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Clofibrate effect on catecholamine-induced metabolic changes in humans
Clofibrate
Effect
on Catecholamine-induced
Metabolic Changes in Humans ByDONALDB. HUNNINGHAKE AND DANIEL L. AZARNOFF Clofibrate administration (25 mg./Kg.) for 7 days decreases the plasma FFA in normal human volunteers. At this dosage, the drug also inhibits the increase in plasma FFA induced by epinepbrine (2 pg./mm. or 6.15 pg./Kg./min.) inf~sions, but not by norepinepbrine (11.5 pg./Kg./min.) nor isoproterenol (9.08
pg./Kg./min.) infusions. The mechanisms of these effects was not elucidated. Clofibrate had no effect on the plasma glucose or cardiovascular responses induced by the various catecholamines. (Metabolism 17: No- 7, Jub, 588-595, 1968)
THYL P-CHLOROPHENOXYISOBUTYMTE (clofibrate) has been shown to lower serum cholesterol and triglycerides in humans,ls2s3 but its mode of action is unknown. Clofibrate inhibits cholesterol biosynthesis in the rat,495 but whether this mechanism is of significance in humans is not certain. Fractional turnover rates of cholesterol-Cl4 in humans following clofibrate administration are decreased, suggesting inhibition of cholesterol or lipoprotein synthesis or a change in pool size.6 The fatty acids of the plasma triglycerides are primarily derived from the plasma FFA fraction.’ The molar ratio of FFA to albumin appears to control the rate of triglyceride synthesis in Ehrlich ascites tumor cells and, similarly, increased lipolysis in adipose tissue with the resultant rise of FFA transported in plasma can increase hepatic triglyceride synthesis8 Rifkind has recently reported that clofibrate lowers the fasting levels of FFA in mans This finding, coupled with the knowledge that clofibrate is more effective in lowering serum triglycerides than serum cholesterol, has raised the question of whether a significant part of the hypolipidemic effect of clofibrate might be explained by an effect on FFA metabolism. Clofibrate in combination with androsterone also suppresses the rise in plasma FFA following epinephrine injections in the rat and dog10 although clofibrate does not inhibit norepinephrine-induced rises in plasma FFA in humans.‘r Nicotinic acid, which also lowers serum cholesterol and triglycerides in humans, reduces fasting levels of FFAI” and inhibits norepinephrine-induced
E
From the Departments of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, Kansas. Received for publication November 24, 1967. DONALD B. HUNNINGHAKE, M.D.: VSPHS Fellow in Clinical Pharmacology, Instructor in Medicine, University of Kansas Medical Center, Kansas City, Kansas. Present address: Department of Pharmacology, University of Minnesota School of Medicine, Minneapolis, Minn. DANIEL L. AZARNOFF, M.D.: Burroughs Wellcome Scholar in Clinical Pharmacology, Associate Professor of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, Kansas.
CLOFIBRATE
589
EFFE(;T
0 CONTROL .CLOFIBRATE
Fig. l.-Clofibrate inhibition of epinephrineinduced changes of plasma FFA. Mean changes plus and minus standard error of mean are recorded for ten volunteers infused with l-epinephrine, 2 pg./ min., for 15 minutes.
2JJQ/YIN
30 MINUTES
15
rises in humans.lr clofibrate
The present
study was undertaken
on catecholamine-induced
hypotriglyceridemic
60
45
lipolysis
to determine
as a possible
the effect of
explanation
for its
effect. METHOD
After healthy
a thorough
discussion
adult male and female
of the purpose volunteers
and procedures
were studied.
The
of this
volunteers
investigation, were 2240
of age, ate a regular diet, and only one was more than 10 per cent overweight. randomly
received
either
clofibrate,
1.5-2.0
Gm. (25 mg./Kg.),
or placebo
Each
subject
(corn oil) orally
daily in four divided doses for one week prior to the test and served as his own control later study, at least one week later. The last dose of medication to the catecholamine mine infusions. collection
The
infusion. general
is as previously
et al.14 modification
Each
method described.
volunteer
participated
of study including 13
Plasma
of the DoIe method.
