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Non-cholesterol sterols and faecal elimination of cholesterol during statin and fibrate treatment
Atherosclerosis, 97 (1992) S73480 0 1992 Elsevier Scientific Publishers
Ireland, Ltd. All rights reserved. 0021-9150/92/$05.00
s73
Printed and Published in Ireland ATHERO 04847
Non-cholesterol sterols and faecal elimination of cholesterol during statin and fibrate treatment T.A. Miettinen”, H. Vanhanena, J.-P. Ojalab and M.J. Tikkanen” “Second, bThird and CFirst Department of Medicine, University of Helsinki, SF-00290 Helsinki (Finland)
Summary
A simvastatin-induced lowering of low density lipoprotein (LDL)-cholesterol levels was associated with persistent reductions in the proportions of serum precursor sterol (A’-cholestenol, desmosterol and lathosterol) to cholesterol in familial (FH) and non-familial (non-FH) hypercholesterolaemia for up to 1 year. The changes were reduced by the addition of colestipol, probably due to up-regulation of simvastatininhibited cholesterol synthesis. Plant sterol (campesterol and sitosterol) levels were slightly increased. By contrast, a gemfibrozil-induced reduction in LDL-cholesterol was associated with an increase in the precursor sterol proportion, particularly in the FH subjects, while cholestanol and plant sterol proportions were markedly decreased; subsequent simvastatin treatment resulted in opposite changes in the non-cholesterol sterol proportions, suggesting that the reduction in cholesterol levels caused by gemtibrozil was associated with cholesterol malabsorption and consequent stimulation of cholesterol synthesis, while the more extensive serum cholesterol reduction by simvastatin was due to inhibition of cholesterol synthesis. The third placebo-controlled study showed that cholesterol absorption was reduced only by gemfibrozil and not by pravastatin or pravastatin together with gemtibrozil, that changes in serum non-cholesterol sterols caused by treatments were related to those in faecal elimination and absorption of cholesterol, and that the combined pravastatin and gemfibrozil treatment favourably altered the serum lipoprotein pattern without harmful side-effects.
Key words: Simvastatin;
Gemfibrozil;
Pravastatin;
Introduction
Gemtibrozil and other tibrates have significant hypolipidaemic effects, especially in hypertriglyceridaemia and combined hyperlipoproteinaemia, Correspondence to: Professor ment of Medicine, University Finland.
T.A. Miettinen, Second Departof Helsinki, SF-00290 Helsinki,
Hypercholesterolaemia;
Serum lipids; Serum cholesterol
commonly raising high density lipoprotein (HDL)cholesterol in these lipid disorders, as well as in primary hypercholesterolaemia [ 1,2]. Statins (inhibitors of cholesterol synthesis) are, on the other hand, potent hypocholesterolaemic drugs, also decreasing serum triglyceride levels and raising HDL-cholesterol levels moderately [3,4]. Drug combinations are frequently needed in many cases for treatment of familial hypercholesterolaemia
Patients and methods
both fat and cholesterol. In all cases, serum levels of cholesterol were above 6.2 mmol/l and those of triglycerides were less than 4.25 mmol/l. The simvastatin dose was increased from 5-10 mg to 40 mg/day for up to 52 weeks (Fig. 1). Three of the FH subjects received an additional lo- 15 g/day of colestipol during the last 20 weeks. The mean ages of the non-FH group and FH group were 50 and 49 years, respectively, and the mean body weights 80 and 72 kg, respectively. Blood samples for measurement of serum total and HDL-cholesterol, total triglycerides, squalene, non-cholesterol sterols and safety parameters (serum transaminases and creatine kinase) were taken at baseline and at weeks 6, 32 and 52. The patients also participated in a multicentre study comparing simvastatin to gemfibrozil [2]. They were followed up in an open extension study for up to 1 year after the initial 1Zweek study.
Simvastatin
Gemfibrozil
(FH) or familial combined hyperlipoproteinaemia (FCH). A combination of statin with resin is effective for FH, while a combination of statin with fibrate would be logical for FCH provided that side-effects could be avoided [5]. In this study the hypolipidaemic mechanisms of statins and gemfibrozil, given singly and in combination, were investigated. Serum lipids and the synthesis, absorption and faecal elimination of cholesterol were monitored during treatment with simvastatin, pravastatin and getibrozil (both alone and in combination with pravastatin). Faecal steroids and serum levels of cholesterol precursors were used to monitor cholesterol synthesis [6,7]. Cholesterol absorption was monitored by double isotope techniques and using relevant serum plant sterol levels [8,10].
study
In the first study, simvastatin was administered to 10 non-FH patients (all men) and 5 FH patients (3 men and 2 women), after a 4-week diet low in
and simvastatin
study
The second study involved 8 non-FH patients (all men) and 8 FH patients (6 men and 2 women). After a 4-week placebo ‘run-in’ period with reduc-
150
1
l
S, 40 mg/day
S, S-10 mgiday I
0
I
I
0
6
I
I
1
I
32 Weeks
S, 40 mg/day (+ Cal, 1 gin 3 cases)
52
of treatment
Fig. 1. Relative changes of LDL-cholesterol (LDL) and non-cholesterol sterols during simvastatin (S) and colestipol (Col) treatment. Combined values of FH (n = 5) and non-FH (n = 10) patients from Table 1. Co1 given to only 3 of 15 patients. Ca, campesterol; Si, sitosterol; Cha, cholestanol; A*-ch, As-cholestenol; La, lathosterol; D, desmosterol. *P < 0.05 for change from basal values.
study were taken at baseline and at weeks 4, 6, 12 and 32. The patients were Finnish participants of an international multicentre study [2]. Patients receiving gemtibrozil were switched to simvastatin at the end of the comparative study.
ed fat and cholesterol intake, the subjects were initially treated with gemfibrozil, 600 mg twice daily for 12 weeks and then with simvastatin, 40 mg once daily for an additional 20 weeks (Fig. 2). The mean ages of the non-FH group and FH group were 53 and 35 years, respectively, and the mean body weights 78 and 68 kg, respectively. Blood samples for the variables mentioned in the first
Pravastatin, gemfibrozil and their combination In the third study, 38 hyperlipidaemic (serum
\ ‘\ ‘.
A
Ax-Cholestenol
0
Lathosterol
0
Desmosterol
0
Squalene
Gemfibrozil,
t
1.2 g/day
A
Campesterol
0
Sitosterol
‘\
,
Simvastatin,
40 mgiday
I
??Cholestanol
0
I
0
LDL-Cholesterol
I
I
4
6 Weeks
I
I
12
32
of treatment
Fig. 2. Relative changes of LDL-cholesterol, squalene and non-cholesterol sterols during gemfibrozil and simvastatin treatments. Combined values of FH (n = 8) and non-FH (n = 8) patients from Table 2. *P < 0.05 for change from baseline.
