Original Article
Influence of Pravastatin, a Specific Inhibitor of HMG-CoA Reductase, on Hepatic Metabolism of Cholesterol
N Engl J Med 1990; 323:224-228July 26, 1990
- Abstract
- Abstract
Background.
Inhibitors of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are now used frequently to treat hypercholesterolemia. We studied the effects of specific inhibition of cholesterol synthesis by one of these agents (pravastatin) on the hepatic metabolism of cholesterol in patients with gallstone disease who were scheduled to undergo cholecystectomy.
Methods.
Ten patients were treated with pravastatin (20 mg twice a day) for three weeks before cholecystectomy; 20 patients not treated served as controls. A liver specimen was obtained from each patient at operation, and the activities of rate-determining enzymes in cholesterol metabolism as well as low-density-lipoprotein (LDL)-receptor binding activity were determined.
Results.
Pravastatin therapy reduced plasma total cholesterol by 26 percent and LDL cholesterol by 39 percent (P<0.005). Serum levels of free lathosterol, a precursor of cholesterol whose concentration reflects the rate of cholesterol synthesis in vivo, decreased by 63 percent (P<0.005), indicating reduced de novo biosynthesis of cholesterol. Microsomal HMG-CoA reductase activity, when analyzed in vitro in the absence of the inhibitor, was increased 11.8-fold (1344±311 vs. 105±14-pmol per minute per milligram of protein in the controls; P<0.001). The expression of LDL receptors was increased by 180 percent (P<0.005), whereas the activities of cholesterol 7α-hydroxylase (which governs bile acid synthesis) and of acyl-coenzyme A:cholesterol O-acyltransferase (which regulates cholesterol esterification) were unaffected by treatment.
Conclusions.
Inhibition of hepatic HMG-CoA reductase by pravastatin results in an increased expression of hepatic LDL receptors, which explains the lowered plasma levels of LDL cholesterol. (N Engl J Med 1990; 323: 224–8.)
Media in This Article
Table 1
Base-Line Clinical Characteristics of Patients Given Pravastatin before Cholecystectomy and Patients Not Given Pravastatin (Mean and Range).Table 2
Plasma Lipid Levels in the Study Groups (Mean ±SEM).Article Activity
- Article
THE elevation of plasma low-density lipoprotein (LDL) cholesterol levels is recognized as a primary risk factor for the development of atherosclerosis and coronary heart disease.1 , 2 Therefore, much interest has been focused on the regulation of plasma LDL levels and on possible ways to influence the synthesis and elimination of LDL.3 A lowering of total and LDL cholesterol can reduce the incidence of ischemic heart disease and retard the development of coronary atherosclerosis.4 5 6 However, some hypolipidemic drugs are not tolerated by all patients or are not effective enough. The recent introduction of competitive inhibitors of the rate-limiting enzyme in cholesterol biosynthesis, microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, offers promise for the treatment of hypercholesterolemia.7
The LDL-lowering effect of HMG-CoA reductase inhibitors reported in healthy subjects8 and patients with heterozygous familial hypercholesterolemia9 10 11 12 appears to be the result of stimulated catabolism of LDL.10 This increased elimination is believed to be the consequence of the induction of hepatic LDL receptors, as demonstrated in animals.13 , 14 Moreover, by inhibiting cholesterol synthesis, these drugs might interfere with hepatic formation of plasma lipoproteins and could also reduce plasma LDL levels in this way.7
So far, however, the effects of HMG-CoA reductase inhibitors on the activity of HMG-CoA reductase and LDL-receptor expression in human liver have not been described. Furthermore, the effects of such therapy on the activity of other important enzymes in cholesterol metabolism in human liver, such as cholesterol 7α-hydroxylase (the rate-limiting enzyme in bile acid synthesis) and acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, the regulatory enzyme in cholesterol esterification), have not been investigated. In the present study we examined the effects of pravastatin, an inhibitor of HMG-CoA reductase in hepatic and intestinal cells,15 on hepatic cholesterol metabolism in patients scheduled to undergo cholecystectomy for gallstones. The results showed that the inhibition of HMG-CoA reductase induced by pravastatin is associated with a great compensatory increase in HMG-CoA reductase activity in vitro and a concomitant stimulation of LDL-receptor binding activity, resulting in a reduction in plasma LDL levels.
