Effects of clofibrate on cholesterol metabolism The effects of clofibrate on cholesterol metabolism were studied in one normolipidemic and 5 hyperlipidemic patients by sterol balance methods. In 2 patients, the effects of discontinuation of treatment were also assessed. Clofibrate reduced all classes of plasma lipids. The percentage reduction in triglycerides correlated weil (r = 0.99) with pretreatment levels. On the other hand, percentage reductions in plasma cholesterol, free fatty acids, and phospholipids did not correlate with pretreatment levels but correlated significantly with the fall in plasma triglycerides. In 3 patients, in whom plasma cholesterol was labeled by intravenous infusion of radioactive cholesterol, the specific activity (SA) of plasma cholesterol was determined at frequent intervals. Clofibrate reduced the rate offall ofplasma cholesterol SA while discontinuation of clofibrate increased rate of decline of plasma cholesterol SA. Since clofibrate did not affect the absorption of dietary cholesterol, the changes in plasma cholesterol SA were in all probability related to changes in endogenous synthesis of cholesterol. In addition to the above changes in the specific activity slopes, there were acute increases in plasma cholesterol specific activity which could be explained only by mobilization of tissue cholesterol from the slowly exchangeable pool immediately after the beginning of treatment. The effect of clofibrate on the excretion of fecal neutral and acidic steroids was variable and was probably dependent on the balance of its effect on the turnover of plasma lipoproteins on one hand and the mobilization of tissue cholesterol on the other.
B. J. Kudchodkar, Ph.D., H. S. Sodhi, M.D., Ph.D., L. Horlick, M.D., and Dean T. Mason, M.D. Davis and Sacramento, Calif., and Saskatoon, Saskatchewan, Canada The Laboratories of Lipid Research, Section of Cardiovascular Medicine, Departments of Medicine and Physiology, University of California at Davis School of Medicine and Sacramento Medical Center and University of Saskatchewan
The effects of clofibrate on the matabolism of plasma lipids have been investigated in many
Supported in part by research grants from the Medical Research Counci1 of Canada; the Canadian and Saskatchewan Heart Foundation; the Ayerst Laboratories; Research Program Project Grant HL 14780 from The National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Md.; and Califomia Chapters of The American Heart Association, Dallas, Texas. Received for publication Feb. 17, 1977. Accepted for publication April 23, 1977. Reprint requests to: H. S. Sodhi, M.D., Ph.D., Section of Cardiovascu1ar Medicine, University of California, School of Medicine, Davis, Calif. 95616.
154
species.v- 34 and today it is probably the most comrnonly prescribed drug for treatment of hyperlipidemia. Extensive studies on its actions in experimental animals': 2 and in man'': 22 have failed to provide a consensus on the mechanisms of its hypolipidemic effects. Grundy and associates'" published comprehensive data on the effects of clofibrate on different parameters of cholesterol metabolism in man. We have carried out extensive studies with this drug, some of which have already been reported.P: 28-30 We present in this paper new data collected by
Volume 22 Number 2
Clofibrate and cholesterol metabolism
155
Table I. Clinical data Subject Age Weight (kg) No. Sex (yr)
Percent ofnormal weight*
Daily calorie intake
Cholesterol intake (mg/day)
ß-Sitosterol intake (mg/day)
M
47
76.8
103
2,390
361
289
2
M
19
68.7
109
2,239
235
222
3
M
68
75.1
108
2,405
404
344
3a 4
M M
69 40
75.4 82.2
108 115
2,605 2,417
372 765
229 392
5
M
53
65.5
109
2,475
290
315
6
F
36
70.8
142
1,390
162
207
Diagnosis Angina, myocardial infarction, normolipemic Tendon xanthoma, FH of CHD, t hypercholesterolemia Angina, myocardial infarction , hypertriglyceridemia, hypercholesterolemia Chronic rheumatic heart disease, xanthoma, tuberosum multiplex, hypertriglyceridemia, hypercholesterolemia Angina, myocardial infaretion, hypertriglyceridemia, hypercholesterolemia Obesity, hypertriglyceridemia, hypercholesterolemia
*Ca1culated as weight in kilograms -;- (height in centimeters - 1(0) x 100. tFamily history of coronary heart disease.
cholesterol balance studies and also our view of the mechanisms of the drug's hypotriglyceridemic and hypocholesterolemic action in man. Methods
Patients. These studies were carried out in 6 ambulatory adults on a metabolic ward. Most subjects had hyperlipidemia (cholesterol >250 mg and triglycerides > 150 mg/IOD ml plasma) with or without coronary artery disease. Sex, age, weight, caloric and cholesterol intake, and other clinical data are listed in Table 1. Unpublished data from earlier studies'" in 8 subjects are also included in this report to facilitate comprehensive discussion on the drug action. Diet. The subjects were given solid food diets of constant composition described in our previous publications.P: 29 During the period of investigation no signifieant ehanges in body weight were noted. Chromic oxide was started 2 wk before starting the fecal collections." Experimental design. [4_ 14C] Cholesterol (Amersham/Searle, Ontario) was purified and used to label endogenous eholesterol in 3 subjects (2, 5, and 6) as described earlier.v': 29 The balance studies were started after the decline in specifie activity (SA) of plasma eholesterol became exponential, and they included two
phases-a control period lasting 9 days and a treatment period (0.5 Gm of clofibrate 4 times daily) of 9 or 12 days. Subject 5 had been on clofibrate for 2 yr prior to the experimental studies, and, therefore, in this case the treatment period preceded the contro!. Subject 3 was re-examined after 6 mo of continuous treatment and the study was continued for a second control period after the treatment was stopped. Analyses of plasma and fecallipids. Plasma cholesterol," triglycerides.s" free fatty acids, (FFA)17 and phospholipids," as weIl as fecal neutral'? and acidic metabolites-" 16 of cholesterol, were isolated and determined. The fecal recoveries of steroid were corrected by the determinations of feeal recoveries of ingested ß-sitosterol and chromic oxide.? Fecal bile acids were also estimated individually by gas liquid ehromatography. The details of these procedures have been deseribed in our previous publications.P: 16. 29 Plasma volume was assumed to be 4.5% of the body weight. 8 Total cholesterol in the plasma pool was obtained by multiplying concentration and plasma volume using standard methods of statistical analysis. 24 Results
Plasma lipids. The effects of clofibrate on plasma lipids are shown in Tables II and III.
156
Kudchodkar et al.
Table 11. Effect
Clinical Pharmaeology and Therapeuties
0/ c!ofibrate
on concentrations
0/ plasma
lipids (mean ± SD)*
Cholesterol (mg/ZOO ml) Free Patient No.
No treatment
1 2 3 3a 4 5 6
77 ::':: 4 173 ::':: 3 89 ::':: 2 100::,:: 4 77 ::':: 4
Ester No treatment
Treatment
74 ::':: 162 ::':: 77 ::':: 82 ::':: 80 ::':: 95 ::':: 61 ::'::
2 5t 5t 6 6t 5 6t
No treatment
-t 88 ::':: 4 100::':: 6
Treatment
135 ::':: 9 133 ::':: 333 ::':: 6 311 ::':: 170::':: 4 156 ::':: 163 ::':: 185 ::':: 7 150 ::':: 206::,:: 187 ::':: 6 149 ::'::
Triglycerides (mg /100 ml) No treatment
5 IOt 7t 10 181 ::':: 14t 9t 13 233 ::':: 11t 6t
No treatment
Treatment
130 ::':: 60 ::':: 189 ::':: 213 ::':: 478 ::':: 15 265 ::':: 187 ::':: 588 ::':: 25 310 ::':: 146 ::':: 8 65 ::':: 6 242 ::':: 9
6 3 31t 16 74t 6 42t
No treatment
271 ::':: 23t 213 ::':: 1St
*Mean ± SD of 4 determinations during each period. tTests not performed. :j:p < 0.05.
Table 111. Effect 0/ c!ofibrate on concentrations 0/ free fatty acids (mean ± SD)* Free fatty acids (mEq/L)
Subjectt F. P. H. P. L. H. S. 1. A. M. T. R. P. P. P. B.
No treatment
323::':: 656::':: 460::':: 423 ::':: 542::':: 814::':: 375::':: 531::'::
69 81 58 101 46 83 41 54
Treatment
183 ::':: 380 ::':: 312 ::':: 250 ::':: 345 ::':: 425 ::':: 275 ::':: 285 ::'::
64t 57t 55t 95t 95t 92t 67t 59t
*Mean ± SD of 4 determinations during each period. tEffect of clotibrate on other plasma lipids has been given in earlier publication.!" :j:p < 0.05.