S76
cholesterol > 6.0 mrnol/l and triglycerides < 4.0 mmol/l after a dietary period) patients were randomized after a 4-week diet low in both fat and cholesterol to placebo (n = lo), pravastatin (n = 9), gemfibrozil (n = 9) or pravastatin together with gemfibrozil (n = 10). The dose of pravastatin was 40 mg/day, that of gernfibrozil 1200 mg/day and the treatment lasted for 6 weeks. The mean ages of the placebo, pravastatin, gernlibrozill pravastatin and gemfibrozil groups were 56, 52, 53 and 57 years, respectively, and the mean body weights were 77, 75,78 and 78 kg, respectively. All subjects tolerated the treatments well and there were no transaminase or creatine kinase elevations above two or three times the upper normal limits. Blood samples for the variables mentioned in the first study were taken before and at the end of the treatments, with values at the two time points taken as means of three determinations. The safety parameters were measured at 2-week intervals. In addition to the sernm lipids, faecal steroids and cholesterol absorption were measured at both time points. Laboratory methods and measurements Serum total cholesterol, triglycerides and HDLcholesterol were analyzed with commercial kits (Boehringer Diagnostica, Germany) and LDLcholesterol was calculated according to the Friedewald formula [ll]. Serum squalene and total non-cholesterol sterols were determined by the gas-liquid chromatography (GLC) method described previously [lo]. These values are expressed relative to cholesterol (mmol/mol of cholesterol) to exclude the effect of lipoprotein concentration on the levels of these precursors. In the third study, cholesterol absorption was measured with an oral 7-day [ “C]cholesterol[3H]sitosterol feeding method [12] prior to randomization and again during the last 7 days of treatment. Stools were collected for the last 3 days of the relevant measurement weeks of the study for the determination of cholesterol absorption efficiency and of faecal output of bile acids and neutral sterols with GLC [13]. Statistical analyses Paired samples were analysed by paired t-test and changes by t-test. Correlations were calculated
by the least squares method. All statistical tests were two-tailed, with results taken as significant when P < 0.05. Results Simvastatin study Simvastatin reduced total and LDL-cholesterol by 39% and 51%, respectively, in the non-FH group and by 40% and 46%, respectively, in FH groups using the maximal dose (Table 1, Fig. 1). An additional 5% fall in the serum LDLcholesterol was achieved by the addition of colestipol to the treatment of some of the FH patients. In both groups, the serum proportions of squalene were not consistently changed, while of A’cholestenol, those desmosterol and lathosterol were reduced below their baseline levels by increasing the dose of simvastatin. The corresponding mean reductions in the two combined groups were 35%, 70% and 53%. The absolute and relative reductions were similar in the two groups, suggesting similar reductions in cholesterol synthesis. The levels of cholestanol and sitosterol were not consistently changed. The campesterol content rose significantly in both groups by about 28% at week 32. The addition of colestipol to simvastatin from week 32 ameliorated the reductions of cholesterol precursor sterols. Thus, the reduction in the content of lathosterol was significantly less at week 52 than at week 32. Gemfibrozil and simvastatin study Gemtibrozil reduced serum levels of total and LDL-cholesterol and triglycerides in both groups by less than 20% (Table 2, Fig. 2) and increased HDL-cholesterol by over 20%. The switch to treatment with simvastatin reduced the total and LDLcholesterol, decreased triglyceride and increased HDL-cholesterol levels more than gemlibrozil (data not shown). The serum proportions of A8-cholestenol and lathosterol increased markedly in the FH group and to a lesser extent in the non-FH group during treatment with gemlibrozil. The desmosterol value was unaltered in the FH group and even reduced in the non-FH group. The proportions of cholestanol and plant sterols were decreased by
TABLE 1 BASAL VALUES AND CHANGES IN SERUM TOTAL AND LDL-CHOLESTEROL, CHOLESTANOL AND PLANT STEROLS DURING SIMVASTATIN TREATMENTa
CHOLESTEROL
PRECURSORS,
Cholesterol values (mean + SE.) are in mmolil, other values (mean f SE.) are given as lo2 mmol/mol of cholesterol. FH (n = 5) Untreated
Cholesterol LDL-cholesterol Squalene A%holestenol Desmosterol Lathosterol Cholestanol Campesterol Sitosterol
12.1 f 10.5 f 13 f 23 f 45 f 132 f 114 f 246 f 192 f
1.0 0.9 3 5 6 34 14 45 36
non-FH (n = 10) Simvastatina
Untreated
32 weeks
52 weeks
-4.8 -4.8 -9.0 -19.0 -73.2 -1.2 -3.0 -3.0
-5.1 -5.3 -4.6 -5.6 -17.4 -33.2 -12.2 +71.8 +5.8
f 0.5* f 0.5* f f f f f f
2.8’ 5.1* 23.6+ 9.6 22.6 22.6
* f i f f f f f f
Simvastatin 32 weeks
0.7’ 0.9* 2.0 4.5 7.5 30.6 6.8 36.6 7.2
8.4 ?? 0.2 6.3 f 0.2 17 f 2 25 f 3 75 f 13 147 ?? 14 106 f 12 214 f 24 161 f 17
-3.3 -3.2 +I.5 -8.3 -48.9 -75.4 -4.4 +63.4 +5.6
f f f f * + f * *
52 weeks 0.3* 0.2* 5.9 3.1; 13.4’ 9.2* 3.5 26.2+ 12.1
-3.1 -3.0 -7.3 -47.5 -82.0 +I.1 +23.8 +9.4
f f
0.3* 0.3*
f f f f f f
3.6 11.8* 8.2* 10.5 15.7 11.5
aThree of the FH subjects had additional colestipol (10 g/day) from weeks 33 to 52. *P < 0.05 for changes from basal values of studies shown in Fig. 1.
about 40% in each group. The switch to simvastatin reduced the values of A8-cholestenol to the basal level and those of lathosterol significantly below the basal level, but had no effect on the values of desmosterol. Cholestanol and plant sterol proportions returned to the basal levels; all
of the changes significant.
