Effect of candicidin on cholesterol and bile acid metabolism in the rat

Sterol metabolism studies were carried out in rats maintained on a diet containing a polyene antibiotic, candicidin, (30 mg/kg/day) for 2-1/2 months. Compared to the controls, the candicidintreated animals had a smaller food intake and weight gain during this period. There was no difference between the 2 groups in serum cholesterol levels, biliary cholesterol or bile acid concentrations. However, in the experimental group, liver cholesterol content decreased by 27% and hepatic HMG-CoA reductase increased by 36%. Candicidin administration produced an 84% increase in neutral sterol output without change in bile acid output. Cholesterol absorption was reduced 80% by candicidin feeding. The weight of ventral prostate was reduced 33% by candicidin administration. Prostatic HMG-CoA reductase levels were 3 times higher than those of the liver, but enzyme activity was unchanged by candicidin treatment.     

Bile acid metabolism in mammals: IX. Conversion of chenodeoxycholic acid to cholic acid by isolated perfused rat liver

Current dogma of bile acid synthesis in mammals insists that hydroxylation of the ring structure at C-12 precedes side chain oxidation, and that chenodeoxycholic acid is not converted to cholic acid under normal conditions. This report concerns the conversion of chenodeoxycholic acid to cholic acid by isolated, perfused rat liver. Results indicate that isolated perfused rat liver has a definite, but limited, capacity for synthesis of cholic acid from chenodeoxycholic acid.     

Age-related changes in the metabolism of cholesterol in rat liver microsomes

The effects of aging on the hepatic metabolism of cholesterol were studied in 1-, 6- and 24-month-old male Sprague-Dawley rats. Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, which regulates cholesterol biosynthesis, decreased from 835±144 (SEM) pmol/min/mg protein in the youngest group to 219±34 and 205±53 pmol/min/mg protein (p<0.001) in the 6- and 24-month-old groups, respectively. Cholesterol 7α-hydroxylase activity, which governs bile acid synthesis, was gradually reduced from 70±14 pmol/min/mg protein in the 1-month-old group to 32±7 and 16±3 pmol/min/mg protein (p<0.05) in the 6- and 24-month-old groups, respectively. Acyl coenzyme A:cholesterol acyltransferase activity, which catalyzes the esterification of cholesterol, averaged 431±47 and 452 ±48 pmol/min/mg protein in the 1- and 6-month-old groups, respectively, and was increased to 585±55 pmol/min/mg protein (p<0.05) in the 24-month-old group. The level of total cholesterol showed an age-related increase from 1.56±0.16 mg/g liver in the 1-month-old group to 1.70±0.15 and 2.20±0.19 mg/g liver (p<0.05) in the 6- and 24-month-old groups, respectively. The increase was mainly caused by an accumulation of esterified cholesterol. We conclude that a marked decrease in HMG-CoA reductase occurs between 1 and 6 months of age; thereafter the enzyme activity stays unchanged. The activity of cholesterol 7α-hydroxylase decreases progressively and drastically with age, whereas the capacity for esterifying cholesterol increases slightly. We speculate that the reduced conversion of cholesterol to bile acids may be one explanation of the age-related increase of plasma cholesterol seen in rats.     

Perfluorodecanoic acid and lipid metabolism in the rat

Alterations in lipid metabolism were axamined in adult male Sprague-Dawley rats seven days after a single intraperitoneal injection of perfluorodecanoic acid (PFDA; 20, 40 or 80 mg/kg). Because PFDA treatment caused a dose-related reduction in feed intake, the response of vehicle-treated rats pair-fed to those receiving PFDA was monitored to distinguish direct effects of the perfluorinated fatty acid from those secondary to hypophagia. Carcass content of lipid phosphorus and free cholesterol decreased in dose-dependent fashion in both PFDA-treated and pair-fed rats. Carcass triacylglycerols diminished in a similar manner, yet PFDA-treated rats at each dose had a higher concentration of neutral acylglycerols than their vehicle-treated, pair-fed counterparts. In vehicle-treated, pair-fed rats at the 80 mg/kg dose level, lipid phosphorus and free cholesterol as a proportion of carcass fat increased, whereas the share of the triacyl-glycerols declined. Because of the higher concentration of triacylglycerols in the carcass of rats treated with 80 mg/kg PFDA, enrichment of lipid phosphorus and free cholesterol in carcass fat was less than in their pair-fed partners. The amount of lipid phosphorus and free cholesterol per hepatocyte was similar in both PFDA-treated rats and their pair-fed partners. Liver triacyl-glycerols were markedly increased in PFDA-treated rats. A similar but less extensive augmentary effect of PFDA on hepatic esterified cholesterol was found. Concentration of triacylglycerols in plasma was not elevated in PFDA-treated rats, in spite of hepatic accumulation of esterified compounds. Also, the plasma level of free fatty acids and 3-hydroxybutyrate was similar in all treatment groups, including those receiving PFDA. Thus, the administration of PFDA appears to divert fatty acids from oxidation toward esterification in the liver.     