The mean reductions in plasma cholesterol and phospholipids were 12% and 13%, so that the ratio of cholesterol to phospho1ipids in plasma remained unaltered by the treatment with clofibrate. The mean reduction in plasma FFA (39%) was greater than the reduction in any other lipid, including triglycerides (27%). The percentage reduction in triglycerides correlated well with pretreatment values (r = 0.99), whereas correlations between decreases in plasma cholesterol, phospholipids, and FFA and pretreatment levels were rather poor (r = 0.13; 0.39; 0.24). The reductions in all of
these lipids correlated significantly (r = 0.96; 0.82; 0.56) with changes in the levels of plasma triglycerides. Immediately after starting the treatment, the decline in the concentration of plasma free cholesterol was greater than that of plasma cholesterol esters, but after 12 to 15 days the difference in the decrease of plasma free and esterified cholesterol was only 1%. During the 6 mo of continuous treatment für Patient 3, the levels of plasma cholesterol and triglycerides did not change significantly. On withdrawal of clofibrate, however, plasma lipids began to rise rapidly and in 9 to 12 days significant increases had already occurred in Patients 3 and 5. Fecal excretion 0/ cholesterol and its metabolites. Total neutral steroids. Responses of fecal steroids to clofibrate treatment were variableas increase in total fecal neutral steroids in 3 of 6 subjects (2, 3, and 6) and a decrease in those in the remaining 3 (Table IV). The changes were insignificant in all but Patient 2. In one (patient 3), the total fecal neutral steroids remained greater than control values even after 6 mo of treatment. Discontinuation of treatment in this subject led to a nonsignificant decrease. In Subject 5, the total fecal neutral steroids increased significantly when the drug was stopped after 2 yr of continued treatment. Fecal neutral steroids derived only from endogenous choles-
Clofibrate and cholesterol metabolism
Volume 22 Number 2
Phospholipids (mg/IOO ml) No treatment
No treat-
Treat-
ment
ment
208 ± 10 399 ± 18 303 ± 6 300 ±
9
274 ±
7
207 348 272 281 243 313 220
±
8
± 12 ± 18
± 12 ± 22:j: ± 3 ± 17:j:
31O±11:j: 327 ± 11
terol were estimated in 3 subjects (2, 5, and 6), and changes in them were similar to those for total fecal neutral steroids. Fecal acidic steroids. Clofibrate decreased the fecal excretion of bile acids in all but one patient. Patient 2 showed an increase during the first 6 days, but the excretion during the next 6 days was similar to that during the control period. The decrease in fecal bile acids, however, was significant only in 2 subjects (4 and 6). Continuation of treatment for 6 mo failed to alter the fecal output of bile acids in Patient 3. In Subject 5, stopping clofibrate after 2 yr of treatment significantly increased bile acid excretion in the feces (Table IV). Total endogenous steroids. Total metabolites of endogenous cholesterol were measured in 3 patients (2, 5, and 6); they were significantly increased in Patient 2, increased, but not significantly, in Patient 5, and not changed in Patient 6. Withdrawal of clofibrate after 2 yr of treatment in Patient 5 increased the fecal metabolites of endogenous cholesterol significantly. In these 3 patients (2, 5, and 6), the excretion of total endogenous steroids paralleled the excretion of total fecal steroids of both endogenous and dietary origin. In the rernaining 3 subjects (l, 3, and 4) cholesterol absorption was not determined. Since clofibrate did not affect the absorption of dietary cholesterol in other subjects, it was assumed that even in these subjects the changes in the total fecal neutral steroids reflected the changes in the metabolites of endogenous cholesterol. When examined at
157
the end of 6 mo of treatment with clofibrate, Patient 3 showed increased excretion of fecal steroids over that of the control period. Stopping treatment in this patient reverted total steroid excretion to the control levels. Decreases in the excretion of total endogenous steroids were seen in Patients land 4, although the difference was significant only in Patient 4. Fecal excretion 0/ individual bile acids. Individual bile acids in fecal pools were estimated in 3 of these 6 patients and in 8 others studied similarly. These data have not been previously published, and since they are of considerable interest they are given in Table V. Excretion of lithocholic, deoxycholic, and other unidentified bile acids was significantly increased by clofibrate only in those subjects (F. G., H. P., and L. H.) who had significant increases in total fecal acidic steroids. Similarly, significant decreases in the excretion of individual bile acids were found only in Patient 4 who had a significant decrease in the excretion of total bile acids. Of the remaining 7 subjects, in 6 there was reduction in fecallithocholic acid and in I (T. R.) a slight increase. A significant decrease in fecallithocholic acid was also observed after 6 mo of treatment in Patient 3. The changes in the excretion of deoxycholic acid and other bile acids were variable and insignificant. In general, clofibrate decreased the ratio of lithocholic to deoxycholic acid significantly (Table V). Absorption 0/ dietary cholesterol. Cholesterol was a natural component of various food items and its intake in the diet varied between 162 and 290 mg/day (Table VI). Cholesterol absorption was determined in 3 of the 6 patients (2, 5, and 6). Treatment with clofibrate had no effect on it; mean absorption was 39% before and 41 % after initiation of the treatment with clofibrate. In Subject 5, discontinuation of the treatment caused no change in absorption of dietary cholesterol. Cholesterol balance. During the steady-state conditions of the control period, the negative balance, i.e., the difference in the amounts of cholesterol ingested and the amounts of cholesterol and its metabolites excreted in the feces, represented amounts of cholesterol synthesized in the body. The negative balance seen im-
158
Kudchodkar et al.
Clinical Pharmacology and Therapeutics
Table IV. Effect of clofibrate on fecal excretion of steroids (mean ± SD) Neutral steroids (mg/day) Subjeet
2 3 3a 4 5 6
Period
n
No treatment Treatment No treatment Treatment No treatment Treatment Treatment No treatment No treatment Treatment Treatment No treatment No treatment Treatment
3 4 3 4 3 4 3 3 3 3 3 4 3 4
I
Total
830 ± 792 ± 494 ± 631 ± 778± 835 ± 862 ± 794 ± 1,623 ± 1,373 ± 1,044 ± 1,184 ± 1,447 ± 1,493 ±
90 120 24 45 50 35 53 71 164 98 26 47t 23 113
Acidie steroids (mg/day)
Endogenous
* 355 ± 27 495 ± 40
870 1,006 1,347 1,396
± 30 ± 43t ± 24 ± 114
440 409 154 202 290 267 271 280 370 194 317 400 504 426
± ± ± ± ± ± ± ± ± ± ± ± ± ±
67 71 23 42 20 12 24 21 84 27 22 24t 49 27t
Total steroids (mg/day)
1,270 ± 1,200 ± 647± 834 ± 1,067 ± 1,101 ± 1,133 ± 1,074 ± 1,993 ± 1,567 ± 1,362 ± 1,583 ± 1,950 ± 1,919 ±
157 142 25 86 33 28 73 54 245 116t 44 57t 52 137
Total endogenous steroids (mg lday]
509 ± 697 ±
1,187 1,406 1,851 1,822
29 81
± 47 ± 52t ± 51 ± 138
n: number of 3-day feeal pools analyzed. *Tests not performed. tp < 0.05.
mediately after the start of the treatment, however, refiects the sum of cholesterol derived from tissue and plasma pools as weil as from synthesis (the new synthetic rate). Significant increase in negative balance was seen in Patient 2 and a significant decrease in Patient 4. Patients 1 and 6 showed only minor decreases. In Patient 3 a modest increase was apparent immediatelyon start of the treatment, and this was also seen after 6 mo of therapy. Stopping treatment in this subject further increased the negative balance, although the increase was not significant. Stopping of the treatment in Patient 5, who was on clofibrate for 2 yr, also increased the negative balance. Specijic activity (SA) of plasma cholesterol. Immediately after clofibrate was given to Patients 2 and 6, there was a temporary upswing in the SA slope. The acute increase in the SA lasted for about 72 hr, and it was followed by a prompt decline which resumed the exponential pattern of decay. In both patients the slope during the treatment was less than the slope before the start of the treatment. Conversely, in Patient 5, discontinuation of clofibrate increased the rate of decline of plasma cholesterol specific activity (Fig. I).
Discussion
In control conditions plasma free fatty acids constitute the sole precursor for hepatic synthesis of very low-density lipoprotein (VLDL) triglycerides.r! Therefore areduction in plasma FFA of such magnitude (=39%) would be expected to cause a secondary decrease in hepatic synthesis and secretion into plasma of VLDL triglycerides. Clofibrate, however, also causes a marked increase in hepatic synthesis of fatty acids, II and during the treatment period a significant fraction of fatty acids in VLDL triglycerides are derived from liver. 4, 28 Thus the effect of reduction in plasma FFA (on plasma triglycerides) is blunted by the increase in synthesis and incorporation of hepatic fatty acids into VLDL triglycerides. This may at least in part account for differences in the reductions in plasma levels of FFA (39%) and triglycerides (29%). Although there is some evidence to the contrary'" most studies suggest that (despite increased lipogenesis in the liver) clofibrate decreases the hepatic secretion of VLDL into plasma."- 20 There is also evidence suggesting that clofibrate increases the efficiency of clearance of plasma triglycerides. 6,28 Thus while the general mechanisms of clofibrate's hypotri-
Clofibrate and eholesterol metabolism
Volume 22 Number 2
159
Table V. Effeet of cZofibrate on the feeal excretion of various bile acids (mean ± SD)