in
gemtibrozil
levels
were
Pravastatin, gemfibrozil and their combination
Preliminary results from the third study (Table 3) indicated that, compared to the placebo group,
TABLE 2 BASAL VALUES AND CHANGES IN SERUM TOTAL AND LDL-CHOLESTEROL, CHOLESTEROL PRECURSORS, CHOLESTANOL AND PLANT STEROLS DURING GEMFIBROZIL AND SIMVASTATIN TREATMENTS Cholesterol values (mean f SE.) are in mmolil, other values (mean * SE.) are given as IO2 mmol/mol of cholesterol. FH (n = 8)
Cholesterol LDL-cholesterol Squalene A*-cholestenol Desmosterol Lathosterol Cholestanol Campesterol Sitosterol
non-FH (n = 8)
Untreated
Gemtibrozil (12 weeks)
Simvastatin (32 weeks)
Untreated
Gemfibrozil (12 weeks)
Simvastatin (32 weeks)
12.4 * 10.5 f 18 f 14 f 38 f 110 f 102 * 230 f 134 f
-1.7 -1.8 -9.6 +11.3 +5.1 +27.1 -50.0 -68.6 -46.0
-5.6 -5.7 -9.3 +2.6 -3.5 -55.5 -7.3 24.9 +a.9
8.3 f 0.5 6.2 * 0.4 17 * 3 20 f 2 63 * 9 140 f 16 93 ?? 14 205 * 31 140 f 22
-1.1 f 0.4* -1.0 ?? 0.42 -1.6 f 3.4 +7.6 f 3.3 -17.1 f 7.4 +17.8 * 12.5 -44.3 f 7.6. -70.6 * 11.5’ -65.9 f 9.7.
-3.0 -2.9 -7.6 +0.3 -21.8 -64.6 +5.6 +6.9 -12.3
0.7 0.6 3 2 16 12 13 15 12
f f f f f f * f f
0.4’ 0.2* 2.6* 2.8* 5.0 6.0* 7.9’ 12.9* 12.7’
f OS*? ?? 0.4*t
f 3.2’ f 3.0 f 7.5 ?? 10.5*t f 4.9t * 21.97 f 9.27
*P C 0.05 for changes from basal values of studies shown in Fig. 2. PP < 0.05 for difference from gemtibrozil.
* f f f f * f + +
0.4*t 0.4*t 1.6* 3.5 10.8 9.4*t 8.3t 15.2t 13.0t
S78 TABLE 3 PLACEBO-RELATED PERCENTAGE CHANGES IN CHOLESTEROL ABSORPTION EFFICIENCY (%) AND SERUM LIPIDS, SQUALENE AND NONCHOLESTEROL STEROLS BY PRAVASTATIN AND GEMFIBROZIL Pravastatin Absorption efficiency
Gemfibrozil
Gemtibrozil + pravastatin
+4
-23*t
-22*t -32.t 0
-1o*t
-29*t
-2 +12*
-32.t +13*t
Triglycerides
-9
-31’
-372
Squalene A8cholestenol Desmosterol Lathosterol Cholestanol Campesterol Sitosterol
-18* -18 -24 -27.t +3 +20*t +23
-19 +96*t +9 +32*t -31*t -22 -2o*t
+22 -10 -27*t -36*t -20’7 -7 -3
Cholesterol LDL-cholesterol HDL-cholesterol
0
*P < 0.05 for change from baseline. tP < 0.05for change vs. placebo
Bile acids
pravastatin reduced the levels of total and LDLcholesterol and triglycerides significantly and had no effect on HDL-cholesterol. The values for gemtibrozil were - lO%, -2%, -3 1% and + 12%, respectively, and for the combined pravastatin and gemfibrozil treatment -29%, -32%, -37% and + 13%, respectively. Squalene, cholestenol, desmosterol and lathosterol values were lowered by 18-27% during the pravastatin treatment. The respective sterol values increased 9-96% with gemfibrozil and decreased lo-36% with the combined drug treatment. The proportions of cholestanol and plant sterols were increased by pravastatin and decreased by getibrozil and changes were less marked than with the combined treatment. Cholesterol absorption efficiency (Table 3) and faecal output of bile acids were decreased and the faecal output of neutral sterols enhanced, so that the net cholesterol elimination was increased by gemf’lbrozil (Fig. 3). Other treatments had no consistent effect on faecal steroids. There was, however, a significant association between faecal steroid elimination and the serum level of lathosterol in the total study population before randomization, during treatment and between the
Neutral sterols
Total steroids
Fig. 3. Changes in faecal steroids by pravastatin (PRA), gemtibrozil (GE) and combined pravastatin plus gemtibrozil treatments. Changes in total steroids are identical to changes in cholesterol synthesis. All changes are related to basal values and changes in placebo treatment. *P < 0.05 for change from baseline; tP -C0.05 for change vs. placebo.