Methods
Patients and Treatment
We studied 30 patients of normal weight who were scheduled to undergo cholecystectomy because of gallstone disease. Patients with evidence of hepatic, cardiovascular, renal, or metabolic dysfunction, as well as those with a history of allergy or excessive ethanol consumption, were excluded. Ten patients were treated with pravastatin sodium (E.R. Squibb, New Brunswick, N.J.; 20 mg twice a day) for three weeks before operation (Table 1Table 1
Base-Line Clinical Characteristics of Patients Given Pravastatin before Cholecystectomy and Patients Not Given Pravastatin (Mean and Range).); the other 20 served as controls (no pravastatin treatment). The patients who were asked to participate as recipients of pravastatin were selected at random, but no formal matching or strict protocol for randomization was followed. The women in the pravastatin-treated group were postmenopausal or had become sterile through surgery; inability to conceive was the only criterion followed in selecting female patients for treatment. The control group, which included eight postmenopausal women, was intended to be twice the size of the pravastatin group, to obtain a reliable range of control values for the enzyme assays.16 In the pravastatin group, routine blood samples were obtained after an overnight fast on three occasions: before the administration of the drug and after one week and three weeks of treatment (i.e., on the day before operation). For the control group, data obtained on the day before operation are reported; lipoprotein values were determined in only 12 of the 20 patients. The last dose of pravastatin was taken at 9 p.m. on the day before operation.Informed consent was obtained from each patient, and the study was approved by the Ethics Committee at Huddinge University Hospital.
Experimental Procedures
All operations were performed between 8 and 9 a.m. after a 12-hour fast. Standardized anesthesia was administered, with induction by thiopental and continuous treatment with nitrous oxide, diazepam, and fentanyl.16 Immediately after the abdomen was opened, a biopsy specimen (2 to 4 g) was taken from the left lobe of the liver and placed in ice-cold homogenizing medium; the preparation of microsomes was started in the laboratory within 10 minutes. A small specimen of the liver was examined histologically.
The cystic duct was clamped, and gallbladder bile was obtained by aspiration. If the gallbladder was not functioning, its contents were not analyzed. Hepatic bile was obtained from six pravastatin-treated patients and nine controls by puncturing the common bile duct with a thin needle. Cholecystectomy was performed without any complications. All biochemical assays were performed without knowledge of the status of each patient with regard to pravastatin treatment.
Assays
Plasma cholesterol and triglycerides were assayed by enzymatic methods (Boehringer Mannheim, Mannheim, Federal Republic of Germany). Lipoproteins were analyzed by a combination of ultracentrifugation and precipitation.17 , 18 In brief, plasma was spun at 35,000 rpm for 18 hours at 4°C in a Contron Centrikon T-2060 ultracentrifuge equipped with a 45.6 rotor (density, 1.006 g per milliliter). The tubes were sliced, and the supernatant fraction as well as the infranatant were analyzed for cholesterol and triglyceride content. A portion of the infranatant was treated with phosphotungstic acid to precipitate proteins containing apolipoprotein B and was analyzed as described above. For the analyses of apolipoproteins AI and B, immunoturbidometric methods were used (Orion Diagnostica, Espoo, Finland). Serum levels of lathosterol and free cholesterol were measured by isotope dilution-mass spectrometry after the addition of 2H-labeled internal standards.19
The liver biopsy specimens were minced and homogenized in a Potter-Elvehjem homogenizer with a loosely fitting Teflon pestle, in nine volumes of 50 mM TRIS-hydrochloride buffer, pH 7.4, containing 0.3 M sucrose, and either 50 mM sodium chloride or 50 mM sodium fluoride (HMG-CoA reductase assay), 10 mM dithiothreitol, 10 mM EDTA (cholesterol 7α-hydroxylase and HMG-CoA reductase assays), or 1 mM EDTA (ACAT assay). Dithiothreitol was omitted from the assay for ACAT. The homogenate was centrifuged at 20,000×g for 15 minutes at 4°C. The supernatant was centrifuged at 100,000×g for 60 minutes and then again for another 60 minutes (except in the assay for ACAT). The microsomal content of protein was determined according to the method of Lowry et al.20 The concentrations of free and total cholesterol were determined by isotope dilution-mass spectrometry as described previously.21 , 22
Microsomal HMG-CoA reductase activity was assayed by measuring the conversion of [14C]HMG-CoA to mevalonate.16 The activity of cholesterol 7α-hydroxylase was determined with a method based on isotope dilution-mass spectrometry.23 The assay of ACAT activity was performed by adding [14C]oleoyl coenzyme A and measuring the amount converted to cholesteryl oleate.24 The coefficients of variation for the three enzyme assays were 7 percent, 10 percent, and 7 percent, respectively.