FFA
Plasma
clofibrate
spectrophotometric method of Barrett and Thorp,m’ technique utilizing the Auto-analyzer,16 and triglycerides
at a
was given two hours prior
in only one series of catechola-
catecholamine
levels
29 years
infusion
were determined levels
cholesterol
and sample by the Trout
were determined by
the
ferric
by the chloride
by the method of Azarnoff .rr
*One ml. of serum is acidified with 0.5 ml. 3N WC1 and extracted with 5 ml. isooctaneethanol (95:5v/v). The absorption of the acid is determined at 226 mu. The authors are indebted to Dr. J. M. Thorp for making the method available before publication.
590
HUNNINGHAKE
AND AZARNOFF
o CONTROL
T
. CLOFIBRATE
/h
Fig. 2.-Clofibrate inhibition of epinephrineinduced changes of plasma FFA. Same as Fig. 1 except six volunteers infused with 1-epinephrine, 0.15 pg./Kg./min., for 15 minutes.
15
30 MINUTES
45
$0
RESULTS
Plasma FFA are expressed as peq./L. net change from the baseline values. In the analysis of FFA by the Dole procedure, approximately 43 per cent of p-chlorophenoxyisobutyric acid is extracted and titrated,Q while 30 per cent is extracted and titrated in the Trout modification of the Dole procedure.ll Our own data corroborate the latter estimate, Clofibrate has a plasma half-life in humans of approximately 12 hours,l* and the blood levels of clofibrate should not change significantly during our one hour study period. Serial measurements of clofibrate levels during the baseline and test period actually revealed no significant change, Hence, expressing the FFA as net change from baseline values should give an accurate estimate of the absolute changes in FFA irrespective of the effect of clofibrate on the analytical procedure. Figure 1 illustrates the FFA responses in ten subjects receiving a 15-minute infusion of epinephrine at the rate of 2pg./min. for 15 minutes. The mean peak rise in the placebo group was 1,008 ,ueq.lL. compared to 562 peq./L. in the clofibrate-treated group. The inhibition in the mean peak rise of plasma FFA was 46 per cent (p = < 0.05). When six patients were infused with epinephrine, O.l5pg./Kg./ min. for 15 minutes (Fig. 2), the mean peak rise in the placebo group was 977 peq.lL. compared to 737 peq./L. after clofibrate admin-
CLOFIBRATE
s91
EFFECT
Q CONTROL lCLOFIBRATE
I
Fig. 3.-Clofibrate effect on isoproterenol-induced changes in plasma
FFA. Lack of effect on mean FFA in six volunteers receiving dl isoproterenol, 0.03 pg./Kg./min. for 15 minutes.
15
30
45
60
MINUTES
istration which is a mean inhibition of 25 per cent (p = <0.05). Although the degree of inhibition appears to be greater in the group receiving the smaller dose of epinephrine, the degree of inhibition between the two epinephrine groups was not significantly different (p = > 0.2). Six patients were infused with isoproterenol, 0.03 pg./Kg./min. for 15 minutes (Fig. 3), and mean peak rises in plasma FFA of 497 and 469 peq.lL. in the placebo and clofibrate-treated groups respectively were observed. The 6 per cent inhibition was not significant (p = > 0.15). Figure 4 illustrates the results of seven patients infused with norepinephrine, 11.5 pg./min. for 15 minutes. The mean peak rise of 803 peq./L. in the placebo group and 726 ,ueq./L. following clofibrate was not signiEcantly different (p = > 0.1). The titrated baseline FFA values were 490 and 612 ,ueq./L. for control and periods was 150 and 141 mg./lOO ml., respectively, and is not a significant clofibrate-treated groups respectively. The mean clofibrate concentration in the latter was 174 mg./L. If a correction is made on the basis of 30 per cent clofibrate (equivalent weight = 214.7) being titrated as FFA, the corrected FFA values for the clofibrate-treated group would be 371 peq./L. The mean fasting plasma glucose values for the control and clofibrate-treated groups were 84 and 82 mg./lOO ml., respectively. The mean peak rises in plasma glucose are recorded in Table 1, and no significant differences were noted following clofibrate administration in any of the catecholamine groups. The cardiovascular responses are also listed in Table 1; no significant differences
592
HIJNNINGHAIU?
AND
AZARNOFF
oCONTROL
l CLOFlBRATE
1
Fig. I.-Clofibrate effect on norepinephrine-induced changes in plasma FFA. Lack of effect on mean FFA in seven volunteers receiving l-norepinephrine, 11.5 pg./min. for 15 minutes.