s79
respective changes in faecal steroids and serum lathosterol caused by the four treatment procedures. Similar correlations were obtained for the other precursors, indicating that changes in the serum proportions of cholesterol precursors, especially those of lathosterols, reflected changes in cholesterol synthesis measured by the faecal elimination of cholesterol (data not shown). Variation in cholesterol absorption efficiency was inversely correlated with variation in faecal steroid output and negatively correlated with cholesterol precursors in serum. However, cholesterol absorption efficiency was positively correlated with cholestanol and plant sterols in serum. Variations in the levels of cholestanol and plant sterols were negatively correlated with respective faecal steroid values (data not shown). Discussion The present results clearly show that simvastatin and pravastatin consistently reduce the levels of cholesterol precursor sterols in serum, particularly the largest fraction, lathosterol. A reduction in the level of lathosterol has been reported earlier for lovastatin and pravastatin [14,16]. Reductions in the smaller fractions, A8-cholestenol and desmosterol, can be detected less consistently, partly due to methodological problems. Reductions in squalene values appear to be the least consistent. However, levels of methylated precursor sterols of cholesterol, located between squalene and A8-cholestenol in the cholesterol synthesis pathway, are also reduced during pravastatin treatment, indicating that statins, by inhibiting P-hydroxy-P-methylglutaryl-CoA (HMG-CoA) reductase, consequently lower serum levels of all precursor sterols. These reductions probably reflect inhibition of hepatic HMG-CoA reductase activity. A decrease in lathosterol was related to a decrease in cholesterol synthesis, as measured by the sterol balance technique [14,16]. These findings show, for the first time, that gemfibrozil significantly increases faecal elimination of cholesterol and serum levels of the cholesterol sterols and decreases levels of precursor cholestanol and plant sterols. These observations indicate that cholesterol synthesis is enhanced as a consequence of increased biliary cholesterol secre-
tion and impaired cholesterol absorption. The latter, together with increased cholesterol synthesis by gemfibrozil in humans, has not previously been demonstrated, though reduced faecal bile acid and increased neutral sterol outputs have been demonstrated during gemtibrozil treatment [ 17,181, in agreement with the present results. Increased in vivo hepatic sterol biosynthesis during gemflbrozil treatment has previously been demonstrated in rat studies [19]. In agreement with earlier work [ 14,151, serum cholestanol and plant sterols were positively correlated with cholesterol absorption efficiency. However, by contrast to gemfibrozil, statins commonly increased the values of serum cholestanol and plant sterols, a consequence of reduced biliary sterol secretion in the presence of unaltered cholesterol absorption efficacy. Biliary cholesterol secretion and subsequent absorption of cholesterol were reduced on an unmodified diet during pravastatin treatment in FH patients [16]. Pravastatin had no effect on cholesterol absorption in the present series and tended only to reduce faecal elimination and synthesis of cholesterol. According to unpublished results from this department, cholesterol absorption is positively correlated with fat intake, suggesting that the diet low in fat and cholesterol used in the present study, or the presence of FH in previous work [16], may explain the different effects of pravastatin on cholesterol absorption in the two series. Cholesterol absorption efficiency is decreased in cholesterol-fed patients during lovastatin treatment [20]. The combination of pravastatin with gemfibrozil had a beneficial effect on the lipoprotein pattern without the incidence of any harmful muscle problems or other treatment-related sideeffects. The decrease in cholesterol absorption was not detectable during the combined drug treatment, possibly because the gemtibrozil-induced increase in biliary cholesterol secretion and intestinal pool size was prevented by pravastatin. Thus, in the absence of a change in cholesterol absorption, serum cholesterol reduction was mainly due to inhibition of cholesterol synthesis as indicated by reduced serum precursor sterol levels, despite the fact that faecal steroids tended only to decrease. Inhibited cholesterol synthesis and activity, probably enhanced LDL receptor
S80
associated with altered very low density lipoprotein metabolism [29], might explain the changes in lipoprotein levels.
11
References 1 Vega, G.L. and Grundy, S.M., Gemtibrozil therapy in primary hypertriglyceridemia associated with coronary heart disease, J. Am. Med. Assoc., 253 (1985) 2398. 2 Tikkanen, M.J., Bocanegra, T.S., Walker, J.F. and Cook, T., The Simvastatin Study Group, Am. J. Med., 87 (Suppl. 4A) (1989) 48s. 3 Gnmdy, SM., HMG-CoA-reductase inhibitors for treatment of hypercholesterolemia, N. Engl. J. Med., 319 (1988) 24. 4 Bradford, R.H., Shear, C.L., Chremos, A.N., Franklin, F.A., Nash, D.T., Hurley, D.P., Dujovne, C.A., Pool, J.L., Schnaper, H., Hesney, M. and Langendiirfer, A., Expanded clinical evaluation of lovastatin (EXCEL) study results: III. Efficacy in modifying lipoproteins and implications for managing patients with moderate hypercholesterolemia, Am. J. Med., 91 (Suppl IB) (1991) 18s. 5 Miettinen, T.A., Combined drug treatment of hyperlipidaemia, Curr. Op. Lipids, 1 (1990) 39. 6 Miettinen, T.A., Detection of changes in human cholesterol metabolism, Ann. Clin. Res., 2 (1970) 300. I Miettinen, T.A. and Koivisto, P., Non-cholesterol sterols and bile acid production in hypercholesterolemic patients with heal bypass. In: Paumgartner, G., Stiehl, A. and Gerok, W. (Eds.), Bile Acids and Cholesterol in Health and Disease, MTP Press Ltd, Lancaster, 1983, p. 183. 8 Tilvis, R. and Miettinen, T.A., Serum plant sterols and their relation to cholesterol absorption, Am. J. Clin. Nutr., 43 (1986) 92. 9 Miettinen, T.A., Tilvis, R.S. and Kesaniemi, Y.A., Serum cholestanol and plant sterol levels in relation to cholesterol metabolism in middle-aged men, Metabolism, 38 (1989) 136. 10 Miettinen, T.A., Tilvis, R.S. and Kesaniemi, Y.A., Serum plant sterols and cholesterol precursors reflect cholesterol
12
13
14
15
16
17
18
19
20
absorption and synthesis in volunteers of a randomly selected male population, Am. J. Epidemiol., 131 (1990) 20. Friedewald, W.T., Levy, R.I. and Fredrickson, S.S., Estimation of the concentrations of low-densitylipoprotein cholesterol in plasma without use of the preparative ultracentrifuge, Clin. Chem., 18 (1972) 499. Crouse, J. and Grundy, S.M., Evaluation of continuous isotope feeding method of measurement of cholesterol absorption in man, J. Lipid Res., 6 (1978) 411. Miettinen, T.A., Gas-liquid chromatographic determination of fecal neutral sterols using a capillary column, Clin. Chim. Acta, 124 (1982) 245. Kempen, H.J.M., Clam, J.F.C., Leuven, J.A.G., van der Voort, H.A. and Katan, M.B., Serum lathosterol concentration is an indicator of whole-body cholesterol synthesis in humans, J. Lipid Res., 29 (1988) 1149. Reihner, E., Rudling, M., Stihlberg, D., Berglund, L., Ewerth, S., Bjiirkhem, I., Einarsson, K. and Angelin, B., Influence of pravastatin, a specific inhibitor of HMGCoA reductase, on hepatic metabolism of cholesterol, N. Engl. J. Med., 323 (1990) 224. Vanhanen, H., Kesaniemi, H.A. and Miettinen, T.A., Pravastatin lowers serum cholesterol, cholesterol precursor sterols, fecal steroids and cholesterol absorption in man, Metabolism, in press. Kesaniemi, Y.A. and Grundy, S.M., Influence of gemfibrozil and cloiibrate on metabolism of cholesterol and plasma triglycerides, J. Am. Med. Assoc., 251 (1984) 2241. Vega, G.L. and Grundy, SM., Gemfibrozil therapy in primary hypertriglyceridemia associated with coronary heart disease: effects of metabolism of low-density lipoproteins, J. Am. Med. Assoc., 253 (1984) 2398. Maxwell, R.E., Nawrocki, J.W. and Uhlendorf, P.D., Some comparative effects of gemfibrozil, clofibrate, bezafibrate, cholestyramine and compactin on sterol metabolism in rats, Atherosclerosis, 48 (1983) 195. Miettinen, T.A., Tikkanen, M.J., Helve, E. and Ojala, J.P., Inhibition of dietary cholesterol absorption during lovastatin (Mevinolin) treatment, Drug Invest., 2 (Suppl. 2) (1990) 29.