The heparin-sensitive binding of LDL to liver-tissue homogenates was determined with a filter assay as described elsewhere.25 , 26 Liver homogenates were stored at — 20°C and analyzed at the same time to avoid interassay variations. The results, expressed in nanograms of [125I]LDL bound per milligram of protein, represent heparin-sensitive binding (total binding minus binding after incubation with heparin) when 240 μg of a protein homogenate was incubated with 50 μg of [125I]LDL protein per milliliter (specific activity, 525 cpm per nanogram). The nonspecific (heparin-resistant) binding was about 55 percent of total. The coefficient of variation of the assay was 9 percent.
Gallbladder bile was extracted with chloroform:methanol 2:1 (vol/vol), and the chloroform phase was analyzed with respect to cholesterol27 and phospholipids.28 The total bile acid concentration was determined by an enzymatic method29 in another aliquot of bile. The relative concentrations of cholesterol, bile acids, and phospholipids were expressed as molar percentages of total biliary lipids. The cholesterol saturation of bile was calculated according to the technique of Carey.30 Bile acid composition was determined by gas-liquid chromatography.31
Pravastatin and its metabolite SQ 31,906 were extracted from serum and bile and purified with C18 disposable solid-phase columns. Their concentrations were determined with a capillary gas chromatography-mass spectrometry technique. (These analyses were performed at the Squibb Institute Laboratories, Princeton, N.J.)
Results
Treatment with pravastatin resulted in a marked decrease in plasma total cholesterol after one week. At the time of operation, plasma cholesterol levels were reduced by 26 percent (P<0.005) in the pravastatin group (Table 2Table 2
Plasma Lipid Levels in the Study Groups (Mean ±SEM).), and plasma triglyceride levels by 15 percent (P<0.01). LDL cholesterol and apolipoprotein B concentrations were decreased by 39 percent (P<0.005) and 29 percent (P<0.005), respectively, whereas the concentration of high-density lipoprotein cholesterol tended to be increased.Lathosterol is a steroid precursor of cholesterol that can be measured in serum. Its serum concentration has been shown to reflect whole-body cholesterol production in vivo.33 To determine whether the effect of pravastatin was related to inhibition of cholesterol synthesis, we measured serum lathosterol and cholesterol in nine of the pravastatin-treated patients. The serum free lathosterol concentration was decreased in all nine patients, from a mean value of 2.4±0.3 μg per milliliter to 0.9±0.1 μg per milliliter (63 percent; P<0.005) after three weeks of pravastatin treatment. The ratio of free lathosterol to free cholesterol in serum was reduced by 47 percent (P<0.01), from 3.8±0.4 to 2.0±0.3. The data for the 10th patient were lacking because a blood sample obtained during treatment was missing.