Subject
3 3a 4
F. G. H. P. L. H.
S. J. A.M. T. R. P. P. P. B. Mean ± SO
Period
Number 0/ estimations
No treatment Treatment No treatment Treatment Treatment No treatment No treatment Treatment No treatment Treatment No treatment No treatment Treatment No treatment Treatment No treatment Treatment No treatment Treatment No treatment Treatment No treatment No treatment Treatment No treatment No treatment Treatment
3 4 3 3 3 3 3 3 3 5 4 4 4 4 4 4 4 4 5 3 5 3 3 6 3 3 5
Lithocholic acid (mg/day)
179 159 110 94 90 99 J58 60 51 78 40 147 192 120 172 178 120 62 44 122 131 141 144 101 105 106 95
No treatment Treatment No treatment
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
35 30 6 4 5* 6 38 8 7 23 8 20 30 39 17 9 21 13 10 30 36 38 36 30 30 10 39
Deoxycholic acid (mg/day)
218 218 151 143 15/ 148 180 116 37 86 30 116 31/ 75 124 14/ 142 95 72 18/ 203 228 200 195 170 155 163
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
43 32 12 5 11 9 30 14 4 20 12 18 71 25 27 12 30 14 23 64 75 48 49 36 29 15 30
Other bile acids (mg/day)
43 ± 33 ± 29 ± 30 ± 30 ± 33 ± 32 ± 18 ± 6± 27 ± 9 ± 19 ± 44 ± 19 ± 45 ± 23 ± 16 ± 16 ± 12 ± 8 ± 11 ± 20 ± 39 ± 32 ± 32 ± 20 ± 20 ±
22 10 2 3 8 6 11 5 4 9 2 3 16 8 23 11 6 3 4 5 6 8 10 10 3 9 13
Lithocholic / deoxycholic
0.821 0.729 0.729 0.657 0.596* 0.669 0.878 0.517* 1.378 0.907* 1.333* 1.268 0.617* 1.600 1.387* 1.262 0.845* 0.652 0.611 0.674 0.645 0.618 0.720 0.518 0.618 0.684 0.583 0.969 ± 0.34 0.717 ± 0.24* 0.809 ± 0.35
*p < 0.05.
glyceridemic action are reasonably clear the same is not true of its hypocholesterolemic action. The hypocholesterolemic action of clofibrate is generally attributed to a decrease in the hepatic synthesis of cholesterol. It was first shown in rats by Avoy and associates? and in man by Sodhi and co-workers'" that clofibrate inhibited cholesterogenesis between acetate and mevalonate. Our data on the changes in plasma cholesterol SA decay are consistent with this. During the control period, entry of unlabeled cholesterol (from de novo synthesis and from absorption) into the readily miscible pool is the most important determinant of the rate of the exponential decay in plasma cholesterol SA. Al-
though isotopic exchange with higher SA tissue pools of cholesterol would tend to decrease the rate of decay, its effects are relatively unimportant. It has also been shown that clofibrate does not alter the rates of isotopic exchange of free cholesterol between tissues and plasma.P" Previous studies-'- 29 as weIl as our investigations indicate that clofibrate does not alter the absorption of dietary cholesterol. Thus, a decrease in the rate of decay of plasma cholesterol SA may be attributed to either inhibition of endogenous synthesis or continuous mobilization of higher SA cholesterol from tissues into plasma or both. While there is evidence (as indicated below) for mobilization of tissue cholesterol into plasma.P- 29 inhibition of endogenous synthesis
160
Kudchodkar et al.
Clinical Pharmacology and Therapeutics
Table VI. Effect of clofibrate on absorption of dietary cholesterol and on cholesterol balance
Subject
2 3 3a 4 5 6
Period
Cholesterol intake (mglday)
No treatment Treatment No treatment Treatment No treatment Treatment Treatment No treatment No treatment Treatment Treatment No treatment No treatment Treatment
361 361 235 235 404 404 372 372 765 765 290 290 162 162
Cholesterol absorbed* %
40 ± 2 (3)t 42 ± 2 (4)
40 ± 1 (3) 39 ± 3 (4) 38 ± 1 (3) 40±2(4)
I
(mglday)
95 ± 4 98 ± 6
115 113 62 65
± 4 ± 7
± 2 ± 3
(mg lday}
Cholesterol balance (mglday)
1,270 1,200 647 834 1,067 1,101 1,074 1,133 1,993 1,567 1,362 1,583 1,950 1,919
-909 -839 -412 -599:1: -663 -697 -702 -761 -1,228 -802:1: -1,072 -1,293:1: -1,788 -] ,757
Total fecal steroids
*Mean ± SO. tNumber of 3-day fecal pools analyzed. +p < 0.05.
appears to be the more important factor in this regard. Subject 5 received clofibrate for 2 yr and thus could be assumed to be in a reasonably steady state. When treatment was stopped, there was a prompt increase in the decay of plasma cholesterol SA which was associated with an increase in negative cholesterol balance. These changes can be explained only by the drug's effect on synthesis of cholestero!. There is considerable evidence which suggests that treatment with clofibrate can mobilize tissue cholesterol.P: 29 We have shown, however, that mobilization of tissue cholesterol is neither a unique nor a primary action of clofibrate. Administration of other hypocholesterolemic drugs is also associated with prompt mobilization of tissue cholesterol, an effect which appears secondary to the falling levels of plasma cholestero!. 29 In 1973 we had suggested that the cholesterol mobilized from tissue pools is not held in plasma compartment, but it is rapidly excreted by the liver as biliary cholestero!. 26. 29 In steady-state conditions (i.e., in absence of mobilization of tissue cholesterol) the synthetic rate of cholesterol equals the fecal losses of neutral and acidic metabolites of endogenous cholestero!. The cholesterol which is catabolized in the Iiver is partitioned into biliary cholesterol and biliary bile acids , the ratios of which remain more or less the same in steady-
state conditions.P According to our model for cholesterol metabolism, 25, 32 areduction in the endogenous synthesis of cholesterol would be reflected in proportionate decrease in biliary excretion of both cholesterol and bile acids. If there is any mobilization of tissue cholesterol, it will be reflected primarily as an increase in biliary cholesterol. Thus the changes observed in biliary (or fecal) cholesterol and bile acids would reflect the balance of the drug ' s effects on endogenous synthesis and tissue mobilization. Our previous studies had suggested that during the few days following the administration of clofibrate, approximately 100 to 300 mg of tissue cholesterol is mobilized per day, and these amounts did not appear to be related to the type of hyperlipoproteinemia.t" On the other hand, we have shown that synthesis of cholesterol was much greater in patients with hypertriglyceridemia (with or without hypercholesterolemia) than in patients with normal plasma triglycerides. 27 With the same degree of inhibition, therefore, the net reduction in synthesis in hypertriglyceridemic subjects and its effects on biliary or fecal cholesterol would be much greater than those in patients without hypertriglyceridemia. Since mobilization of tissue cholesterol is similar in all subjects, the increases in biliary cholesterol or fecal neutral steroids are more Iikely to become apparent in
Va/urne 22 Number 2
hypercholesterolemic patients than in hypertriglyceridemic patients. Although observations of Grundy and colleagues-" are not in complete agreement with ours, there is considerable support for our hypothesis. Even the data of Grundy and co-workers showed that, in general, the lower the synthetic rate during the control period, the greater the increase in fecal excretion of neutral steroids on treatment. MitcheU and Murchison-' observed increases in fecal neutral steroids in only 9 of 21 subjects on treatment with clofibrate, while significant reduction in serum cholesterol occurred in all subjects. Subjects 2 and 3 of our study, who had increased fecal neutral steroids, had among the lowest synthetic rates during control periods . According to our hypothesis, 26, 31 the biliary or fecal excretion of cholesterol cannot be considered to represent mechanisms of hypocholesterolemic action of clofibrate. The changes in the fecal excretion of cholesterol are secondary to changes in synthesis and mobilization of tissue cholesterol. Similarly, changes in the fecal excretion of bile acids, according to this hypothesis, are assumed to be secondary to the changes in synthesis of endogenous cholesterol. Most subjects had decreased fecal bile acids on clofibrate. Furthermore, the decrease was greater in hypertriglyceridemic subjects than in those with only hypercholesterolemia. 1ncreased excretion of fecal bile acids after discontinuation of clofibrate after 2 yr of continued treatment also indicated that release of inhibition of cholesterol synthesis was associated with increased bile acid excretion. Clofibrate treatment, however, did increase the excretion of fecal bile acids in Subject 2. This increase was confined only to the first 6 days of treatment. Such transient increases were also seen in some of our previous patients treated with clofibrate. 15 Miettinen" also found increased fecal excretion of bile acids during the first few days after the starting of the treatment with a similar drug [4-4'-(isopropylidenedithio) bis (2, 6-dibutylphenol)]; the increments tended to disappear despite continued treatment. These transient increases may be explained by the transient increase in clearance of plasma lipoproteins caused by clofibrate during the early treatment period, the period as-
Clofibrate and cholesterol metabolism
161
S.A. PLASMA CHOLESTEROL DPM/mg
1~~~l
NO Rx F.T.R. 0.0119
600
F.T.R.' 0.Q10
CHANGE-17%
~
400
2
Rx
.~
t
Rx , , , , , , 0606672788490
200
DAYS
1~~~t ~NOIRX 600
5
Rx F.T. R.