15
-200
30 MINUTES
45
t
I
Table l.-Effect
of Clojibrate Peak Rise in Plasma Glucose
Ct,api”““cl~~~te
on Glucose and Cardiooasculur Increase in Mean Heart Rate (per cent& $b ate Contml 0 r
Changes
Change in Mean B.P. (mm. Hg)t Clofibrate control
Epinephrine, S16
+16
$19
+15
-5
-3
Epinephrine, 0.15 #g./Kg./min.
+46
t41
+27
+29
$5
14
Isoproterenol, 0.03 pg./Kg./min.
+ 3
+3
+39
f45
+2
-1
2 pg./mm
*Expressed as mean peak rise from baseline values. +A mean rate for the infusion period was calculated and the values shown are per cent change from baseline values. SMean change from baseline of mean blood pressure (diastolic + l/3 pulse pressure) during catecholamine infusions.
were noted following clofibrate administration in either heart rate or mean blood pressure during the various catechofamine infusions. The mean serum cholesterol level for the control and clofibrate treatment periods was 150 and 141 mg.1100 ml., respectively, and is not a significant change (p = > 0.05). A non-signi&ant reduction of the glyceride glycerol levels following clofibrate administration was noted with mean values of 6.5 and 6.0 mg./lOO ml. for the control and clofibrate-treated series (p = >
593
CLOFIJ3RATJT EFFECC
0.05).
It should be emphasized that these are low normal values even before of clofibrate, and a lack of effect after a week does not constitute evidence for a lack of efficacy.
the administration
DISCUSSION
Clofibrate inhibits the epinephrine-induced rise in plasma FFA in humans, but no significant inhibitions of the norepinephrine or isoproterenol-induced rises in plasma FFA were demonstrated. These data agree with the findings of Duncan and Best who were unable to demonstrate inhibition in norepinephrine-induced rises in plasma FFA in man,ll and Thorp’s findings of inhibition in the plasma rise in plasma FFA in the rat and dog after epinephrine injection by clofibrate. lo The lowering of baseline FFA values after clofibrate corroborate Rifkind’s results.9 The mechanism of the inhibition of the epinephrine-induced rises of plasma FFA by clofibrate is not clear in this study. Thorp has suggested that clofibrate’s action may be mediated by a competition for anionic binding sites normally occupied by FFA, thyroxine, and other physiological endogenous substrates.lO Even though clofibrate has been demonstrated, in humans, to displace thyroxine19 and warfarin ( non-competitively)20 from their plasma protein binding sites, these observations do not appear to adequately explain our findings, Since the rises in plasma FFA produced by norepinephrine and epinephrine were similar, one would have expected inhibition in both the epinephrine and norepinephrine groups if clofibrate was competing with fatty acid for protein binding sites. It has previously been demonstrated in vitro that clofibrate does not effectively compete with fatty acids for binding sites on bovine serum albumin or rat plasma proteins.“’ Many acidic drugs utilize 1 or 2 primary binding sites on the albumin molecule,ls and may have secondary and tertiary binding sites available to them. Clofibrate was present in an average concentration of 174 pg./ml. serum or approximately 8 x 10h4 M. In vitro studies by Thorp utilizing bovine serum albumin suggest that clofibrate is about 96 per cent bound to plasma proteins at these concentrations.22 Albumin is present in concentrations of about 7 x 1P4 14, and hence, clofibrate binding is consistent with 1 or 2 sites on the albumin molecule. Although FFA are bound primarily to albumin and the presence of this acceptor is necessary for in vitro studies of FFA release following lipolysis, in vivo small amounts of FFA may also be bound to erythrocytes, low density, and high density lipoproteins, and the latter also increase their binding of FFA as plasma levels increase markedly above norma1.23 In vitro studies by Goodman suggests that 3 classes of binding sites are available for FFA transport.24 There are 2 binding sites in the first class, approximately 5 in the second, and more than 5 in the third class with decreasing specificity of the binding for each of the above classes. The affinity of the weakest group is 102-lo3 which is approximately the same as the primary affinity of most acidic drugs. Thus, with increasing FFA concentrations. many, non-specific binding sites are avail-
HUNNINGHAKE
594
AND
AZARNOFF