Since these results indicated that pravastatin inhibited cholesterol synthesis in vivo, we proceeded to study its effect on the activity of hepatic HMG-CoA reductase. When human liver microsomes were incubated with increasing concentrations of pravastatin in vitro before the assay of HMG-CoA reductase, the activity of the enzyme was progressively reduced; inhibition was approximately 50 percent when concentrations of the drug were 0.5 to 1.0 μg per liter (data not shown). However, when assayed directly under standard conditions (when presumably most or all of the inhibitor had been removed), the activity of the microsomal HMG-CoA reductase in the pravastatin-treated patients was increased 11.8 times (Table 3Table 3
Hepatic Microsomal Enzyme Activities in the Study Groups (Mean ±SEM).) (1344±311 vs. 105± 14 pmol per minute per milligram of protein; P<0.001). The HMG-CoA reductase in human liver is present in two forms: one dephosphorylated and catalytically active, and the other phosphorylated and inactive.16 When the standard procedure is used, inactive enzyme becomes activated during the preparation of microsomes. To determine whether the proportion of enzyme active in vivo was reduced by pravastatin, we also assayed the enzyme activity in 10 patients from each study group, in microsomes prepared in sodium fluoride (which prevents such activation). However, the relative proportion of HMG-CoA reductase initially present in the active form was not significantly changed in the pravastatin group (29 percent vs. 45 percent). This indicates that a considerable increase in the amount of potentially active enzyme protein occurred in response to pravastatin treatment.In spite of the marked effects of pravastatin on the expression of HMG-CoA reductase in human liver, there was no evidence that the drug had any effect on the activity of the rate-limiting enzyme of bile acid synthesis, cholesterol 7α-hydroxylase (Table 3), or on the activity of ACAT, the regulating enzyme of cholesterol esterification.
To determine directly whether the pravastatin-induced suppression of hepatic cholesterol biosynthesis affected the expression of LDL receptors in the human liver, we measured the heparin-sensitive binding of Radio-labeled LDL to liver homogenates. Because of the amount of liver tissue required, binding could be measured only in the samples from five pravastatin-treated patients and seven untreated patients. These subgroups did not differ from the two study groups in their response to treatment as reflected by changes in LDL cholesterol levels or HMG-CoA reductase activity. LDL-receptor expression was consistently increased in all five treated patients by an average of 1.8-fold during pravastatin treatment (6.2±0.7 vs. 2.2±0.3 ng per milligram of protein; P<0.005). When the LDL-receptor binding was evaluated in relation to plasma LDL cholesterol concentrations in the pravastatin and control groups combined, there was a strong inverse correlation between these variables (Rs = -0.81, P<0.005).
Since cellular cholesterol content is considered to be an important regulator of the uptake of LDL by LDL receptors,34 we analyzed the amount of total and free cholesterol in liver homogenates and microsomes. In the homogenates from pravastatin-treated patients, there was a significant decrease in both total cholesterol (28±2 vs. 38±2 nmol per milligram of protein; P<0.01) and free cholesterol (21±1 vs. 30±2 nmol per milligram of protein; P<0.005). In contrast, there was no difference between the two groups in the concentrations of total or free cholesterol in the microsomal fractions (data not shown). This finding probably reflects the sensitive homeostatic regulation of cholesterol concentrations in human liver membranes.
When the biliary lipid composition of gallbladder bile was analyzed, the concentrations of bile acid, phospholipid, and cholesterol did not differ in the pravastatin and control groups (Table 4Table 4
Biliary Lipid Levels in Gallbladder Bile from the Study Groups (Mean ±SEM).). However, the level of cholesterol saturation tended to be lower in the pravastatin group (P = 0.06). The composition of hepatic bile (data not shown) did not differ between the groups except in the level of cholesterol saturation, which tended to be lower in the pravastatin-treated patients (118 percent vs. 156 percent). The pattern of bile acids in gallbladder bile was changed after pravastatin treatment in that the proportion of chenodeoxycholic acid was significantly reduced (Table 4).At operation — that is, approximately 12 hours after the final dose of pravastatin (20 mg) — the mean serum concentration of pravastatin (all 10 patients) was 1.1±0.2 ng per milliliter, a level comparable to values observed in similarly treated patients with hypercholesterolemia. No metabolites of pravastatin were detected in serum. In the two patients studied, pravastatin and its major metabolite (SQ 31,906) were present in very high concentrations in gallbladder bile (pravastatin, 19,830 and 42,800 ng per milliliter; SQ 31,906, 2740 and 3860 ng per milliliter). Despite these high concentrations, simultaneously measured levels of pravastatin in the serum were low (1.6 and 1.6 ng per milliliter).
No adverse drug experiences were reported, and clinical and laboratory examinations did not reveal any drug-related side effects. Histologic examination of the liver biopsy specimens showed slight fatty infiltration in 4 of the 10 pravastatin-treated patients and in 9 of the 20 controls. Signs of mild chronic inflammation were observed in one control.