Uptake and metabolism of dolichol and cholesterol in perfused rat liver

The uptake of dolichol and cholesterol by perfused rat liver was studied. When these radioactive lipids were incorporated into egg phosphatidylcholine liposomes, both dolichol and cholesterol appeared initially in the supernatant and in the microsomal fraction and, later on, in the mitochondrial-lysosomal fraction. The lipids taken up were esterified to some extent, but no phosphorylation of dolichol occurred. Incorporation of dolichol and cholesterol into lipoproteins increased the efficiency of uptake, which was receptor-mediated in this case. Accumulation of these lipids occurred in lysosomes followed by a transport to the endoplasmic reticulum (ER). Both labeled dolichol and cholesterol appeared in the bile. In the case of dolichol, the majority of this radioactivity was not associated with the original substance itself, and probably represented lipid-soluble catabolites. In the case of cholesterol, most of the radioactivity was associated with bile acids. It appears that, in contrast to the receptor-mediated uptake of lipoproteins from the perfusate, the uptake of liposomal lipids involves a different mechanism. After association with the plasma membrane, the lipids enter into the cytoplasm and are transported to the ER and later to the lysosomes.     

2-Aminoethylphosphonic acid metabolism in the rat

Time course studies of the incorporation of radioactive 2-aminoethylphosphonic acid (AEP) into the tissues of rats demonstrated that maximum incorporation into the liver lipids occurred within 12 to 30 hr after injection, compared to 2 to 3 hr for the incorporation of phosphorylethanolamine. Little incorporation of AEP was observed in the other tissues investigated (heart, lung, spleen, adipose, kidney). The AEP was incorporated to the greatest extent into 1,2-diacylglyceryl-aminoethylphosphonate (diacylglyceryl-AEP), the phosphonate analogue of phosphatidylethanolamine, with some incorporation into the lyso derivative. Diacylglycerol-AEP apparently was not further metabolized by the rat; no methylation of diacylglyceryl-AEP to phosphonolecithin was observed. Subcellular fractionation was performed on the livers of rats who received³H-AEP 12, 30, 36, and 48 hr prior to sacrifice. The greatest amount of radioactivity was recovered in the soluble fractions. Lipid extraction was performed on the subcellular fractions, and most of the radioactivity present in the lipids was found in the microsomal fraction, with the next highest recovery in the mitochondrial and nuclear fractions. This work is based upon a thesis submitted by J.C.-J. to the Graduate College of the University of Illinois at the Medical Center in partial fulfillment of the requirements for the Ph.D. degree in Biological Chemistry. Present Address: Department of Biochemistry, Northwestern University Medical Center, Chicago, Illinois, 60611.     

Effects of perfluorodecanoic acid onde novo fatty acid and cholesterol synthesis in the rat

Perfluorodecanoic acid (PFDA) is a peroxisome proliferator that causes a dose-dependent (20–80 mg/kg) increase in hepatic triacylglycerol and cholesteryl ester levels in the rat. We hypothesized that PFDA may cause an increase in thede novo synthesis of fatty acids and cholesterol in this species, which would explain observed effects. The incorporation of³H₂O into tissue lipids was examined 7 days after rats received vehicle or 20 or 80 mg/kg of PFDA. PFDA treatment decreased the rate of synthesis of cholesterol and fatty acids in the liver and in epididymal fat pad. At a PFDA dose (20 mg/kg) that decreasedde novo synthesis of fatty acids and cholesterol, there was no effect on the concentration of fatty acids and cholesterol in the liver, epididymal fat pads, and plasma. We conclude that PFDA induced fatty liver is due to either a decrease in the oxidation of fatty acids in the liver, or an impairment of triacylglycerol catabolism and/or export from the liver, and is not the result of an increase inde novo synthesis of fatty acids and cholesterol.     