NO Rx F.T.R. 0.0139 0.0107 CHANGE' 30% 300, ,,' 04854606672 DAYS
400
1000J 800
600~
6 NO Rx
1 F.T.R. 0.0216
4001
1
300
Rx' TREATMENT F.T. R.' FRACTIONAL TURNOVER RATE
Rx
F.T.R; 0.0154
~-29%
2001
+ , R,X, , 150-J-,>----,-~~~'--~~ o 48 54 60 66 72 78 DAYS
Fig. 1. Specific activity-time curves of plasma cholesterol after pulse labeling during control and treatment (Rx) with c1ofibrate. On treatment, Patients 2 and 6 showed acute increases in the specific activity which were followed by a decreased rate of fall in specific activity. Stopping treatment in Patient 5 increased the rate of decline in specific activity. sociated with the greatest rate of faU in plasma lipids. If it is assumed that there were no differences in the absorption of bile acids in the intestinal lumen before and during clofibrate treatment, the fecal excretion of lithocholic acid and deoxycholic acids reftect the relative rates of synthesis of chenodeoxycholic acid and cholic acid, respectively, during the two periods. Clofibrate treatment affected the synthesis of chenodeoxycholic acid more than that of cholic acid. Previous studies have shown that clofibrate treatment decreases the synthesis of cholic acid and chenodeoxycholic acid, but the effects are inconsistent and may depend on the type of hyperlipemia." In conclusion, acute affects of clofibrate treatment include increased clearance of plasma VLDL which , in combination with its well-
162
Kudchodkar et al.
sustained inhibitory action of hepatic synthesis and secretion of VLDL, is responsible for the changes in plasma lipids and lipoproteins. As might be predicted from our hypothesis, 26, 31 the sustained decrease in tumover of plasma VLDL is responsible for decreased hepatic synthesis of cholesterol and a decrease in its catabolism. The falling levels of plasma cholesterol stimulate mobilization of tissue cholesterol, which is promptly excreted from the body as fecal neutral steroids. The fecal excretion of neutral steroids, however, represents a balance between the cholesterol derived from tissueplasma pools and from the catabolism of Iipoproteins. The changes in fecal excretion of bile acids are not influenced by mobilization of tissue cholesterol and are thus dependent primarily on catabolism of lipoprotein cholesterol. We gratefully acknowledge the help of Carolyn Clifford, Bonnie Fulton, and Leslie Silvernail.
References I. Adams, L. L., Webb, W. W., and Fallon, 1. H.: Inhibition of hepatic triglyceride formation by clofibrate, J. Clin. Invest. 50:2339-2346, 1971. 2. Avoy, D. R., Swyryd, E. A., and Gould, R. G.: Effects of aIpha-parachlorophenoxyisobutyryl ethyl ester (CPIB) with and without aldosterone on cholesterol biosynthesis in rat liver, J. Lipid Res. 6:369-376, 1965. 3. Badzio, T., and Boczon, H.: The determination of free and esteri fied cholesterol in blood after separation by thin-layer chromatography, Clin. Chim. Acta 13:794-797, 1966. 4. Barter, P., Nestei, P. 1., and Carroll, K. F.: Precursors of plasma triglyceride fatty acid in humans. Effects of glucose consumption, clofibrate administration and alcoholic fatty liver, Metabolism 21:117-123, 1972. 5. Bartlett, G. R.: Phosphorus assay in column chromatography, J. Biol. Chem. 234:466-468, 1959. 6. Bierman, E. L., Brunzell, 1. 0., Bagdade, J. 0., Lerner, R. L., Hazard, W. R., and Porte, 0., Jr.: On the mechanism of action of Atromid-S on triglyceride transport in man, Trans. Assoc. Am. Physicians 83:211-224, 1970. 7. Bolin, D. W., King, R. P., and Klosterman, E. W.: A simplified method for the determination of chromic oxide (Cr203) when used as an index substance, Science 116:634-635, 1952. 8. Edelman, I. S., and Liebman, J.: Anatomy of body water and electrolytes, Am. J. Med. 27:256-277, 1959.
Clinical Pharmacology and Therapeutics
9. Einarsson, K., Hellstron, K., and Kallner, M.: The effect of clofibrate on the elimination of cholesterol as bile acids in patients with hyperlipoproteinemia Type 11 and IV, Eur. J. Clin. Invest. 3:345-351, 1973. 10. Gould, R. G., Swyryd, E. A., Avoy, 0., and Coan, B.: The effect of o-p-chlorophenoxyisobutyrate on the synthesis and release into plasma of lipoproteins in rats, Prog. Biochem. Pharmacol. 2:345-357, 1967. 11. Gould, R. G., Swyryd, E. A., Coan, B. J., and Avoy, D. R.: Effects of chlorophenoxyisobutyrate (CPIB) on the liver composition and triglyceride synthesis in rats. J. Atheroscler. Res. 6:555-564, 1966. 12. Grundy, S. M., Ahrens, E. H., Jr., and Miettinen, T. A.: Quantitative isolation and gas liquid chromatographic analysis of total fecal bile acids, J. Lipid Res. 6:397-410, 1965. 13. Grundy, S. M., Ahrens , E. 1., Jr., Salen, G., Schreibman, P. H., and Nestei, P. 1.: Mechanisms of action of clofibrate on cholesterol metabolism in patients with hyperlipidemia, J. Lipid Res. 13:531-551, 1972. 14. Havel, R. 1., Felts, 1. M., and VanDuyne, C. M.: Formation and fate of endogenous triglycerides in blood plasma of rabbits, J. Lipid Res. 3:297 -308, 1962. 15. Horlick, L., Kudchodkar, B. J., and Sodhi, H. S.: Mode of action of chlorophenoxyisobutyric acid on cholesterol metabolism in man, Circulation 43:299-309, 1971. 16. Kudchodkar, B. J., Sodhi, H. S., and Horlick, L.: Comparison of isotopic and gas liquid chromatographic methods for estimating fecal bile acids, Clin. Chim. Acta 41:47-54, 1972. 17. Lauwerys, R. R.: Calorimetric determination of free fatty acids, Anal. Biochem. 32:331-333, 1969. 18. Miettinen, T. A.: Mode of action of a new hypocholesteraemic drug (DH-581) in familial hypercholesteremia, Atherosclerosis 15: 163-176, 1972. 19. Miettinen, T. A., Ahrens, E. H., Jr., and Grundy, S. M.: Quantitative isolation and gas liquid chromatographic analysis of total dietary and fecal neutral steroids, 1. Lipid Res. 6:411424, 1965. 20. Mishkel, M. A., and Webb, W. F.: The mechanisms underlying the hypolipidemic effects of Atromid-S, nicotinic acid and benzmalecene-I, Biochem. Pharmacol. 16:897-905, 1967. 21. MitchelI, W. 0., and Murchison, L. E.: The effect of clofibrate on serum and fecal lipids, Clin. Chim. Acta. 36:153-161,1972. 22. Nestei, P. J., Hirsch, Z., and Couzens, E.: The effect of chlorophenoxyisobutyric acid and ethinyl estradiol on cholesterol tumover, J. Clin. Invest. 44:891-896, 1965. 23. Oliver, M. F.: The current status of ethyl
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24. 25. 26.
27.
28.
29.
30.
chlorophenoxyisobutyrate (Atrornid-S), Prog. Biochem. Pharmacol. 2:315-324, 1967. Snedecor, G. S., and Cochran, W. G.: Statistical methods, Ed. 6, Ames, Iowa, 1968, The Iowa State University Press. Sodhi, H. S.: A new perspective on cholesterol metabolism in man, Perspect. Biol. Med. 18:477-485, 1975. Sodhi, H. S., and Kudchodkar, B. J.: Correlating metabolism of plasma and tissue cholesterol with that of plasma lipoproteins, Lancet 1:513519, 1973. Sodhi, H. S., and Kudchodkar, B. J.: Synthesis of cholesterol in hypercholesterolemia and its relationship to plasma triglycerides, Metabolism 22:895-912, 1973. Sodhi, H. S., Kudchodkar, B. 1., and Horlick, L.: Effect of chlorophenoxyisobutyrate on the metabolism of endogenous glycerides in man, Metabolism 20:309-318, 1971. Sodhi, H. S., Kudchodkar, B. J., and Horlick, L.: Hypocholesterolemic agents and mobilization of tissue cholesterol in man, Atherosclerosis 17:1-19,1973. Sodhi, H. S., Kudchodkar, B. J., Horlick, L., and Weder, C. H.: Effects of chlorophenoxyisobutyrate on the synthesis and metabolism of cholesterol in man, Metabolism 20:348-359, 1971.