able and Thorp I8 states that release of FFA into the blood as a result of physiological stimuli may serve to displace protein-bound drug. The inhibition by clofibrate of epinephrine-induced rises in plasma FFA also cannot be satisfactorily explained on the basis of adrenergic blockade. The lipolysis mediated by catecholamines is reported to be a /3-adrenergic effect, with the P-adrenergic blocking agents competitively inhibiting the lipidmobilizing effect of the catecholamines. Although cu-adrenergic blocking agents also inhibit catecholamine-induced lipolysis, this effect is considered nonspecific .25 In a previous study,13 we were able to inhibit epinephrine but not norepinephrine-induced rises in plasma FFA in man with a small dose of butoxamine indicating differences in the sensitivity to various catecholamines. However, the changes in heart rate and blood pressure produced by epinephrine were also blocked by this dose of butoxamine. Dose response curves utilizing smaller doses are needed to clarify the character and specificity of clofibrate on catecholamine-induced rises in plasma FFA. When the level of FFA is corrected for the amount of clofibrate titrated as FFA, a lowering of the baseline FFA is noted. These data agree with the report of Rifkind,g except our plasma levels of clofibrate are higher. The difference is presumably due to our patients receiving the last dose of clofibrate the morning of the test so that our studies were done at peak clofibrate concentrations. This study supplies further confirmatory evidence that clofibrate has an effect on plasma FFA levels. Since our studies were done in normolipemic rather than hyperlipemic individuals, additional investigations are indicated to evaluate whether the effect on FFA is a prominent factor in the hypolipidemic effect of clofibrate. Our preliminary studies in dogs indicate similar effects to those seen in humans. ACKNOWLEDGMENTS This study was supported in part by Grants FR67, HE 07601, and 1 Tl GM 1342 from the U.S. Public Health Service. The clofibrate used in this study was generously supplied by Doctor Jerome Noble of Ayerst Laboratories. The competent technical assistance of Beverly Walker and Wonja Hahn is gratefully acknowledged.
REFERENCES 1. Oliver,
M.
F.:
Reduction
of
serum-
lipid and uric-acid levels by an orally active androsterone. Lancet I: 1321, 1962. 2. Best, M. M., and Duncan, C. H.: Reduction of serum triglycerides and cholesterol by ethyl p-chlorophenoxyisobutyrate (CPIB). Amer. J. Cardiol. 15:230, 1965. 3. Best, M. M., and Duncan, C. H.: Effects of cholesterol-lowering drugs on serum triglycerides. J.A.M.A. 187:37, 1964. 4. Azamoff, D. L., Tucker, D. R., and Barr, G. A.: Studies with ethyl chlorophenoxyisobutyrate (clofibrate). Metabolism 14:959, 1965. 5. Thorp, J. M., and Waring, W. S.: Mod-
ification of metabolism and distribution of lipids by ethyl chlorophenoxvisobutyrate. Nature 194:948. 1962. 6. Nestel, P. J., Hirsch, E. Z., and Couzens, E. A.: The effect of chlorophenoxyisobutyric acid and ethinyl estradiol on cholesterol turnover. J. Clin. Invest. 44:891, 1965. 7. Havel, R. J.: Conversion of plasma free fatty acids into triglycerides of plasma lipoprotein fractions in man. Metabolism 10: 1031, 19Fl. 8. Steinberg, D.: Catecholamine stimulation of fat mobilization and its metabolic consequences. Pharmacol. Rev. 18~217, 1986.
CLOFIBBATE
595
EFFECT
9. Rifkind,
B.
M.:
Effect
of CPIB
ester
determination
of serum lipids.
J. Lab.
Clin.
on plasma free fatty acid levels in man. Metabolism 15:673, 1966. 10. Thorp, J. M.: An experimental approach to the problem of disordered lipid metabolism. J. Atheroscler. Res. 3:351, 1963. 11. Duncan, C. H., Best, hf. M., and Robertson, G. L.: A comparison of the effect of ethyl chlorophenoxyisobutyric acid and nicotinic acid on plasma free fatty acids. Lancet 1:191, 1965. 12. Carlson, L. A., and Oro, L.: The effect of nicotinic acid on the plasma free fatty acids. Demonstration of a metabolic
Med. 60:331, 1962. 18. Thorp, J. M.: The influence of plasma proteins on the action of drugs. In Binns. T. B. (Ed.): Absorption and Distribution of Drugs. Baltimore, Williams and Wilkins Co.,
type of sympathicolysis. Acta Med. Stand. 172:641, 1969. 13. Hunninghake, D. B., Azarnoff, D. L., and Waxman, D.: The effect of butoxamine on catecholamine-induced metabolic changes in humans. Clin. Pharmacol. Ther.