Dietary oxidized cholesterol modulates cholesterol metabolism and linoleic acid desaturation in rats fed high-cholesterol diets

The interactive effect of high dietary levels of oxidized cholesterol on exogenous cholerterol and linoleic acid metabolism was examined in male 4-wk-old Sprague-Dawley rats given high-cholesterol diets. The rats were pair-fed purified diets free of or containing either 0.5% cholesterol alone or both 0.5% cholesterol and 0.5% oxidized cholesterol mixture (containing 93% oxidized cholesterol) for 3 wk. Hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity was reduced in rats given cholesterol alone or both cholesterol and oxidized cholesterol. However, hepatic cholesterol 7α-hydroxylase activity was lowered only when rats were given both cholesterol and oxidized cholesterol, although dietary cholesterol increased this activity. Reflecting this effect, acidic steroid excretion was lowest among the groups of rats given cholesterol and oxidized cholesterol. On the other hand, the activity of hepatic Δ6 desaturase, a key enzyme in the metabolism of linoleic acid to arachidonic acid, was increased in rats given both cholesterol and oxidized cholesterol, although dietary cholesterol alone lowered its activity. As a result, the Δ6 desaturation index, 20∶3n-6+20∶4n-6/18∶2n-6, in liver and serum phosphlipids tended to be higher in the group fed both cholesterol and oxidized cholesterol than in the one fed cholesterol alone. Thus, dietary oxidized cholesterol significantly modulated exogenous cholesterol metabolism and promoted linoleic acid desaturation even when it was given at high levels together with a high cholesterol diet.     

Vaccenic acid metabolism in the liver of rat and bovine

Hepatic metabolism of vaccenic acid (VA), especially its conversion into CLA, was studied in the bovine (ruminant species that synthesizes CLA) and in the rat (model for nonruminant) by using the in vitro technique of liver explants. Liver tissue samples were collected from fed animals (5 male Wistar rats and 5 Charolais steers) and incubated at 37°C for 17 h under an atmosphere of 95% O₂/5% CO₂ in medium supplemented with 0.75 mM of FA mixture and with 55 μM [1-¹⁴C]VA. VA uptake was about sixfold lower in bovine than in rat liver slices (P<0.01). For both species, VA that was oxidized to partial oxidation products represented about 20% of VA incorporated by cells. The chemical structure of VA was not modified in bovine liver cells, whereas in rat liver cells, 3.2% of VA was converted into 16∶0 and only 0.33% into CLA. The extent of esterification of VA was similar for both animal species (70–80% of incorporated VA). Secretion of VA as part of VLDL particles was very low and similar in rat and bovine liver (around 0.07% of incorporated VA). In conclusion, characteristics of the hepatic metabolism of VA were similar for rat and bovine animals, the liver not being involved in tissue VA conversion into CLA in spite of its high capacity for FA desaturation especially in the rat. This indicates that endogenous synthesis of CLA should take place exclusively in peripheral tissues.     

Oral acetylsalicylic acid induces biliary cholesterol secretion in the rat

Several agents can alter biliary cholesterol secretion, critical for cholesterol excretion and gallstone formation. Although salicylate effects on bile formation and gallstones have been studied, biliary lipid secretion has not been measured during oral aspirin treatment. We examined whether oral acetylsalicylic acid affects bile lipid secretion. Three groups of young rats were fed chow for 3 wk. Two of the groups then received aspirin at either 1.67 or 3.33 g/kg diet for 4 d. Serum, hepatic, and bile lipids were measured, as were enzymes of cholesterol synthesis and esterification. With oral aspirin, bile cholesterol secretion increased by 42% and hepatic cholesteryl ester content decreased by 40%. Serum cholesterol and hepatic free cholesterol did not change. To evaluate mechanisms of the cholesterol hypersecretion, hypothyroid animals fed low-fat or fish oil diets and repleted with triiodothyronine were also studied. Aspirin stimulated cholesterol secretion to a degree similar to triiodothyronine. An additive response was seen in fish oilfed rats. Aspirin did not appear to have a primary action on 3-hydroxy-3-methylglutaryl-CoA reductase or acyl CoA:cholesterol acyltransferase activities, and had no direct effect on esterification of cholesterol by isolated hepatocytes. Aspirin may directly increase cholesterol transport into bile or have cell membrane effects which alter cholesterol transport. It remains to be determined whether the observed alterations in bile cholesterol secretion are specific to the rat or also apply to humans.     