Ireland, Ltd. All rights reserved. 0021-9150/92/$05.00
s73
Printed and Published in Ireland ATHERO 04847
Non-cholesterol sterols and faecal elimination of cholesterol during statin and fibrate treatment T.A. Miettinen”, H. Vanhanena, J.-P. Ojalab and M.J. Tikkanen” “Second, bThird and CFirst Department of Medicine, University of Helsinki, SF-00290 Helsinki (Finland)
Summary
A simvastatin-induced lowering of low density lipoprotein (LDL)-cholesterol levels was associated with persistent reductions in the proportions of serum precursor sterol (A’-cholestenol, desmosterol and lathosterol) to cholesterol in familial (FH) and non-familial (non-FH) hypercholesterolaemia for up to 1 year. The changes were reduced by the addition of colestipol, probably due to up-regulation of simvastatininhibited cholesterol synthesis. Plant sterol (campesterol and sitosterol) levels were slightly increased. By contrast, a gemfibrozil-induced reduction in LDL-cholesterol was associated with an increase in the precursor sterol proportion, particularly in the FH subjects, while cholestanol and plant sterol proportions were markedly decreased; subsequent simvastatin treatment resulted in opposite changes in the non-cholesterol sterol proportions, suggesting that the reduction in cholesterol levels caused by gemtibrozil was associated with cholesterol malabsorption and consequent stimulation of cholesterol synthesis, while the more extensive serum cholesterol reduction by simvastatin was due to inhibition of cholesterol synthesis. The third placebo-controlled study showed that cholesterol absorption was reduced only by gemfibrozil and not by pravastatin or pravastatin together with gemtibrozil, that changes in serum non-cholesterol sterols caused by treatments were related to those in faecal elimination and absorption of cholesterol, and that the combined pravastatin and gemfibrozil treatment favourably altered the serum lipoprotein pattern without harmful side-effects.
Key words: Simvastatin;
Gemfibrozil;
Pravastatin;
Introduction
Gemtibrozil and other tibrates have significant hypolipidaemic effects, especially in hypertriglyceridaemia and combined hyperlipoproteinaemia, Correspondence to: Professor ment of Medicine, University Finland.
T.A. Miettinen, Second Departof Helsinki, SF-00290 Helsinki,
Hypercholesterolaemia;
Serum lipids; Serum cholesterol
commonly raising high density lipoprotein (HDL)cholesterol in these lipid disorders, as well as in primary hypercholesterolaemia [ 1,2]. Statins (inhibitors of cholesterol synthesis) are, on the other hand, potent hypocholesterolaemic drugs, also decreasing serum triglyceride levels and raising HDL-cholesterol levels moderately [3,4]. Drug combinations are frequently needed in many cases for treatment of familial hypercholesterolaemia
Patients and methods
both fat and cholesterol. In all cases, serum levels of cholesterol were above 6.2 mmol/l and those of triglycerides were less than 4.25 mmol/l. The simvastatin dose was increased from 5-10 mg to 40 mg/day for up to 52 weeks (Fig. 1). Three of the FH subjects received an additional lo- 15 g/day of colestipol during the last 20 weeks. The mean ages of the non-FH group and FH group were 50 and 49 years, respectively, and the mean body weights 80 and 72 kg, respectively. Blood samples for measurement of serum total and HDL-cholesterol, total triglycerides, squalene, non-cholesterol sterols and safety parameters (serum transaminases and creatine kinase) were taken at baseline and at weeks 6, 32 and 52. The patients also participated in a multicentre study comparing simvastatin to gemfibrozil [2]. They were followed up in an open extension study for up to 1 year after the initial 1Zweek study.
Simvastatin
Gemfibrozil
(FH) or familial combined hyperlipoproteinaemia (FCH). A combination of statin with resin is effective for FH, while a combination of statin with fibrate would be logical for FCH provided that side-effects could be avoided [5]. In this study the hypolipidaemic mechanisms of statins and gemfibrozil, given singly and in combination, were investigated. Serum lipids and the synthesis, absorption and faecal elimination of cholesterol were monitored during treatment with simvastatin, pravastatin and getibrozil (both alone and in combination with pravastatin). Faecal steroids and serum levels of cholesterol precursors were used to monitor cholesterol synthesis [6,7]. Cholesterol absorption was monitored by double isotope techniques and using relevant serum plant sterol levels [8,10].
study
In the first study, simvastatin was administered to 10 non-FH patients (all men) and 5 FH patients (3 men and 2 women), after a 4-week diet low in
and simvastatin
study
The second study involved 8 non-FH patients (all men) and 8 FH patients (6 men and 2 women). After a 4-week placebo ‘run-in’ period with reduc-
150
1
l
S, 40 mg/day
S, S-10 mgiday I
0
I
I
0
6
I
I
1
I
32 Weeks
S, 40 mg/day (+ Cal, 1 gin 3 cases)
52
of treatment
Fig. 1. Relative changes of LDL-cholesterol (LDL) and non-cholesterol sterols during simvastatin (S) and colestipol (Col) treatment. Combined values of FH (n = 5) and non-FH (n = 10) patients from Table 1. Co1 given to only 3 of 15 patients. Ca, campesterol; Si, sitosterol; Cha, cholestanol; A*-ch, As-cholestenol; La, lathosterol; D, desmosterol. *P < 0.05 for change from basal values.
study were taken at baseline and at weeks 4, 6, 12 and 32. The patients were Finnish participants of an international multicentre study [2]. Patients receiving gemtibrozil were switched to simvastatin at the end of the comparative study.
ed fat and cholesterol intake, the subjects were initially treated with gemfibrozil, 600 mg twice daily for 12 weeks and then with simvastatin, 40 mg once daily for an additional 20 weeks (Fig. 2). The mean ages of the non-FH group and FH group were 53 and 35 years, respectively, and the mean body weights 78 and 68 kg, respectively. Blood samples for the variables mentioned in the first
Pravastatin, gemfibrozil and their combination In the third study, 38 hyperlipidaemic (serum
\ ‘\ ‘.
A
Ax-Cholestenol
0
Lathosterol
0
Desmosterol
0
Squalene
Gemfibrozil,
t
1.2 g/day
A
Campesterol
0
Sitosterol
‘\
,
Simvastatin,
40 mgiday
I
??Cholestanol
0
I
0
LDL-Cholesterol
I
I
4
6 Weeks
I
I
12
32
of treatment
Fig. 2. Relative changes of LDL-cholesterol, squalene and non-cholesterol sterols during gemfibrozil and simvastatin treatments. Combined values of FH (n = 8) and non-FH (n = 8) patients from Table 2. *P < 0.05 for change from baseline.