The effect of various drugs on the binding of warfarin-1°C to human albumin. Aiothem. Pharmacol. 16:1219, 1967. 21. Barrett, A. M.: The effect of chlorophenoxyisobutyric acid on the release of frer fatty acids from isolated adipose tissue in vitro. Brit. J. Pharmacol. 26:363, 1966.
7:470, 1966. 14. Trout, D. L., Estes, E. J., Jr., and Friedberg, S. J.: Titration of free fatty acids of plasma, a study of current methods and a new
modification.
J.
Lipid
Res.
1:199,
1960. 15. Barrett,
A.
and
M.,
Thorp,
J.
M.:
Unpublished. 16. Walter, and Levine,
D., Block,
agent to improve tion
K., Jarrett,
J. B.: Use of a single
of serum
the automated
total
Symposia
on
Chemistry,
New
cholesterol.
Automation York,
in
Mediad
J.. Jr.. color re-
determinaTechnicon Analytical Inc.,
1965,
p. 345. 17. Azarnoff,
D. L.:
Micromethod
for the
1964, 19. lone, from
p. 64. Chang, Y. H., Pinson, R., Jr., and XlaM. H.: Displacement of L-th\-roxine its binding proteins in human, dog,
and rat plasma by a-(p-chlorophenoxy) isobutyric acid. Biochem. Pharmacol. 16:20S3, 1967. 20. Solomon, H. M., and Schrogie. J. J.:
22. Thorp, J. M.: Experimental evaluation of an orally active combination of androsterone with ethyl chlorophenoxyisob~ltyrate. Lancet 1:1323, 1962. 23. Fredrickson, D. R., and Gordon, R. S.. Jr.: Transport of fatty acids. Physiol. He\-. 38:585, 1958. 24. Goodman, D. S.: The interaction of human serum albumin with long-chain fatt\ acid anions. J. Amer. Chem. Sot. 80:389-7, 1958. 25. Wenke, M., Lincova, D., Cepelik, J., Cernohorsky, S., and Hynie, S.: Some aspects of the beta adrenergic blocking drugs on adrenergic lipid mobilization. Ann. N. I’. Acad. Sci. 139:860, 1967.
Effect
on Catecholamine-induced
Metabolic Changes in Humans ByDONALDB. HUNNINGHAKE AND DANIEL L. AZARNOFF Clofibrate administration (25 mg./Kg.) for 7 days decreases the plasma FFA in normal human volunteers. At this dosage, the drug also inhibits the increase in plasma FFA induced by epinepbrine (2 pg./mm. or 6.15 pg./Kg./min.) inf~sions, but not by norepinepbrine (11.5 pg./Kg./min.) nor isoproterenol (9.08
pg./Kg./min.) infusions. The mechanisms of these effects was not elucidated. Clofibrate had no effect on the plasma glucose or cardiovascular responses induced by the various catecholamines. (Metabolism 17: No- 7, Jub, 588-595, 1968)
THYL P-CHLOROPHENOXYISOBUTYMTE (clofibrate) has been shown to lower serum cholesterol and triglycerides in humans,ls2s3 but its mode of action is unknown. Clofibrate inhibits cholesterol biosynthesis in the rat,495 but whether this mechanism is of significance in humans is not certain. Fractional turnover rates of cholesterol-Cl4 in humans following clofibrate administration are decreased, suggesting inhibition of cholesterol or lipoprotein synthesis or a change in pool size.6 The fatty acids of the plasma triglycerides are primarily derived from the plasma FFA fraction.’ The molar ratio of FFA to albumin appears to control the rate of triglyceride synthesis in Ehrlich ascites tumor cells and, similarly, increased lipolysis in adipose tissue with the resultant rise of FFA transported in plasma can increase hepatic triglyceride synthesis8 Rifkind has recently reported that clofibrate lowers the fasting levels of FFA in mans This finding, coupled with the knowledge that clofibrate is more effective in lowering serum triglycerides than serum cholesterol, has raised the question of whether a significant part of the hypolipidemic effect of clofibrate might be explained by an effect on FFA metabolism. Clofibrate in combination with androsterone also suppresses the rise in plasma FFA following epinephrine injections in the rat and dog10 although clofibrate does not inhibit norepinephrine-induced rises in plasma FFA in humans.‘r Nicotinic acid, which also lowers serum cholesterol and triglycerides in humans, reduces fasting levels of FFAI” and inhibits norepinephrine-induced
E
From the Departments of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, Kansas. Received for publication November 24, 1967. DONALD B. HUNNINGHAKE, M.D.: VSPHS Fellow in Clinical Pharmacology, Instructor in Medicine, University of Kansas Medical Center, Kansas City, Kansas. Present address: Department of Pharmacology, University of Minnesota School of Medicine, Minneapolis, Minn. DANIEL L. AZARNOFF, M.D.: Burroughs Wellcome Scholar in Clinical Pharmacology, Associate Professor of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, Kansas.