Effect of glucose administration on cholesterol and bile acid metabolism in rats

Glucose administered to fasted rats caused a marked stimulation in hepatic cholesterogenesis and cholesterol 7 alpha-hydroxylation, and an increase in biliary excretion of cholesterol and total bile acids. The excretion of cholic acid was not incluenced during the first few hr after glucose administration, but was significantly increased after 5 hr. Chenodeoxycholic acid showed a similar change, but the increase was only ca. one tenth of that of cholic acid. The excretion of deoxycholic acid was markedly increased by 1 hr, but gradually decreased thereafter. Pretreatment with neomycin abolished the increase in deoxycholic acid by fasting and glucose administration. Other bile acid components showed no significant change. It thus was presumed that cholesterol endogenously synthesized in the liver was metabolized mainly to cholic acid. In contrast, exogenous cholesterol was metabolized mainly to chenodeoxycholic acid. During the period of the acute enhancement of cholic acid formation from the endogenous cholesterol, biliary excretion of deoxycholic acid was increased. This probably occurred through the depression of 7 alpha-rehydroxylation of deoxycholic acid, or through the enhancement of microbial formation of deoxycholic acid in the lumen, and through the increase of intestinal absorption.     

Effects of dietary cholesterol upon bile acid metabolism in guinea pig

Cholesterol fed guinea pigs develop a hemolytic anemia accompanied by high cholesterol concentrations in the liver, plasma, and red cells. We have studied the bile acid metabolism of guinea pigs fed a diet with or without cholesterol in a search for the factor(s) which prevent adequate control of their body cholesterol pool and, therefore, its pathological consequences. The results show that in the cholesterol fed guinea pig the synthesis (and excretion) of bile acids was at least three times greater than in controls. This is the result of a doubling of the fractional turnover rate and a smaller increase in the pool size. The major increase of the bile acid pool was in the liver. The main bile acid in gall bladder bile and small intestines was chenodeoxycholic acid, with smaller amounts of 7-ketolithocholic and ursodeoxycholic acids. In the caecum, large intestines, and feces, the major bile acid was lithocholic acid.     

Dietary lipid,fatty acid synthesis and cholesterol metabolism in aging rats

Male and female rats were fed diets containing 2% of calories as corn oil or that plus 40% of calories as beef tallow or corn oil. After 3, 6, 12 and 18 months groups were given 4-¹⁴C-cholesterol ip, and feces were collected for 9 days. Just prior to necropsy³H-acetate was administered ip. Samples of serum, liver, heart and carcass were obtained for analysis. Concentrations of fatty acids and cholesterol, synthesis of those and recovery of ring-labeled steroid are reported. Mortality from acute respiratory disease was very high in male rats fed beef tallow or low fat diets and very low in those fed the corn oil diet. In females, only beef tallow diet resulted in a high mortality rate, and this was lower and at a later age than in males. The most notable effects of age were in relation to fatty acid synthesis and presence of¹⁴C-acidic steroid in the carcass. In 3-month-old rats both fats depressed fatty acid synthesis in comparison to the low fat diet. At later ages beef fat ceased to depress fatty acid synthesis in both sexes. Corn oil continued to depress fatty acid synthesis up to 12 months in males and 18 months in females. The presence of¹⁴C-acidic steroid in carcass was substantial in 6-month-old rats and constituted ca. 40% of recovered¹⁴C in 18-month-old rats. The possibility that the increase in acetate incorporation into fatty acids with age in fat feeding is related to chain elongation rather than de novo synthesis is discussed. Both the presence and amount of acidic steroid in the carcass are notable and may be of importance in constructing models of cholesterol turnover. Presented in part at the AOCS Sterol Symposium, April 1970, and the Federation of American Societies for Experimental Biology, April 1971. Scientific Series Paper No. 1536, Colorado Agricultural Experiment Station.     