Clofibrate and cholesterol metabolism
163
31. Sodhi, H. S., Kudchodkar, B. 1., Borhani, N., and Mason, D. T.: Reappraisal of the mechanisms for control of plasma cholesterol concentrations, Artery 3:120-133,1977. 32. Sodhi, H. S., Kudchodkar, B. J., Mason, D. T., and Borhani, N. 0.: Relationship between metabolism of cholesterol and the turnover of plasma lipoproteins, Proceedings of the Fourth International Symposium on Atherosclerosis, Tokyo, 1976. (In press.) 33. Stanley, M., and Cheng, S.: Excretion from the gut and gastrointestinal exchange studied by means of the inert indicator method, Am. J. Dig. Dis. 2:628-642, 1957. 34. Thorp, J. M., and Waring, W. S.: Modification of metabolism and distribution of lipids by ethyl chlorophenoxyisobutyrate, Nature 194:948-949, 1962. 35. VanHandel, E., and Zilversrnit, D. B.: Micromethod for direct determination of serum triglycerides, J. Lab. Clin. Med. 50: 152-157, 1957. 36. Wolfe, B. M., Kane, 1. P., Havel, R. J., and Brewster, H. P.: Mechanism of the hypolipemic effect of clofibrate in postabsorptive man, J. Clin. Invest. 52:2146-2159, 1973.
B. J. Kudchodkar, Ph.D., H. S. Sodhi, M.D., Ph.D., L. Horlick, M.D., and Dean T. Mason, M.D. Davis and Sacramento, Calif., and Saskatoon, Saskatchewan, Canada The Laboratories of Lipid Research, Section of Cardiovascular Medicine, Departments of Medicine and Physiology, University of California at Davis School of Medicine and Sacramento Medical Center and University of Saskatchewan
The effects of clofibrate on the matabolism of plasma lipids have been investigated in many
Supported in part by research grants from the Medical Research Counci1 of Canada; the Canadian and Saskatchewan Heart Foundation; the Ayerst Laboratories; Research Program Project Grant HL 14780 from The National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Md.; and Califomia Chapters of The American Heart Association, Dallas, Texas. Received for publication Feb. 17, 1977. Accepted for publication April 23, 1977. Reprint requests to: H. S. Sodhi, M.D., Ph.D., Section of Cardiovascu1ar Medicine, University of California, School of Medicine, Davis, Calif. 95616.
154
species.v- 34 and today it is probably the most comrnonly prescribed drug for treatment of hyperlipidemia. Extensive studies on its actions in experimental animals': 2 and in man'': 22 have failed to provide a consensus on the mechanisms of its hypolipidemic effects. Grundy and associates'" published comprehensive data on the effects of clofibrate on different parameters of cholesterol metabolism in man. We have carried out extensive studies with this drug, some of which have already been reported.P: 28-30 We present in this paper new data collected by
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Clofibrate and cholesterol metabolism
155
Table I. Clinical data Subject Age Weight (kg) No. Sex (yr)
Percent ofnormal weight*
Daily calorie intake
Cholesterol intake (mg/day)
ß-Sitosterol intake (mg/day)
M
47
76.8
103
2,390
361
289
2
M
19
68.7
109
2,239
235
222
3
M
68
75.1
108
2,405
404
344
3a 4
M M
69 40
75.4 82.2
108 115
2,605 2,417
372 765
229 392
5
M
53
65.5
109
2,475
290
315
6
F
36
70.8
142
1,390
162
207
Diagnosis Angina, myocardial infarction, normolipemic Tendon xanthoma, FH of CHD, t hypercholesterolemia Angina, myocardial infarction , hypertriglyceridemia, hypercholesterolemia Chronic rheumatic heart disease, xanthoma, tuberosum multiplex, hypertriglyceridemia, hypercholesterolemia Angina, myocardial infaretion, hypertriglyceridemia, hypercholesterolemia Obesity, hypertriglyceridemia, hypercholesterolemia
*Ca1culated as weight in kilograms -;- (height in centimeters - 1(0) x 100. tFamily history of coronary heart disease.
cholesterol balance studies and also our view of the mechanisms of the drug's hypotriglyceridemic and hypocholesterolemic action in man. Methods
Patients. These studies were carried out in 6 ambulatory adults on a metabolic ward. Most subjects had hyperlipidemia (cholesterol >250 mg and triglycerides > 150 mg/IOD ml plasma) with or without coronary artery disease. Sex, age, weight, caloric and cholesterol intake, and other clinical data are listed in Table 1. Unpublished data from earlier studies'" in 8 subjects are also included in this report to facilitate comprehensive discussion on the drug action. Diet. The subjects were given solid food diets of constant composition described in our previous publications.P: 29 During the period of investigation no signifieant ehanges in body weight were noted. Chromic oxide was started 2 wk before starting the fecal collections." Experimental design. [4_ 14C] Cholesterol (Amersham/Searle, Ontario) was purified and used to label endogenous eholesterol in 3 subjects (2, 5, and 6) as described earlier.v': 29 The balance studies were started after the decline in specifie activity (SA) of plasma eholesterol became exponential, and they included two
phases-a control period lasting 9 days and a treatment period (0.5 Gm of clofibrate 4 times daily) of 9 or 12 days. Subject 5 had been on clofibrate for 2 yr prior to the experimental studies, and, therefore, in this case the treatment period preceded the contro!. Subject 3 was re-examined after 6 mo of continuous treatment and the study was continued for a second control period after the treatment was stopped. Analyses of plasma and fecallipids. Plasma cholesterol," triglycerides.s" free fatty acids, (FFA)17 and phospholipids," as weIl as fecal neutral'? and acidic metabolites-" 16 of cholesterol, were isolated and determined. The fecal recoveries of steroid were corrected by the determinations of feeal recoveries of ingested ß-sitosterol and chromic oxide.? Fecal bile acids were also estimated individually by gas liquid ehromatography. The details of these procedures have been deseribed in our previous publications.P: 16. 29 Plasma volume was assumed to be 4.5% of the body weight. 8 Total cholesterol in the plasma pool was obtained by multiplying concentration and plasma volume using standard methods of statistical analysis. 24 Results
Plasma lipids. The effects of clofibrate on plasma lipids are shown in Tables II and III.
156
Kudchodkar et al.
Table 11. Effect
Clinical Pharmaeology and Therapeuties
0/ c!ofibrate
on concentrations
0/ plasma
lipids (mean ± SD)*
Cholesterol (mg/ZOO ml) Free Patient No.
No treatment
1 2 3 3a 4 5 6
77 ::':: 4 173 ::':: 3 89 ::':: 2 100::,:: 4 77 ::':: 4
Ester No treatment
Treatment
74 ::':: 162 ::':: 77 ::':: 82 ::':: 80 ::':: 95 ::':: 61 ::'::
2 5t 5t 6 6t 5 6t
No treatment
-t 88 ::':: 4 100::':: 6
Treatment
135 ::':: 9 133 ::':: 333 ::':: 6 311 ::':: 170::':: 4 156 ::':: 163 ::':: 185 ::':: 7 150 ::':: 206::,:: 187 ::':: 6 149 ::'::
Triglycerides (mg /100 ml) No treatment
5 IOt 7t 10 181 ::':: 14t 9t 13 233 ::':: 11t 6t
No treatment
Treatment
130 ::':: 60 ::':: 189 ::':: 213 ::':: 478 ::':: 15 265 ::':: 187 ::':: 588 ::':: 25 310 ::':: 146 ::':: 8 65 ::':: 6 242 ::':: 9
6 3 31t 16 74t 6 42t
No treatment
271 ::':: 23t 213 ::':: 1St
*Mean ± SD of 4 determinations during each period. tTests not performed. :j:p < 0.05.
Table 111. Effect 0/ c!ofibrate on concentrations 0/ free fatty acids (mean ± SD)* Free fatty acids (mEq/L)
Subjectt F. P. H. P. L. H. S. 1. A. M. T. R. P. P. P. B.
No treatment
323::':: 656::':: 460::':: 423 ::':: 542::':: 814::':: 375::':: 531::'::
69 81 58 101 46 83 41 54
Treatment
183 ::':: 380 ::':: 312 ::':: 250 ::':: 345 ::':: 425 ::':: 275 ::':: 285 ::'::
64t 57t 55t 95t 95t 92t 67t 59t
*Mean ± SD of 4 determinations during each period. tEffect of clotibrate on other plasma lipids has been given in earlier publication.!" :j:p < 0.05.
