleukodistrophy. In an initial liver biopsy, a reduced volume fraction of peroxisomes was found (Vv.=.012) after a morphometric analysis, initiating treatment with clofibrate at a dose of 1.5 g/d. The administration of clofibrate was maintained for 7 yr. Liver biopsies were taken after 2, 4, and 7 yr, to follow the peroxisomal response. Results demonstrated a 500% increase in peroxisomal Vv. (.060) after 2 yr of treatment, compared with the pretreatment Vv. In subsequent biopsies, the peroxisomal Vv. value was maintained at 225 and 183% increases above the pretreatment biopsy (.027 and .022, respectively).     

Effect of Clofibrate on Blood Viscosity in Intermittent Claudication

Sixty-two patients with intermittent claudication associated with peripheral arterial diseases were treated with clofibrate, 2 g daily, for a minimum of six months. Progress was compared with that in a similar pretreatment period and also with that of a matched untreated control group of 27 patients. The most striking effect of clofibrate was a steep and sustained fall in whole-blood viscosity measured over a wide range of shear rates. This was associated with a significant fall in abnormally raised initial plasma-fibrinogen levels. An increased proportion of patients on treatment showed evidence of clinical improvement. Clofibrate had no effect on the susceptibility of red blood cells to autoxidation but it led to a significant shift in the red cell fatty acid pattern.     

Enhancement of rat liver catalase activity dietary cholesterol

The effects of a fat-supplemented diet and clofibrate (ethylchlorophenoxyisobutirate) upon serum lipids and liver catalase activity were studied in male rats. A butter-supplemented diet produced a striking increase of serum triglycerides but did not affect the liver catalase activity. Cholesterol (1%, w/w), added to the butter supplemented diet markedly increased liver catalase activity. This diet produced a hypercholesterolemic state higher than that induced by a butter-supplemented diet only, although the hypertriglyceridemic effect was less pronounced. Clofibrate given a butter-supplemented diet produced a marked increase of liver catalase activity (about four-fold). When clofibrate is administered with the cholesterol-supplemented diet, the increment observed in the liver catalase activity was the same as that induced with the cholesterol supplemented diet alone. Clofibrate, in either lipid-rich diet, failed to induce a hypocholesterolemic response, although a clear hypotrigliceridemic effect was evident. This effect appears to be potentiated with clofibrate and the cholesterol supplemented diet. Thus the increment in liver catalase activity induced by dietary cholesterol and clofibrate seems to be related to a hypotriglyceridemic effect which gives support to a role of liver peroxisomes in lipid metabolism. The role that liver catalase would play, in this regard, remains unclear from these results.     

The effect of clofibrate feeding on the NADP-linked dehydrogenases activity in rat tissue

Administration of clofibrate to the rat increased several fold the activity of malic enzyme in the liver. Clofibrate treatment resulted also in an increased activity of the hepatic hexose monophosphate shunt dehydrogenases but was without effect on NADP-linked isocitrate dehydrogenase. The increased activity of malic enzyme in the liver resulting from the administration of clofibrate was inhibited by ethionine and puromycin, which suggests that de novo synthesis of the enzyme protein did occur as the result of the drug action. In contrast to the liver malic enzyme, the enzyme activity in kidney cortex increased only two-fold, whereas in the heart and skeletal muscle the activity was not affected by clofibrate administration.     

Hepatic enzymes, CoASH and long-chain acyl-CoA in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.     

The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats.

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.     

Renin-Aldosterone System in Osmoregulatory Reactions of Healthy Subjects in Response to Desmopressin

Systemic regulation of osmotic and ionic homeostasis was studied in healthy male volunteers after oral administration of desmopressin. Endogenous secretion of the antidiuretic hormone was inhibited by a water load (WL, 2% of the body mass). Desmopressin exerted an antidiuretic effect. In addition, the WL portion excreted during 4 h decreased and the urine osmolality at peak diuresis increased with the absence of osmotically free water. At maximum diuresis, the ratio between concentrations of osmotically active substances in the urine and in the blood was high, which reflected an intense antidiuretic effect. Desmopressin progressively decreased the rate of sodium excretion owing to a change of sodium reabsorption in the kidneys. The WL increased the level of aldosterone and the activity of renin in blood plasma 1.5 h after its administration. Contrary to the control series, desmopressin stimulated the renin-angiotensin-aldosterone system only by the end of the 4-h observation period. A significant negative correlation between the aldosterone level and the rate of sodium excretion was observed 3 h after the beginning of testing (r = ?0.76). Thus, under conditions of water loading, desmopressin had a specific antidiuretic effect involving systemic mechanisms of ion regulation.     

Hepatic release of carnitine: effect of increased concentration by clofibrate treatment.

The release of carnitine is an important metabolic function of the liver. In the present study, we have investigated the effect of increased carnitine concentration on the hepatic release of carnitine. Hepatic carnitine concentration was increased in rats by clofibrate treatment. Release of carnitine was investigated as its efflux from perfused liver and its secretion into bile. A significantly smaller proportion of the hepatic pool of carnitine was released into the perfusion medium when carnitine concentration was increased by clofibrate treatment. However, the amount of carnitine released (nmol/g liver) was comparable to that of control rats. Increased carnitine concentration by clofibrate treatment also did not affect the rate of biliary secretion of carnitine. In control rats, nearly 50% of the released carnitine, in both the perfusion medium and bile, was acylcarnitine whereas in clofibrate-treated rats 35% of the released carnitine was acylcarnitine. Release into the perfusion medium was the major route for the hepatic export of carnitine. We conclude that when hepatic carnitine concentration is increased by clofibrate treatment, a smaller proportion of the hepatic carnitine pool is released, but the amount of carnitine released (nmol/g liver) is not greatly different than that from control animals.     

Increase in hepatic catalase and glycerol phosphate dehydrogenase activities on administration of clofibrate and clofenapate to the rat

1. Clofenapate (methyl 2-[4-(p-chlorophenyl)phenoxy]-2-methylpropionate) fed to the rat in the diet increased the content of mitochondrial protein in the liver by 50-60%. In this respect it resembled the related compound clofibrate (ethyl alpha-p-chlorophenoxyisobutyrate), which is widely used as an antihypercholesterolaemic drug. 2. Both compounds when fed to the rat enhanced the activity of alpha-glycerol phosphate dehydrogenase in the liver mitochondria manyfold, but were without effect on the enzyme in the soluble fraction. 3. On the other hand, the catalase activity in the supernatant fraction increased twofold after administration of the drugs. The mitochondrial catalase activity showed a consistent decrease. 4. It was unlikely that under the influence of the drug the increase in catalase activity took place in the mitochondrial particles and was leached into the cytosol during isolation. 5. The increase in catalase activity in the cytosol under the influence of the drug is best explained on the assumption that peroxisomes which contain this enzyme, and which are known to increase on administration of the drug, were broken during the process of cellular fractionation and released the enzyme into the cytosol. 6. All the above effects shown by both drugs were fully reversed when drugs were withdrawn from the diet. 7. Clofenapate was effective in bringing about the above changes when administered to the animal at one-hundredth the concentration of clofibrate.