The effects of clofibrate and bezafibrate on cholesterol metabolism in the liver of the male rat

Fibric acid derivatives are used to treat hyperlipidemia and have wide ranging effects on lipid metabolism. The action of these compounds on cholesterol esterification, catalyzed by acyl-coenzyme A:cholesterol acyltransferase (ACAT), has been quite widely studied, but their effect on cholesteryl ester hydrolysis and the enzyme neutral cholesteryl ester hydrolase (nCEH) has been largely ignored. Male rats were therefore fed for 10 d on a standard chow diet supplemented with either clofibrate or bezafibrate, to study their effects on plasma lipid levels and hepatic cholesterol metabolism. Plasma triacyglycerols were not significantly altered by these diets, but bezafibrate significantly lowered plasma cholesterol levels (29.7%,P<0.01). When expressed per unit weight of DNA, both fibrates reduced the hepatic content of triacylglycerol, cholesterol and cholesteryl esters (40, 18.7, 16.5 and 66.7, 28.6, 34.2% for clofibrate and bezafibrate, respectively). ACAT activity was significantly reduced by both drugs, but clofibrate (65% inhibition) was more effective than bezafibrate (35% inhibition). The most dramatic effect of the diets was a marked increase in the activity of both the microsomal and the cytosolic nCEH. When expressed on a whole liver basis, the effect of bezafibrate on the cytosolic enzyme (13.6-fold increase in activity) was much greater than that of clofibrate (4.8-fold increase). Increases in the activity of a cytosolic protein that inhibits the activity of nCEH were also noted, but these changes were relatively small. The results suggest that the activation of nCEH, in combination with the inhibition in ACAT activity, contributes to a decrease in the cholesteryl ester content of the liver which may influence the secretion of very low density lipoprotein.     

Fish oil prevents change in arachidonic acid and cholesterol content in rat caused by dietary cholesterol

Rats were fed diets high in either saturated fat (beef tallow) or α-linolenic acid (linseed oil) or eicosapentaenoic and docosahexaenoic acids (fish oil) with or without 2% cholesterol supplementation. Consumption of linseed oil and fish oil diets for 28 days lowered arachidonic acid content of plasma, liver and heart phospholipids. Addition of 2% cholesterol to diets containing beef tallow or linseed oil lowered 20∶4ω6 levels but failed to reduce 20∶4ω6 levels when fed in combination with fish oil. Feeding ω3 fatty acids lowered plasma cholesterol levels. Addition of 2% cholesterol to the beef tallow or linseed oil diet increased plasma cholesterol concentrations but not when fish oil was fed. Feeding the fish oil diet reduced the cholesterol content of liver, whereas feeding the linseed oil diet did not. Dietary cholesterol supplementation elevated the cholesterol concentration in liver in the order: linseed oil > beef tallow > fish oil (8.6-, 5.5-, 2.6-fold, respectively). Feeding fish oil and cholesterol apparently reduced 20∶4ω6 levels in plasma and tissue lipids. Fish oil accentuates the 20∶4ω6 lowering effect of dietary cholesterol and appears to prevent accumulation of cholesterol in plasma and tissue lipids under a high dietary load of cholesterol.     

Regulation of the metabolism of linoleic acid to arachidonic acid in rat hepatocytes

When 5×10⁶ hepatocytes were incubated for 40 min with from 0.15 to 0.60 mM [1-¹⁴C]linoleic acid, [1-¹⁴C]6,9,12-octadecatrienoic acid, or [1-¹⁴C]8,11,14-eicosatrienoic acid, there was a concentration-dependent acylation of radioactive metabolites into both triglycerides and phospholipids. When the concentration of either [1-¹⁴C]linoleic acid or [1-¹⁴C]8,11,14-eicosatrienoic acid exceeded 0.3 mM, there was no further increase in the metabolism of either fatty acid to other (n−6) metabolites. When the concentration of [1-¹⁴C]6,9,12-octadecatrienoic acid exceeded 0.15 mM, there was an apparent substrate-induced inhibition in its metabolism to 8,11,14-eicosatrienoic acid. With all three substrates (0.3 mM), there was time-dependent metabolism to other (n−6) acids. Cells then were incubated simultaneously with 0.3 mM [1-¹⁴C]linoleic acid along with 0.15 to 0.45 mM 6,9,12-octadecatrienoic acid or 8,11,14-eicosatrienoic acid. These exogenous nonradioactive (n−6) acids suppressed but did not abolish the conversion of [1-¹⁴C]linoleate to radioactive arachidonate. These findings suggest that some linoleate is converted to arachidonate without intracellular mixing of 6,8,12-octadecatrienoic or 8,11,14-eicosatrienoic acids. This hypothesis is supported by the finding that exogenous linoleate did not markedly affect the metabolism of [1-¹⁴C]6,9,12-octadecatrienoic or [1-¹⁴C]8,11,14-eicosatrienoic acid by microsomal chain elongating or desaturating enzymes.     