The mean reductions in plasma cholesterol and phospholipids were 12% and 13%, so that the ratio of cholesterol to phospho1ipids in plasma remained unaltered by the treatment with clofibrate. The mean reduction in plasma FFA (39%) was greater than the reduction in any other lipid, including triglycerides (27%). The percentage reduction in triglycerides correlated well with pretreatment values (r = 0.99), whereas correlations between decreases in plasma cholesterol, phospholipids, and FFA and pretreatment levels were rather poor (r = 0.13; 0.39; 0.24). The reductions in all of
these lipids correlated significantly (r = 0.96; 0.82; 0.56) with changes in the levels of plasma triglycerides. Immediately after starting the treatment, the decline in the concentration of plasma free cholesterol was greater than that of plasma cholesterol esters, but after 12 to 15 days the difference in the decrease of plasma free and esterified cholesterol was only 1%. During the 6 mo of continuous treatment für Patient 3, the levels of plasma cholesterol and triglycerides did not change significantly. On withdrawal of clofibrate, however, plasma lipids began to rise rapidly and in 9 to 12 days significant increases had already occurred in Patients 3 and 5. Fecal excretion 0/ cholesterol and its metabolites. Total neutral steroids. Responses of fecal steroids to clofibrate treatment were variableas increase in total fecal neutral steroids in 3 of 6 subjects (2, 3, and 6) and a decrease in those in the remaining 3 (Table IV). The changes were insignificant in all but Patient 2. In one (patient 3), the total fecal neutral steroids remained greater than control values even after 6 mo of treatment. Discontinuation of treatment in this subject led to a nonsignificant decrease. In Subject 5, the total fecal neutral steroids increased significantly when the drug was stopped after 2 yr of continued treatment. Fecal neutral steroids derived only from endogenous choles-
Clofibrate and cholesterol metabolism
Volume 22 Number 2
Phospholipids (mg/IOO ml) No treatment
No treat-
Treat-
ment
ment
208 ± 10 399 ± 18 303 ± 6 300 ±
9
274 ±
7
207 348 272 281 243 313 220
±
8
± 12 ± 18
± 12 ± 22:j: ± 3 ± 17:j:
31O±11:j: 327 ± 11
terol were estimated in 3 subjects (2, 5, and 6), and changes in them were similar to those for total fecal neutral steroids. Fecal acidic steroids. Clofibrate decreased the fecal excretion of bile acids in all but one patient. Patient 2 showed an increase during the first 6 days, but the excretion during the next 6 days was similar to that during the control period. The decrease in fecal bile acids, however, was significant only in 2 subjects (4 and 6). Continuation of treatment for 6 mo failed to alter the fecal output of bile acids in Patient 3. In Subject 5, stopping clofibrate after 2 yr of treatment significantly increased bile acid excretion in the feces (Table IV). Total endogenous steroids. Total metabolites of endogenous cholesterol were measured in 3 patients (2, 5, and 6); they were significantly increased in Patient 2, increased, but not significantly, in Patient 5, and not changed in Patient 6. Withdrawal of clofibrate after 2 yr of treatment in Patient 5 increased the fecal metabolites of endogenous cholesterol significantly. In these 3 patients (2, 5, and 6), the excretion of total endogenous steroids paralleled the excretion of total fecal steroids of both endogenous and dietary origin. In the rernaining 3 subjects (l, 3, and 4) cholesterol absorption was not determined. Since clofibrate did not affect the absorption of dietary cholesterol in other subjects, it was assumed that even in these subjects the changes in the total fecal neutral steroids reflected the changes in the metabolites of endogenous cholesterol. When examined at
157
the end of 6 mo of treatment with clofibrate, Patient 3 showed increased excretion of fecal steroids over that of the control period. Stopping treatment in this patient reverted total steroid excretion to the control levels. Decreases in the excretion of total endogenous steroids were seen in Patients land 4, although the difference was significant only in Patient 4. Fecal excretion 0/ individual bile acids. Individual bile acids in fecal pools were estimated in 3 of these 6 patients and in 8 others studied similarly. These data have not been previously published, and since they are of considerable interest they are given in Table V. Excretion of lithocholic, deoxycholic, and other unidentified bile acids was significantly increased by clofibrate only in those subjects (F. G., H. P., and L. H.) who had significant increases in total fecal acidic steroids. Similarly, significant decreases in the excretion of individual bile acids were found only in Patient 4 who had a significant decrease in the excretion of total bile acids. Of the remaining 7 subjects, in 6 there was reduction in fecallithocholic acid and in I (T. R.) a slight increase. A significant decrease in fecallithocholic acid was also observed after 6 mo of treatment in Patient 3. The changes in the excretion of deoxycholic acid and other bile acids were variable and insignificant. In general, clofibrate decreased the ratio of lithocholic to deoxycholic acid significantly (Table V). Absorption 0/ dietary cholesterol. Cholesterol was a natural component of various food items and its intake in the diet varied between 162 and 290 mg/day (Table VI). Cholesterol absorption was determined in 3 of the 6 patients (2, 5, and 6). Treatment with clofibrate had no effect on it; mean absorption was 39% before and 41 % after initiation of the treatment with clofibrate. In Subject 5, discontinuation of the treatment caused no change in absorption of dietary cholesterol. Cholesterol balance. During the steady-state conditions of the control period, the negative balance, i.e., the difference in the amounts of cholesterol ingested and the amounts of cholesterol and its metabolites excreted in the feces, represented amounts of cholesterol synthesized in the body. The negative balance seen im-
158
Kudchodkar et al.
Clinical Pharmacology and Therapeutics
Table IV. Effect of clofibrate on fecal excretion of steroids (mean ± SD) Neutral steroids (mg/day) Subjeet
2 3 3a 4 5 6
Period
n
No treatment Treatment No treatment Treatment No treatment Treatment Treatment No treatment No treatment Treatment Treatment No treatment No treatment Treatment
3 4 3 4 3 4 3 3 3 3 3 4 3 4
I
Total
830 ± 792 ± 494 ± 631 ± 778± 835 ± 862 ± 794 ± 1,623 ± 1,373 ± 1,044 ± 1,184 ± 1,447 ± 1,493 ±
90 120 24 45 50 35 53 71 164 98 26 47t 23 113
Acidie steroids (mg/day)
Endogenous
* 355 ± 27 495 ± 40
870 1,006 1,347 1,396
± 30 ± 43t ± 24 ± 114
440 409 154 202 290 267 271 280 370 194 317 400 504 426
± ± ± ± ± ± ± ± ± ± ± ± ± ±
67 71 23 42 20 12 24 21 84 27 22 24t 49 27t
Total steroids (mg/day)
1,270 ± 1,200 ± 647± 834 ± 1,067 ± 1,101 ± 1,133 ± 1,074 ± 1,993 ± 1,567 ± 1,362 ± 1,583 ± 1,950 ± 1,919 ±
157 142 25 86 33 28 73 54 245 116t 44 57t 52 137
Total endogenous steroids (mg lday]
509 ± 697 ±
1,187 1,406 1,851 1,822
29 81
± 47 ± 52t ± 51 ± 138
n: number of 3-day feeal pools analyzed. *Tests not performed. tp < 0.05.
mediately after the start of the treatment, however, refiects the sum of cholesterol derived from tissue and plasma pools as weil as from synthesis (the new synthetic rate). Significant increase in negative balance was seen in Patient 2 and a significant decrease in Patient 4. Patients 1 and 6 showed only minor decreases. In Patient 3 a modest increase was apparent immediatelyon start of the treatment, and this was also seen after 6 mo of therapy. Stopping treatment in this subject further increased the negative balance, although the increase was not significant. Stopping of the treatment in Patient 5, who was on clofibrate for 2 yr, also increased the negative balance. Specijic activity (SA) of plasma cholesterol. Immediately after clofibrate was given to Patients 2 and 6, there was a temporary upswing in the SA slope. The acute increase in the SA lasted for about 72 hr, and it was followed by a prompt decline which resumed the exponential pattern of decay. In both patients the slope during the treatment was less than the slope before the start of the treatment. Conversely, in Patient 5, discontinuation of clofibrate increased the rate of decline of plasma cholesterol specific activity (Fig. I).
Discussion
In control conditions plasma free fatty acids constitute the sole precursor for hepatic synthesis of very low-density lipoprotein (VLDL) triglycerides.r! Therefore areduction in plasma FFA of such magnitude (=39%) would be expected to cause a secondary decrease in hepatic synthesis and secretion into plasma of VLDL triglycerides. Clofibrate, however, also causes a marked increase in hepatic synthesis of fatty acids, II and during the treatment period a significant fraction of fatty acids in VLDL triglycerides are derived from liver. 4, 28 Thus the effect of reduction in plasma FFA (on plasma triglycerides) is blunted by the increase in synthesis and incorporation of hepatic fatty acids into VLDL triglycerides. This may at least in part account for differences in the reductions in plasma levels of FFA (39%) and triglycerides (29%). Although there is some evidence to the contrary'" most studies suggest that (despite increased lipogenesis in the liver) clofibrate decreases the hepatic secretion of VLDL into plasma."- 20 There is also evidence suggesting that clofibrate increases the efficiency of clearance of plasma triglycerides. 6,28 Thus while the general mechanisms of clofibrate's hypotri-
Clofibrate and eholesterol metabolism
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159
Table V. Effeet of cZofibrate on the feeal excretion of various bile acids (mean ± SD)
Subject
3 3a 4
F. G. H. P. L. H.
S. J. A.M. T. R. P. P. P. B. Mean ± SO
Period
Number 0/ estimations
No treatment Treatment No treatment Treatment Treatment No treatment No treatment Treatment No treatment Treatment No treatment No treatment Treatment No treatment Treatment No treatment Treatment No treatment Treatment No treatment Treatment No treatment No treatment Treatment No treatment No treatment Treatment
3 4 3 3 3 3 3 3 3 5 4 4 4 4 4 4 4 4 5 3 5 3 3 6 3 3 5
Lithocholic acid (mg/day)
179 159 110 94 90 99 J58 60 51 78 40 147 192 120 172 178 120 62 44 122 131 141 144 101 105 106 95
No treatment Treatment No treatment
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
35 30 6 4 5* 6 38 8 7 23 8 20 30 39 17 9 21 13 10 30 36 38 36 30 30 10 39
Deoxycholic acid (mg/day)
218 218 151 143 15/ 148 180 116 37 86 30 116 31/ 75 124 14/ 142 95 72 18/ 203 228 200 195 170 155 163
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
43 32 12 5 11 9 30 14 4 20 12 18 71 25 27 12 30 14 23 64 75 48 49 36 29 15 30
Other bile acids (mg/day)
43 ± 33 ± 29 ± 30 ± 30 ± 33 ± 32 ± 18 ± 6± 27 ± 9 ± 19 ± 44 ± 19 ± 45 ± 23 ± 16 ± 16 ± 12 ± 8 ± 11 ± 20 ± 39 ± 32 ± 32 ± 20 ± 20 ±
22 10 2 3 8 6 11 5 4 9 2 3 16 8 23 11 6 3 4 5 6 8 10 10 3 9 13
Lithocholic / deoxycholic
0.821 0.729 0.729 0.657 0.596* 0.669 0.878 0.517* 1.378 0.907* 1.333* 1.268 0.617* 1.600 1.387* 1.262 0.845* 0.652 0.611 0.674 0.645 0.618 0.720 0.518 0.618 0.684 0.583 0.969 ± 0.34 0.717 ± 0.24* 0.809 ± 0.35
*p < 0.05.