Discussion
In this open study, we were able to demonstrate directly several important effects of pravastatin on the metabolism of cholesterol in human liver. The potent inhibition of HMG-CoA reductase by low levels of the drug was associated with a significant reduction of serum lathosterol levels. Since the concentration of this cholesterol precursor in serum has been shown to correlate well with the amount of cholesterol synthesized in vivo,33 our observations provide strong evidence of reduced de novo biosynthesis of cholesterol during pravastatin therapy. We also found that HMG-CoA reductase activity measured in vitro in the absence of pravastatin was increased 11.8-fold after three weeks of pravastatin treatment. This increase probably reflects an induced synthesis of enzyme protein, occurring in response to the reduction in cholesterol biosynthesis,35 but may also reflect a concomitant decrease in enzyme degradation, as has been demonstrated in studies of lovastatin (mevinolin).36 , 37
An important finding of the present study was the demonstration of a 1.8-fold increase in the binding activity of hepatic LDL receptors in pravastatin-treated patients as compared with untreated patients. In both normolipidemic subjects38 and patients with heterozygous familial hypercholesterolemia,10 inhibitors of cholesterol synthesis have been shown to enhance the fractional catabolic rate of LDL, although LDL synthesis does not change significantly. These observations suggest that such drugs decrease plasma LDL levels mainly by increasing the number of LDL receptors. This also appears to occur in patients who are being treated with cholestyramine, in whom we were recently able to demonstrate a doubling of the number of hepatic LDL receptors.26 Our finding in the present study of an inverse relation between the plasma concentration of LDL cholesterol and the heparin-sensitive binding of LDL further underlines the importance of the expression of hepatic LDL receptors in the regulation of plasma LDL levels.
There are few studies on the influence of HMG-CoA reductase inhibitors on bile acid synthesis. In one study,39 patients with familial hypercholesterolemia treated with lovastatin had no reduction in the amount of bile acids excreted in feces during therapy. In our present study, we could not demonstrate any change in hepatic cholesterol 7α-hydroxylase activity during pravastatin treatment, indicating that therapeutic doses of this drug probably do not reduce bile acid synthesis. The proportion of chenodeoxycholic acid in gallbladder bile was reduced during pravastatin treatment, but the possible relevance of this finding could not be determined.
Another finding of importance relates to the possible effects of HMG-CoA reductase inhibitors on biliary lipid composition. The demonstration of a greatly increased level of HMG-CoA reductase in the liver of pravastatin-treated patients suggests that, as in rats given lovastatin,40 the secretion of cholesterol into bile may be enhanced in humans. However, there were no significant changes in the concentrations of total bile acid or cholesterol in bile during therapy with pravastatin. Instead, cholesterol saturation tended to be reduced in the treated patients as compared with the controls. This observation is in agreement with a recent report by Duane et al.,41 which indicated that cholesterol saturation of bile is reduced during treatment of hypercholesterolemia with simvastatin.
In summary, our data indicate that pravastatin is a specific inhibitor of hepatic HMG-CoA reductase in humans. The amount of this enzyme was increased in response to the inhibition of its activity. Other enzymes involved in cholesterol metabolism — cholesterol 7α-hydroxylase and ACAT — were not affected by treatment. The inhibition of cholesterol synthesis also clearly induced the expression of hepatic LDL receptors, accounting for the lowered plasma total and LDL cholesterol levels. The lithogenic index of gallbladder bile was not increased, which should be a favorable observation with respect to long-term therapy.
Supported by grants (03X–4793 and 03X–7137) from the Swedish Medical Research Council, the King Gustaf V and Queen Victoria Foundation, the Swedish Society of Medicine, and E.R. Squibb.
We are indebted to Ms. Gunvor Alvelius, Ms. Lisbet Benthin, Ms. Lilian Larsson, Ms. Anita Lövgren, Ms. Ingela Svensson, and Ms. Kristina Söderberg-Reid for expert technical assistance; to Mr. Jan Holm for excellent nursing care; and to Ms. Lena Ericsson for assistance in the preparation of the manuscript.
Source Information
From the Departments of Surgery (E.R., S.E.), Medicine (M.R., D.S., K.E., B.A.), and Clinical Chemistry (L.B., I.B.), Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden. Address reprint requests to Dr. Angelin at the Metabolism Unit, Department of Medicine, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
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