Studies on the in vivo metabolism of retinoic acid in the rat

A time study of the metabolism of 6,7-¹⁴C-retinoic acid after intraperitoneal injection of physiological levels (17 μg, 0.39 μc) into vitamin A deficient rats, which had been repleted with retinoic acid for two weeks up to two days before injection, resulted in a rapid metabolism to more polar compounds in the small intestine and its contents and a slower metabolism to primarily different materials in the liver and kidney. The major route of metabolism resulted in the urinary excretion of 60% of the injected dose in 24 to 27 hr. Urinary metabolites of 15-¹⁴C-retinoic acid were eluted from silicic acid at a similar concentration of solvents as the ring labeled metabolites although only 32% of the injected dose was recovered in 24 hr. Compounds chromatographically similar to the urinary metabolites were observed at various times in the liver, kidney and small intestine plus contents in addition to retinoic acid and other metabolites. The relative amounts of the metabolites in the different tissues studied varied as a function of the tissue and the time of analysis after injection. Most of the radioactivity from all tissues was extractable into methanol. A liver subcellular distribution of the radioactivity derived from the intraperitoneal injection of 650 μg of 6,7-¹⁴C-retinoic acid (25.9 μc) after 3 hr indicated a minimal level of association of radioactivity (150–250 dpm/mg protein) with all fractions and a greater association of radioactivity with the lysosomal-microsomal fraction (300–350 dpm/mg protein) and the 60–100% ammonium sulfate precipitable (750–800 dpm/mg protein) and 100% ammonium sulfate soluble fractions (422 dpm/mg protein) of the soluble supernatant.     

Serum lipoprotein and apoprotein concentrations in 4-(4-chlorophenyl)-2-hydroxytetronic acid and clofibrate-treated cholesterol and cholic acid-fed rats

Influence of clofibrate and an aci-reductone, 4-(4-chlorophenyl)-2-hydroxytetronic acid (CHTA) on lipoproteins and apoproteins was studied in cholesterol- plus cholic acid-fed rats. CHTA (0.4 mmol/kg body wt, twice daily) significantly lowered serum total cholesterol and triglyceride concentrations at both 10 and 16 days, whereas clofibrate at the same dose did not alter serum cholesterol levels, but elevated serum triglyceride concentrations at 16 days. The abnormal cholesterol-rich very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low density lipoproteins (LDL) produced by cholesterol plus cholic acid were significantly reduced in their cholesterol content by treatment with CHTA, a compound having an oxidation reduction potential. Conversely, clofibrate administration increased VLDL-cholesterol with concomitant decreases in IDL- and LDL-cholesterol concentrations. Administration of CHTA to cholesterol- plus cholic acid-fed rats significantly increased concentrations of VLDL and IDL, but had no effect on HDL protein. Both CHTA and clofibrate administration to cholesterol- plus cholic acid-fed rats significantly lowered IDL protein concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies of apoproteins revealed that clofibrate treatment significantly reduced apoC-III and C-II in VLDL, C-II in IDL, and apoA-IV and A-I in HDL. Rats treated with CHTA significantly raised apoC-II and C-III in HDL. Isoelectric focusing (IEF) of VLDL apoproteins showed a significant decrease in apoC-II, C-III-0 and apoC-III-3 in clofibrate-treated animals. Thus, the mechanism for antilipidemic action of the oxidation reduction compound, CHTA, which differs markedly from the prototype drug, clofibrate, is independent from major apoprotein modification.