glyceridemic action are reasonably clear the same is not true of its hypocholesterolemic action. The hypocholesterolemic action of clofibrate is generally attributed to a decrease in the hepatic synthesis of cholesterol. It was first shown in rats by Avoy and associates? and in man by Sodhi and co-workers'" that clofibrate inhibited cholesterogenesis between acetate and mevalonate. Our data on the changes in plasma cholesterol SA decay are consistent with this. During the control period, entry of unlabeled cholesterol (from de novo synthesis and from absorption) into the readily miscible pool is the most important determinant of the rate of the exponential decay in plasma cholesterol SA. Al-
though isotopic exchange with higher SA tissue pools of cholesterol would tend to decrease the rate of decay, its effects are relatively unimportant. It has also been shown that clofibrate does not alter the rates of isotopic exchange of free cholesterol between tissues and plasma.P" Previous studies-'- 29 as weIl as our investigations indicate that clofibrate does not alter the absorption of dietary cholesterol. Thus, a decrease in the rate of decay of plasma cholesterol SA may be attributed to either inhibition of endogenous synthesis or continuous mobilization of higher SA cholesterol from tissues into plasma or both. While there is evidence (as indicated below) for mobilization of tissue cholesterol into plasma.P- 29 inhibition of endogenous synthesis
160
Kudchodkar et al.
Clinical Pharmacology and Therapeutics
Table VI. Effect of clofibrate on absorption of dietary cholesterol and on cholesterol balance
Subject
2 3 3a 4 5 6
Period
Cholesterol intake (mglday)
No treatment Treatment No treatment Treatment No treatment Treatment Treatment No treatment No treatment Treatment Treatment No treatment No treatment Treatment
361 361 235 235 404 404 372 372 765 765 290 290 162 162
Cholesterol absorbed* %
40 ± 2 (3)t 42 ± 2 (4)
40 ± 1 (3) 39 ± 3 (4) 38 ± 1 (3) 40±2(4)
I
(mglday)
95 ± 4 98 ± 6
115 113 62 65
± 4 ± 7
± 2 ± 3
(mg lday}
Cholesterol balance (mglday)
1,270 1,200 647 834 1,067 1,101 1,074 1,133 1,993 1,567 1,362 1,583 1,950 1,919
-909 -839 -412 -599:1: -663 -697 -702 -761 -1,228 -802:1: -1,072 -1,293:1: -1,788 -] ,757
Total fecal steroids
*Mean ± SO. tNumber of 3-day fecal pools analyzed. +p < 0.05.
appears to be the more important factor in this regard. Subject 5 received clofibrate for 2 yr and thus could be assumed to be in a reasonably steady state. When treatment was stopped, there was a prompt increase in the decay of plasma cholesterol SA which was associated with an increase in negative cholesterol balance. These changes can be explained only by the drug's effect on synthesis of cholestero!. There is considerable evidence which suggests that treatment with clofibrate can mobilize tissue cholesterol.P: 29 We have shown, however, that mobilization of tissue cholesterol is neither a unique nor a primary action of clofibrate. Administration of other hypocholesterolemic drugs is also associated with prompt mobilization of tissue cholesterol, an effect which appears secondary to the falling levels of plasma cholestero!. 29 In 1973 we had suggested that the cholesterol mobilized from tissue pools is not held in plasma compartment, but it is rapidly excreted by the liver as biliary cholestero!. 26. 29 In steady-state conditions (i.e., in absence of mobilization of tissue cholesterol) the synthetic rate of cholesterol equals the fecal losses of neutral and acidic metabolites of endogenous cholestero!. The cholesterol which is catabolized in the Iiver is partitioned into biliary cholesterol and biliary bile acids , the ratios of which remain more or less the same in steady-
state conditions.P According to our model for cholesterol metabolism, 25, 32 areduction in the endogenous synthesis of cholesterol would be reflected in proportionate decrease in biliary excretion of both cholesterol and bile acids. If there is any mobilization of tissue cholesterol, it will be reflected primarily as an increase in biliary cholesterol. Thus the changes observed in biliary (or fecal) cholesterol and bile acids would reflect the balance of the drug ' s effects on endogenous synthesis and tissue mobilization. Our previous studies had suggested that during the few days following the administration of clofibrate, approximately 100 to 300 mg of tissue cholesterol is mobilized per day, and these amounts did not appear to be related to the type of hyperlipoproteinemia.t" On the other hand, we have shown that synthesis of cholesterol was much greater in patients with hypertriglyceridemia (with or without hypercholesterolemia) than in patients with normal plasma triglycerides. 27 With the same degree of inhibition, therefore, the net reduction in synthesis in hypertriglyceridemic subjects and its effects on biliary or fecal cholesterol would be much greater than those in patients without hypertriglyceridemia. Since mobilization of tissue cholesterol is similar in all subjects, the increases in biliary cholesterol or fecal neutral steroids are more Iikely to become apparent in
Va/urne 22 Number 2
hypercholesterolemic patients than in hypertriglyceridemic patients. Although observations of Grundy and colleagues-" are not in complete agreement with ours, there is considerable support for our hypothesis. Even the data of Grundy and co-workers showed that, in general, the lower the synthetic rate during the control period, the greater the increase in fecal excretion of neutral steroids on treatment. MitcheU and Murchison-' observed increases in fecal neutral steroids in only 9 of 21 subjects on treatment with clofibrate, while significant reduction in serum cholesterol occurred in all subjects. Subjects 2 and 3 of our study, who had increased fecal neutral steroids, had among the lowest synthetic rates during control periods . According to our hypothesis, 26, 31 the biliary or fecal excretion of cholesterol cannot be considered to represent mechanisms of hypocholesterolemic action of clofibrate. The changes in the fecal excretion of cholesterol are secondary to changes in synthesis and mobilization of tissue cholesterol. Similarly, changes in the fecal excretion of bile acids, according to this hypothesis, are assumed to be secondary to the changes in synthesis of endogenous cholesterol. Most subjects had decreased fecal bile acids on clofibrate. Furthermore, the decrease was greater in hypertriglyceridemic subjects than in those with only hypercholesterolemia. 1ncreased excretion of fecal bile acids after discontinuation of clofibrate after 2 yr of continued treatment also indicated that release of inhibition of cholesterol synthesis was associated with increased bile acid excretion. Clofibrate treatment, however, did increase the excretion of fecal bile acids in Subject 2. This increase was confined only to the first 6 days of treatment. Such transient increases were also seen in some of our previous patients treated with clofibrate. 15 Miettinen" also found increased fecal excretion of bile acids during the first few days after the starting of the treatment with a similar drug [4-4'-(isopropylidenedithio) bis (2, 6-dibutylphenol)]; the increments tended to disappear despite continued treatment. These transient increases may be explained by the transient increase in clearance of plasma lipoproteins caused by clofibrate during the early treatment period, the period as-
Clofibrate and cholesterol metabolism
161
S.A. PLASMA CHOLESTEROL DPM/mg
1~~~l
NO Rx F.T.R. 0.0119
600
F.T.R.' 0.Q10
CHANGE-17%
~
400
2
Rx
.~
t
Rx , , , , , , 0606672788490
200
DAYS
1~~~t ~NOIRX 600
5
Rx F.T. R.