S76
cholesterol > 6.0 mrnol/l and triglycerides < 4.0 mmol/l after a dietary period) patients were randomized after a 4-week diet low in both fat and cholesterol to placebo (n = lo), pravastatin (n = 9), gemfibrozil (n = 9) or pravastatin together with gemfibrozil (n = 10). The dose of pravastatin was 40 mg/day, that of gernfibrozil 1200 mg/day and the treatment lasted for 6 weeks. The mean ages of the placebo, pravastatin, gernlibrozill pravastatin and gemfibrozil groups were 56, 52, 53 and 57 years, respectively, and the mean body weights were 77, 75,78 and 78 kg, respectively. All subjects tolerated the treatments well and there were no transaminase or creatine kinase elevations above two or three times the upper normal limits. Blood samples for the variables mentioned in the first study were taken before and at the end of the treatments, with values at the two time points taken as means of three determinations. The safety parameters were measured at 2-week intervals. In addition to the sernm lipids, faecal steroids and cholesterol absorption were measured at both time points. Laboratory methods and measurements Serum total cholesterol, triglycerides and HDLcholesterol were analyzed with commercial kits (Boehringer Diagnostica, Germany) and LDLcholesterol was calculated according to the Friedewald formula [ll]. Serum squalene and total non-cholesterol sterols were determined by the gas-liquid chromatography (GLC) method described previously [lo]. These values are expressed relative to cholesterol (mmol/mol of cholesterol) to exclude the effect of lipoprotein concentration on the levels of these precursors. In the third study, cholesterol absorption was measured with an oral 7-day [ “C]cholesterol[3H]sitosterol feeding method [12] prior to randomization and again during the last 7 days of treatment. Stools were collected for the last 3 days of the relevant measurement weeks of the study for the determination of cholesterol absorption efficiency and of faecal output of bile acids and neutral sterols with GLC [13]. Statistical analyses Paired samples were analysed by paired t-test and changes by t-test. Correlations were calculated
by the least squares method. All statistical tests were two-tailed, with results taken as significant when P < 0.05. Results Simvastatin study Simvastatin reduced total and LDL-cholesterol by 39% and 51%, respectively, in the non-FH group and by 40% and 46%, respectively, in FH groups using the maximal dose (Table 1, Fig. 1). An additional 5% fall in the serum LDLcholesterol was achieved by the addition of colestipol to the treatment of some of the FH patients. In both groups, the serum proportions of squalene were not consistently changed, while of A’cholestenol, those desmosterol and lathosterol were reduced below their baseline levels by increasing the dose of simvastatin. The corresponding mean reductions in the two combined groups were 35%, 70% and 53%. The absolute and relative reductions were similar in the two groups, suggesting similar reductions in cholesterol synthesis. The levels of cholestanol and sitosterol were not consistently changed. The campesterol content rose significantly in both groups by about 28% at week 32. The addition of colestipol to simvastatin from week 32 ameliorated the reductions of cholesterol precursor sterols. Thus, the reduction in the content of lathosterol was significantly less at week 52 than at week 32. Gemfibrozil and simvastatin study Gemtibrozil reduced serum levels of total and LDL-cholesterol and triglycerides in both groups by less than 20% (Table 2, Fig. 2) and increased HDL-cholesterol by over 20%. The switch to treatment with simvastatin reduced the total and LDLcholesterol, decreased triglyceride and increased HDL-cholesterol levels more than gemlibrozil (data not shown). The serum proportions of A8-cholestenol and lathosterol increased markedly in the FH group and to a lesser extent in the non-FH group during treatment with gemlibrozil. The desmosterol value was unaltered in the FH group and even reduced in the non-FH group. The proportions of cholestanol and plant sterols were decreased by
TABLE 1 BASAL VALUES AND CHANGES IN SERUM TOTAL AND LDL-CHOLESTEROL, CHOLESTANOL AND PLANT STEROLS DURING SIMVASTATIN TREATMENTa
CHOLESTEROL
PRECURSORS,
Cholesterol values (mean + SE.) are in mmolil, other values (mean f SE.) are given as lo2 mmol/mol of cholesterol. FH (n = 5) Untreated
Cholesterol LDL-cholesterol Squalene A%holestenol Desmosterol Lathosterol Cholestanol Campesterol Sitosterol
12.1 f 10.5 f 13 f 23 f 45 f 132 f 114 f 246 f 192 f
1.0 0.9 3 5 6 34 14 45 36
non-FH (n = 10) Simvastatina
Untreated
32 weeks
52 weeks
-4.8 -4.8 -9.0 -19.0 -73.2 -1.2 -3.0 -3.0
-5.1 -5.3 -4.6 -5.6 -17.4 -33.2 -12.2 +71.8 +5.8
f 0.5* f 0.5* f f f f f f
2.8’ 5.1* 23.6+ 9.6 22.6 22.6
* f i f f f f f f
Simvastatin 32 weeks
0.7’ 0.9* 2.0 4.5 7.5 30.6 6.8 36.6 7.2
8.4 ?? 0.2 6.3 f 0.2 17 f 2 25 f 3 75 f 13 147 ?? 14 106 f 12 214 f 24 161 f 17
-3.3 -3.2 +I.5 -8.3 -48.9 -75.4 -4.4 +63.4 +5.6
f f f f * + f * *
52 weeks 0.3* 0.2* 5.9 3.1; 13.4’ 9.2* 3.5 26.2+ 12.1
-3.1 -3.0 -7.3 -47.5 -82.0 +I.1 +23.8 +9.4
f f
0.3* 0.3*
f f f f f f
3.6 11.8* 8.2* 10.5 15.7 11.5
aThree of the FH subjects had additional colestipol (10 g/day) from weeks 33 to 52. *P < 0.05 for changes from basal values of studies shown in Fig. 1.
about 40% in each group. The switch to simvastatin reduced the values of A8-cholestenol to the basal level and those of lathosterol significantly below the basal level, but had no effect on the values of desmosterol. Cholestanol and plant sterol proportions returned to the basal levels; all
of the changes significant.