CLOFIBRATE
589
EFFE(;T
0 CONTROL .CLOFIBRATE
Fig. l.-Clofibrate inhibition of epinephrineinduced changes of plasma FFA. Mean changes plus and minus standard error of mean are recorded for ten volunteers infused with l-epinephrine, 2 pg./ min., for 15 minutes.
2JJQ/YIN
30 MINUTES
15
rises in humans.lr clofibrate
The present
study was undertaken
on catecholamine-induced
hypotriglyceridemic
60
45
lipolysis
to determine
as a possible
the effect of
explanation
for its
effect. METHOD
After healthy
a thorough
discussion
adult male and female
of the purpose volunteers
and procedures
were studied.
The
of this
volunteers
investigation, were 2240
of age, ate a regular diet, and only one was more than 10 per cent overweight. randomly
received
either
clofibrate,
1.5-2.0
Gm. (25 mg./Kg.),
or placebo
Each
subject
(corn oil) orally
daily in four divided doses for one week prior to the test and served as his own control later study, at least one week later. The last dose of medication to the catecholamine mine infusions. collection
The
infusion. general
is as previously
et al.14 modification
Each
method described.
volunteer
participated
of study including 13
Plasma
of the DoIe method.
FFA
Plasma
clofibrate
spectrophotometric method of Barrett and Thorp,m’ technique utilizing the Auto-analyzer,16 and triglycerides
at a
was given two hours prior
in only one series of catechola-
catecholamine
levels
29 years
infusion
were determined levels
cholesterol
and sample by the Trout
were determined by
the
ferric
by the chloride
by the method of Azarnoff .rr
*One ml. of serum is acidified with 0.5 ml. 3N WC1 and extracted with 5 ml. isooctaneethanol (95:5v/v). The absorption of the acid is determined at 226 mu. The authors are indebted to Dr. J. M. Thorp for making the method available before publication.
590
HUNNINGHAKE
AND AZARNOFF
o CONTROL
T
. CLOFIBRATE
/h
Fig. 2.-Clofibrate inhibition of epinephrineinduced changes of plasma FFA. Same as Fig. 1 except six volunteers infused with 1-epinephrine, 0.15 pg./Kg./min., for 15 minutes.
15
30 MINUTES
45
$0
RESULTS
Plasma FFA are expressed as peq./L. net change from the baseline values. In the analysis of FFA by the Dole procedure, approximately 43 per cent of p-chlorophenoxyisobutyric acid is extracted and titrated,Q while 30 per cent is extracted and titrated in the Trout modification of the Dole procedure.ll Our own data corroborate the latter estimate, Clofibrate has a plasma half-life in humans of approximately 12 hours,l* and the blood levels of clofibrate should not change significantly during our one hour study period. Serial measurements of clofibrate levels during the baseline and test period actually revealed no significant change, Hence, expressing the FFA as net change from baseline values should give an accurate estimate of the absolute changes in FFA irrespective of the effect of clofibrate on the analytical procedure. Figure 1 illustrates the FFA responses in ten subjects receiving a 15-minute infusion of epinephrine at the rate of 2pg./min. for 15 minutes. The mean peak rise in the placebo group was 1,008 ,ueq.lL. compared to 562 peq./L. in the clofibrate-treated group. The inhibition in the mean peak rise of plasma FFA was 46 per cent (p = < 0.05). When six patients were infused with epinephrine, O.l5pg./Kg./ min. for 15 minutes (Fig. 2), the mean peak rise in the placebo group was 977 peq.lL. compared to 737 peq./L. after clofibrate admin-
CLOFIBRATE
s91
EFFECT
Q CONTROL lCLOFIBRATE
I
Fig. 3.-Clofibrate effect on isoproterenol-induced changes in plasma
FFA. Lack of effect on mean FFA in six volunteers receiving dl isoproterenol, 0.03 pg./Kg./min. for 15 minutes.