NO Rx F.T.R. 0.0139 0.0107 CHANGE' 30% 300, ,,' 04854606672 DAYS
400
1000J 800
600~
6 NO Rx
1 F.T.R. 0.0216
4001
1
300
Rx' TREATMENT F.T. R.' FRACTIONAL TURNOVER RATE
Rx
F.T.R; 0.0154
~-29%
2001
+ , R,X, , 150-J-,>----,-~~~'--~~ o 48 54 60 66 72 78 DAYS
Fig. 1. Specific activity-time curves of plasma cholesterol after pulse labeling during control and treatment (Rx) with c1ofibrate. On treatment, Patients 2 and 6 showed acute increases in the specific activity which were followed by a decreased rate of fall in specific activity. Stopping treatment in Patient 5 increased the rate of decline in specific activity. sociated with the greatest rate of faU in plasma lipids. If it is assumed that there were no differences in the absorption of bile acids in the intestinal lumen before and during clofibrate treatment, the fecal excretion of lithocholic acid and deoxycholic acids reftect the relative rates of synthesis of chenodeoxycholic acid and cholic acid, respectively, during the two periods. Clofibrate treatment affected the synthesis of chenodeoxycholic acid more than that of cholic acid. Previous studies have shown that clofibrate treatment decreases the synthesis of cholic acid and chenodeoxycholic acid, but the effects are inconsistent and may depend on the type of hyperlipemia." In conclusion, acute affects of clofibrate treatment include increased clearance of plasma VLDL which , in combination with its well-
162
Kudchodkar et al.
sustained inhibitory action of hepatic synthesis and secretion of VLDL, is responsible for the changes in plasma lipids and lipoproteins. As might be predicted from our hypothesis, 26, 31 the sustained decrease in tumover of plasma VLDL is responsible for decreased hepatic synthesis of cholesterol and a decrease in its catabolism. The falling levels of plasma cholesterol stimulate mobilization of tissue cholesterol, which is promptly excreted from the body as fecal neutral steroids. The fecal excretion of neutral steroids, however, represents a balance between the cholesterol derived from tissueplasma pools and from the catabolism of Iipoproteins. The changes in fecal excretion of bile acids are not influenced by mobilization of tissue cholesterol and are thus dependent primarily on catabolism of lipoprotein cholesterol. We gratefully acknowledge the help of Carolyn Clifford, Bonnie Fulton, and Leslie Silvernail.
References I. Adams, L. L., Webb, W. W., and Fallon, 1. H.: Inhibition of hepatic triglyceride formation by clofibrate, J. Clin. Invest. 50:2339-2346, 1971. 2. Avoy, D. R., Swyryd, E. A., and Gould, R. G.: Effects of aIpha-parachlorophenoxyisobutyryl ethyl ester (CPIB) with and without aldosterone on cholesterol biosynthesis in rat liver, J. Lipid Res. 6:369-376, 1965. 3. Badzio, T., and Boczon, H.: The determination of free and esteri fied cholesterol in blood after separation by thin-layer chromatography, Clin. Chim. Acta 13:794-797, 1966. 4. Barter, P., Nestei, P. 1., and Carroll, K. F.: Precursors of plasma triglyceride fatty acid in humans. Effects of glucose consumption, clofibrate administration and alcoholic fatty liver, Metabolism 21:117-123, 1972. 5. Bartlett, G. R.: Phosphorus assay in column chromatography, J. Biol. Chem. 234:466-468, 1959. 6. Bierman, E. L., Brunzell, 1. 0., Bagdade, J. 0., Lerner, R. L., Hazard, W. R., and Porte, 0., Jr.: On the mechanism of action of Atromid-S on triglyceride transport in man, Trans. Assoc. Am. Physicians 83:211-224, 1970. 7. Bolin, D. W., King, R. P., and Klosterman, E. W.: A simplified method for the determination of chromic oxide (Cr203) when used as an index substance, Science 116:634-635, 1952. 8. Edelman, I. S., and Liebman, J.: Anatomy of body water and electrolytes, Am. J. Med. 27:256-277, 1959.
Clinical Pharmacology and Therapeutics
9. Einarsson, K., Hellstron, K., and Kallner, M.: The effect of clofibrate on the elimination of cholesterol as bile acids in patients with hyperlipoproteinemia Type 11 and IV, Eur. J. Clin. Invest. 3:345-351, 1973. 10. Gould, R. G., Swyryd, E. A., Avoy, 0., and Coan, B.: The effect of o-p-chlorophenoxyisobutyrate on the synthesis and release into plasma of lipoproteins in rats, Prog. Biochem. Pharmacol. 2:345-357, 1967. 11. Gould, R. G., Swyryd, E. A., Coan, B. J., and Avoy, D. R.: Effects of chlorophenoxyisobutyrate (CPIB) on the liver composition and triglyceride synthesis in rats. J. Atheroscler. Res. 6:555-564, 1966. 12. Grundy, S. M., Ahrens, E. H., Jr., and Miettinen, T. A.: Quantitative isolation and gas liquid chromatographic analysis of total fecal bile acids, J. Lipid Res. 6:397-410, 1965. 13. Grundy, S. M., Ahrens , E. 1., Jr., Salen, G., Schreibman, P. H., and Nestei, P. 1.: Mechanisms of action of clofibrate on cholesterol metabolism in patients with hyperlipidemia, J. Lipid Res. 13:531-551, 1972. 14. Havel, R. 1., Felts, 1. M., and VanDuyne, C. M.: Formation and fate of endogenous triglycerides in blood plasma of rabbits, J. Lipid Res. 3:297 -308, 1962. 15. Horlick, L., Kudchodkar, B. J., and Sodhi, H. S.: Mode of action of chlorophenoxyisobutyric acid on cholesterol metabolism in man, Circulation 43:299-309, 1971. 16. Kudchodkar, B. J., Sodhi, H. S., and Horlick, L.: Comparison of isotopic and gas liquid chromatographic methods for estimating fecal bile acids, Clin. Chim. Acta 41:47-54, 1972. 17. Lauwerys, R. R.: Calorimetric determination of free fatty acids, Anal. Biochem. 32:331-333, 1969. 18. Miettinen, T. A.: Mode of action of a new hypocholesteraemic drug (DH-581) in familial hypercholesteremia, Atherosclerosis 15: 163-176, 1972. 19. Miettinen, T. A., Ahrens, E. H., Jr., and Grundy, S. M.: Quantitative isolation and gas liquid chromatographic analysis of total dietary and fecal neutral steroids, 1. Lipid Res. 6:411424, 1965. 20. Mishkel, M. A., and Webb, W. F.: The mechanisms underlying the hypolipidemic effects of Atromid-S, nicotinic acid and benzmalecene-I, Biochem. Pharmacol. 16:897-905, 1967. 21. MitchelI, W. 0., and Murchison, L. E.: The effect of clofibrate on serum and fecal lipids, Clin. Chim. Acta. 36:153-161,1972. 22. Nestei, P. J., Hirsch, Z., and Couzens, E.: The effect of chlorophenoxyisobutyric acid and ethinyl estradiol on cholesterol tumover, J. Clin. Invest. 44:891-896, 1965. 23. Oliver, M. F.: The current status of ethyl
Va/urne 22 Number 2
24. 25. 26.
27.
28.
29.
30.
chlorophenoxyisobutyrate (Atrornid-S), Prog. Biochem. Pharmacol. 2:315-324, 1967. Snedecor, G. S., and Cochran, W. G.: Statistical methods, Ed. 6, Ames, Iowa, 1968, The Iowa State University Press. Sodhi, H. S.: A new perspective on cholesterol metabolism in man, Perspect. Biol. Med. 18:477-485, 1975. Sodhi, H. S., and Kudchodkar, B. J.: Correlating metabolism of plasma and tissue cholesterol with that of plasma lipoproteins, Lancet 1:513519, 1973. Sodhi, H. S., and Kudchodkar, B. J.: Synthesis of cholesterol in hypercholesterolemia and its relationship to plasma triglycerides, Metabolism 22:895-912, 1973. Sodhi, H. S., Kudchodkar, B. 1., and Horlick, L.: Effect of chlorophenoxyisobutyrate on the metabolism of endogenous glycerides in man, Metabolism 20:309-318, 1971. Sodhi, H. S., Kudchodkar, B. J., and Horlick, L.: Hypocholesterolemic agents and mobilization of tissue cholesterol in man, Atherosclerosis 17:1-19,1973. Sodhi, H. S., Kudchodkar, B. J., Horlick, L., and Weder, C. H.: Effects of chlorophenoxyisobutyrate on the synthesis and metabolism of cholesterol in man, Metabolism 20:348-359, 1971.
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163
31. Sodhi, H. S., Kudchodkar, B. 1., Borhani, N., and Mason, D. T.: Reappraisal of the mechanisms for control of plasma cholesterol concentrations, Artery 3:120-133,1977. 32. Sodhi, H. S., Kudchodkar, B. J., Mason, D. T., and Borhani, N. 0.: Relationship between metabolism of cholesterol and the turnover of plasma lipoproteins, Proceedings of the Fourth International Symposium on Atherosclerosis, Tokyo, 1976. (In press.) 33. Stanley, M., and Cheng, S.: Excretion from the gut and gastrointestinal exchange studied by means of the inert indicator method, Am. J. Dig. Dis. 2:628-642, 1957. 34. Thorp, J. M., and Waring, W. S.: Modification of metabolism and distribution of lipids by ethyl chlorophenoxyisobutyrate, Nature 194:948-949, 1962. 35. VanHandel, E., and Zilversrnit, D. B.: Micromethod for direct determination of serum triglycerides, J. Lab. Clin. Med. 50: 152-157, 1957. 36. Wolfe, B. M., Kane, 1. P., Havel, R. J., and Brewster, H. P.: Mechanism of the hypolipemic effect of clofibrate in postabsorptive man, J. Clin. Invest. 52:2146-2159, 1973.