in
gemtibrozil
levels
were
Pravastatin, gemfibrozil and their combination
Preliminary results from the third study (Table 3) indicated that, compared to the placebo group,
TABLE 2 BASAL VALUES AND CHANGES IN SERUM TOTAL AND LDL-CHOLESTEROL, CHOLESTEROL PRECURSORS, CHOLESTANOL AND PLANT STEROLS DURING GEMFIBROZIL AND SIMVASTATIN TREATMENTS Cholesterol values (mean f SE.) are in mmolil, other values (mean * SE.) are given as IO2 mmol/mol of cholesterol. FH (n = 8)
Cholesterol LDL-cholesterol Squalene A*-cholestenol Desmosterol Lathosterol Cholestanol Campesterol Sitosterol
non-FH (n = 8)
Untreated
Gemtibrozil (12 weeks)
Simvastatin (32 weeks)
Untreated
Gemfibrozil (12 weeks)
Simvastatin (32 weeks)
12.4 * 10.5 f 18 f 14 f 38 f 110 f 102 * 230 f 134 f
-1.7 -1.8 -9.6 +11.3 +5.1 +27.1 -50.0 -68.6 -46.0
-5.6 -5.7 -9.3 +2.6 -3.5 -55.5 -7.3 24.9 +a.9
8.3 f 0.5 6.2 * 0.4 17 * 3 20 f 2 63 * 9 140 f 16 93 ?? 14 205 * 31 140 f 22
-1.1 f 0.4* -1.0 ?? 0.42 -1.6 f 3.4 +7.6 f 3.3 -17.1 f 7.4 +17.8 * 12.5 -44.3 f 7.6. -70.6 * 11.5’ -65.9 f 9.7.
-3.0 -2.9 -7.6 +0.3 -21.8 -64.6 +5.6 +6.9 -12.3
0.7 0.6 3 2 16 12 13 15 12
f f f f f f * f f
0.4’ 0.2* 2.6* 2.8* 5.0 6.0* 7.9’ 12.9* 12.7’
f OS*? ?? 0.4*t
f 3.2’ f 3.0 f 7.5 ?? 10.5*t f 4.9t * 21.97 f 9.27
*P C 0.05 for changes from basal values of studies shown in Fig. 2. PP < 0.05 for difference from gemtibrozil.
* f f f f * f + +
0.4*t 0.4*t 1.6* 3.5 10.8 9.4*t 8.3t 15.2t 13.0t
S78 TABLE 3 PLACEBO-RELATED PERCENTAGE CHANGES IN CHOLESTEROL ABSORPTION EFFICIENCY (%) AND SERUM LIPIDS, SQUALENE AND NONCHOLESTEROL STEROLS BY PRAVASTATIN AND GEMFIBROZIL Pravastatin Absorption efficiency
Gemfibrozil
Gemtibrozil + pravastatin
+4
-23*t
-22*t -32.t 0
-1o*t
-29*t
-2 +12*
-32.t +13*t
Triglycerides
-9
-31’
-372
Squalene A8cholestenol Desmosterol Lathosterol Cholestanol Campesterol Sitosterol
-18* -18 -24 -27.t +3 +20*t +23
-19 +96*t +9 +32*t -31*t -22 -2o*t
+22 -10 -27*t -36*t -20’7 -7 -3
Cholesterol LDL-cholesterol HDL-cholesterol
0
*P < 0.05 for change from baseline. tP < 0.05for change vs. placebo
Bile acids
pravastatin reduced the levels of total and LDLcholesterol and triglycerides significantly and had no effect on HDL-cholesterol. The values for gemtibrozil were - lO%, -2%, -3 1% and + 12%, respectively, and for the combined pravastatin and gemfibrozil treatment -29%, -32%, -37% and + 13%, respectively. Squalene, cholestenol, desmosterol and lathosterol values were lowered by 18-27% during the pravastatin treatment. The respective sterol values increased 9-96% with gemfibrozil and decreased lo-36% with the combined drug treatment. The proportions of cholestanol and plant sterols were increased by pravastatin and decreased by getibrozil and changes were less marked than with the combined treatment. Cholesterol absorption efficiency (Table 3) and faecal output of bile acids were decreased and the faecal output of neutral sterols enhanced, so that the net cholesterol elimination was increased by gemf’lbrozil (Fig. 3). Other treatments had no consistent effect on faecal steroids. There was, however, a significant association between faecal steroid elimination and the serum level of lathosterol in the total study population before randomization, during treatment and between the
Neutral sterols
Total steroids
Fig. 3. Changes in faecal steroids by pravastatin (PRA), gemtibrozil (GE) and combined pravastatin plus gemtibrozil treatments. Changes in total steroids are identical to changes in cholesterol synthesis. All changes are related to basal values and changes in placebo treatment. *P < 0.05 for change from baseline; tP -C0.05 for change vs. placebo.
s79
respective changes in faecal steroids and serum lathosterol caused by the four treatment procedures. Similar correlations were obtained for the other precursors, indicating that changes in the serum proportions of cholesterol precursors, especially those of lathosterols, reflected changes in cholesterol synthesis measured by the faecal elimination of cholesterol (data not shown). Variation in cholesterol absorption efficiency was inversely correlated with variation in faecal steroid output and negatively correlated with cholesterol precursors in serum. However, cholesterol absorption efficiency was positively correlated with cholestanol and plant sterols in serum. Variations in the levels of cholestanol and plant sterols were negatively correlated with respective faecal steroid values (data not shown). Discussion The present results clearly show that simvastatin and pravastatin consistently reduce the levels of cholesterol precursor sterols in serum, particularly the largest fraction, lathosterol. A reduction in the level of lathosterol has been reported earlier for lovastatin and pravastatin [14,16]. Reductions in the smaller fractions, A8-cholestenol and desmosterol, can be detected less consistently, partly due to methodological problems. Reductions in squalene values appear to be the least consistent. However, levels of methylated precursor sterols of cholesterol, located between squalene and A8-cholestenol in the cholesterol synthesis pathway, are also reduced during pravastatin treatment, indicating that statins, by inhibiting P-hydroxy-P-methylglutaryl-CoA (HMG-CoA) reductase, consequently lower serum levels of all precursor sterols. These reductions probably reflect inhibition of hepatic HMG-CoA reductase activity. A decrease in lathosterol was related to a decrease in cholesterol synthesis, as measured by the sterol balance technique [14,16]. These findings show, for the first time, that gemfibrozil significantly increases faecal elimination of cholesterol and serum levels of the cholesterol sterols and decreases levels of precursor cholestanol and plant sterols. These observations indicate that cholesterol synthesis is enhanced as a consequence of increased biliary cholesterol secre-
tion and impaired cholesterol absorption. The latter, together with increased cholesterol synthesis by gemfibrozil in humans, has not previously been demonstrated, though reduced faecal bile acid and increased neutral sterol outputs have been demonstrated during gemtibrozil treatment [ 17,181, in agreement with the present results. Increased in vivo hepatic sterol biosynthesis during gemflbrozil treatment has previously been demonstrated in rat studies [19]. In agreement with earlier work [ 14,151, serum cholestanol and plant sterols were positively correlated with cholesterol absorption efficiency. However, by contrast to gemfibrozil, statins commonly increased the values of serum cholestanol and plant sterols, a consequence of reduced biliary sterol secretion in the presence of unaltered cholesterol absorption efficacy. Biliary cholesterol secretion and subsequent absorption of cholesterol were reduced on an unmodified diet during pravastatin treatment in FH patients [16]. Pravastatin had no effect on cholesterol absorption in the present series and tended only to reduce faecal elimination and synthesis of cholesterol. According to unpublished results from this department, cholesterol absorption is positively correlated with fat intake, suggesting that the diet low in fat and cholesterol used in the present study, or the presence of FH in previous work [16], may explain the different effects of pravastatin on cholesterol absorption in the two series. Cholesterol absorption efficiency is decreased in cholesterol-fed patients during lovastatin treatment [20]. The combination of pravastatin with gemfibrozil had a beneficial effect on the lipoprotein pattern without the incidence of any harmful muscle problems or other treatment-related sideeffects. The decrease in cholesterol absorption was not detectable during the combined drug treatment, possibly because the gemtibrozil-induced increase in biliary cholesterol secretion and intestinal pool size was prevented by pravastatin. Thus, in the absence of a change in cholesterol absorption, serum cholesterol reduction was mainly due to inhibition of cholesterol synthesis as indicated by reduced serum precursor sterol levels, despite the fact that faecal steroids tended only to decrease. Inhibited cholesterol synthesis and activity, probably enhanced LDL receptor
S80
associated with altered very low density lipoprotein metabolism [29], might explain the changes in lipoprotein levels.