15
30
45
60
MINUTES
istration which is a mean inhibition of 25 per cent (p = <0.05). Although the degree of inhibition appears to be greater in the group receiving the smaller dose of epinephrine, the degree of inhibition between the two epinephrine groups was not significantly different (p = > 0.2). Six patients were infused with isoproterenol, 0.03 pg./Kg./min. for 15 minutes (Fig. 3), and mean peak rises in plasma FFA of 497 and 469 peq.lL. in the placebo and clofibrate-treated groups respectively were observed. The 6 per cent inhibition was not significant (p = > 0.15). Figure 4 illustrates the results of seven patients infused with norepinephrine, 11.5 pg./min. for 15 minutes. The mean peak rise of 803 peq./L. in the placebo group and 726 ,ueq./L. following clofibrate was not signiEcantly different (p = > 0.1). The titrated baseline FFA values were 490 and 612 ,ueq./L. for control and periods was 150 and 141 mg./lOO ml., respectively, and is not a significant clofibrate-treated groups respectively. The mean clofibrate concentration in the latter was 174 mg./L. If a correction is made on the basis of 30 per cent clofibrate (equivalent weight = 214.7) being titrated as FFA, the corrected FFA values for the clofibrate-treated group would be 371 peq./L. The mean fasting plasma glucose values for the control and clofibrate-treated groups were 84 and 82 mg./lOO ml., respectively. The mean peak rises in plasma glucose are recorded in Table 1, and no significant differences were noted following clofibrate administration in any of the catecholamine groups. The cardiovascular responses are also listed in Table 1; no significant differences
592
HIJNNINGHAIU?
AND
AZARNOFF
oCONTROL
l CLOFlBRATE
1
Fig. I.-Clofibrate effect on norepinephrine-induced changes in plasma FFA. Lack of effect on mean FFA in seven volunteers receiving l-norepinephrine, 11.5 pg./min. for 15 minutes.
15
-200
30 MINUTES
45
t
I
Table l.-Effect
of Clojibrate Peak Rise in Plasma Glucose
Ct,api”““cl~~~te
on Glucose and Cardiooasculur Increase in Mean Heart Rate (per cent& $b ate Contml 0 r
Changes
Change in Mean B.P. (mm. Hg)t Clofibrate control
Epinephrine, S16
+16
$19
+15
-5
-3
Epinephrine, 0.15 #g./Kg./min.
+46
t41
+27
+29
$5
14
Isoproterenol, 0.03 pg./Kg./min.
+ 3
+3
+39
f45
+2
-1
2 pg./mm
*Expressed as mean peak rise from baseline values. +A mean rate for the infusion period was calculated and the values shown are per cent change from baseline values. SMean change from baseline of mean blood pressure (diastolic + l/3 pulse pressure) during catecholamine infusions.
were noted following clofibrate administration in either heart rate or mean blood pressure during the various catechofamine infusions. The mean serum cholesterol level for the control and clofibrate treatment periods was 150 and 141 mg.1100 ml., respectively, and is not a significant change (p = > 0.05). A non-signi&ant reduction of the glyceride glycerol levels following clofibrate administration was noted with mean values of 6.5 and 6.0 mg./lOO ml. for the control and clofibrate-treated series (p = >
593
CLOFIJ3RATJT EFFECC
0.05).
It should be emphasized that these are low normal values even before of clofibrate, and a lack of effect after a week does not constitute evidence for a lack of efficacy.
the administration
DISCUSSION
Clofibrate inhibits the epinephrine-induced rise in plasma FFA in humans, but no significant inhibitions of the norepinephrine or isoproterenol-induced rises in plasma FFA were demonstrated. These data agree with the findings of Duncan and Best who were unable to demonstrate inhibition in norepinephrine-induced rises in plasma FFA in man,ll and Thorp’s findings of inhibition in the plasma rise in plasma FFA in the rat and dog after epinephrine injection by clofibrate. lo The lowering of baseline FFA values after clofibrate corroborate Rifkind’s results.9 The mechanism of the inhibition of the epinephrine-induced rises of plasma FFA by clofibrate is not clear in this study. Thorp has suggested that clofibrate’s action may be mediated by a competition for anionic binding sites normally occupied by FFA, thyroxine, and other physiological endogenous substrates.lO Even though clofibrate has been demonstrated, in humans, to displace thyroxine19 and warfarin ( non-competitively)20 from their plasma protein binding sites, these observations do not appear to adequately explain our findings, Since the rises in plasma FFA produced by norepinephrine and epinephrine were similar, one would have expected inhibition in both the epinephrine and norepinephrine groups if clofibrate was competing with fatty acid for protein binding sites. It has previously been demonstrated in vitro that clofibrate does not effectively compete with fatty acids for binding sites on bovine serum albumin or rat plasma proteins.“’ Many acidic drugs utilize 1 or 2 primary binding sites on the albumin molecule,ls and may have secondary and tertiary binding sites available to them. Clofibrate was present in an average concentration of 174 pg./ml. serum or approximately 8 x 10h4 M. In vitro studies by Thorp utilizing bovine serum albumin suggest that clofibrate is about 96 per cent bound to plasma proteins at these concentrations.22 Albumin is present in concentrations of about 7 x 1P4 14, and hence, clofibrate binding is consistent with 1 or 2 sites on the albumin molecule. Although FFA are bound primarily to albumin and the presence of this acceptor is necessary for in vitro studies of FFA release following lipolysis, in vivo small amounts of FFA may also be bound to erythrocytes, low density, and high density lipoproteins, and the latter also increase their binding of FFA as plasma levels increase markedly above norma1.23 In vitro studies by Goodman suggests that 3 classes of binding sites are available for FFA transport.24 There are 2 binding sites in the first class, approximately 5 in the second, and more than 5 in the third class with decreasing specificity of the binding for each of the above classes. The affinity of the weakest group is 102-lo3 which is approximately the same as the primary affinity of most acidic drugs. Thus, with increasing FFA concentrations. many, non-specific binding sites are avail-
HUNNINGHAKE
594
AND
AZARNOFF
able and Thorp I8 states that release of FFA into the blood as a result of physiological stimuli may serve to displace protein-bound drug. The inhibition by clofibrate of epinephrine-induced rises in plasma FFA also cannot be satisfactorily explained on the basis of adrenergic blockade. The lipolysis mediated by catecholamines is reported to be a /3-adrenergic effect, with the P-adrenergic blocking agents competitively inhibiting the lipidmobilizing effect of the catecholamines. Although cu-adrenergic blocking agents also inhibit catecholamine-induced lipolysis, this effect is considered nonspecific .25 In a previous study,13 we were able to inhibit epinephrine but not norepinephrine-induced rises in plasma FFA in man with a small dose of butoxamine indicating differences in the sensitivity to various catecholamines. However, the changes in heart rate and blood pressure produced by epinephrine were also blocked by this dose of butoxamine. Dose response curves utilizing smaller doses are needed to clarify the character and specificity of clofibrate on catecholamine-induced rises in plasma FFA. When the level of FFA is corrected for the amount of clofibrate titrated as FFA, a lowering of the baseline FFA is noted. These data agree with the report of Rifkind,g except our plasma levels of clofibrate are higher. The difference is presumably due to our patients receiving the last dose of clofibrate the morning of the test so that our studies were done at peak clofibrate concentrations. This study supplies further confirmatory evidence that clofibrate has an effect on plasma FFA levels. Since our studies were done in normolipemic rather than hyperlipemic individuals, additional investigations are indicated to evaluate whether the effect on FFA is a prominent factor in the hypolipidemic effect of clofibrate. Our preliminary studies in dogs indicate similar effects to those seen in humans. ACKNOWLEDGMENTS This study was supported in part by Grants FR67, HE 07601, and 1 Tl GM 1342 from the U.S. Public Health Service. The clofibrate used in this study was generously supplied by Doctor Jerome Noble of Ayerst Laboratories. The competent technical assistance of Beverly Walker and Wonja Hahn is gratefully acknowledged.
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