11
References 1 Vega, G.L. and Grundy, S.M., Gemtibrozil therapy in primary hypertriglyceridemia associated with coronary heart disease, J. Am. Med. Assoc., 253 (1985) 2398. 2 Tikkanen, M.J., Bocanegra, T.S., Walker, J.F. and Cook, T., The Simvastatin Study Group, Am. J. Med., 87 (Suppl. 4A) (1989) 48s. 3 Gnmdy, SM., HMG-CoA-reductase inhibitors for treatment of hypercholesterolemia, N. Engl. J. Med., 319 (1988) 24. 4 Bradford, R.H., Shear, C.L., Chremos, A.N., Franklin, F.A., Nash, D.T., Hurley, D.P., Dujovne, C.A., Pool, J.L., Schnaper, H., Hesney, M. and Langendiirfer, A., Expanded clinical evaluation of lovastatin (EXCEL) study results: III. Efficacy in modifying lipoproteins and implications for managing patients with moderate hypercholesterolemia, Am. J. Med., 91 (Suppl IB) (1991) 18s. 5 Miettinen, T.A., Combined drug treatment of hyperlipidaemia, Curr. Op. Lipids, 1 (1990) 39. 6 Miettinen, T.A., Detection of changes in human cholesterol metabolism, Ann. Clin. Res., 2 (1970) 300. I Miettinen, T.A. and Koivisto, P., Non-cholesterol sterols and bile acid production in hypercholesterolemic patients with heal bypass. In: Paumgartner, G., Stiehl, A. and Gerok, W. (Eds.), Bile Acids and Cholesterol in Health and Disease, MTP Press Ltd, Lancaster, 1983, p. 183. 8 Tilvis, R. and Miettinen, T.A., Serum plant sterols and their relation to cholesterol absorption, Am. J. Clin. Nutr., 43 (1986) 92. 9 Miettinen, T.A., Tilvis, R.S. and Kesaniemi, Y.A., Serum cholestanol and plant sterol levels in relation to cholesterol metabolism in middle-aged men, Metabolism, 38 (1989) 136. 10 Miettinen, T.A., Tilvis, R.S. and Kesaniemi, Y.A., Serum plant sterols and cholesterol precursors reflect cholesterol
12
13
14
15
16
17
18
19
20
absorption and synthesis in volunteers of a randomly selected male population, Am. J. Epidemiol., 131 (1990) 20. Friedewald, W.T., Levy, R.I. and Fredrickson, S.S., Estimation of the concentrations of low-densitylipoprotein cholesterol in plasma without use of the preparative ultracentrifuge, Clin. Chem., 18 (1972) 499. Crouse, J. and Grundy, S.M., Evaluation of continuous isotope feeding method of measurement of cholesterol absorption in man, J. Lipid Res., 6 (1978) 411. Miettinen, T.A., Gas-liquid chromatographic determination of fecal neutral sterols using a capillary column, Clin. Chim. Acta, 124 (1982) 245. Kempen, H.J.M., Clam, J.F.C., Leuven, J.A.G., van der Voort, H.A. and Katan, M.B., Serum lathosterol concentration is an indicator of whole-body cholesterol synthesis in humans, J. Lipid Res., 29 (1988) 1149. Reihner, E., Rudling, M., Stihlberg, D., Berglund, L., Ewerth, S., Bjiirkhem, I., Einarsson, K. and Angelin, B., Influence of pravastatin, a specific inhibitor of HMGCoA reductase, on hepatic metabolism of cholesterol, N. Engl. J. Med., 323 (1990) 224. Vanhanen, H., Kesaniemi, H.A. and Miettinen, T.A., Pravastatin lowers serum cholesterol, cholesterol precursor sterols, fecal steroids and cholesterol absorption in man, Metabolism, in press. Kesaniemi, Y.A. and Grundy, S.M., Influence of gemfibrozil and cloiibrate on metabolism of cholesterol and plasma triglycerides, J. Am. Med. Assoc., 251 (1984) 2241. Vega, G.L. and Grundy, SM., Gemfibrozil therapy in primary hypertriglyceridemia associated with coronary heart disease: effects of metabolism of low-density lipoproteins, J. Am. Med. Assoc., 253 (1984) 2398. Maxwell, R.E., Nawrocki, J.W. and Uhlendorf, P.D., Some comparative effects of gemfibrozil, clofibrate, bezafibrate, cholestyramine and compactin on sterol metabolism in rats, Atherosclerosis, 48 (1983) 195. Miettinen, T.A., Tikkanen, M.J., Helve, E. and Ojala, J.P., Inhibition of dietary cholesterol absorption during lovastatin (Mevinolin) treatment, Drug Invest., 2 (Suppl. 2) (1990) 29.