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Effect of pravastatin, an HMG CoA reductase inhibitor, and cholestyramine, a bile acid sequestrant, on lipoprotein particles defined by their apolipoprotein composition
Effect of Pravastatin, an HMG CoA Reductase Inhibitor, and Cholestyramine, a Bile Acid Sequestrant, on Lipoprotein Particles Defined by Their Apolipoprotein Composition J.M.
Bard, H.J. Parra, P. Douste-Blazy,
and J.C. Fruchart
This study compares the effects of cholestyramine (16 g/d) and pravastatin (40 mg/d) on lipoprotein particles defined by their apolipoprotein composition (Lp A-l, Lp A-II:A-I, Lp E:B, and Lp C-lll:Bl. Analysis was performed after 4,B. and 12 weeks of therapy. Low-density lipoprotein (LDL) cholesterol decreased by 26.1% to 35.0% with cholestyramine and 26.2% to 30.7% with pravastatin, while triglycerides decreased slightly with pravastatin therapy and increased slightly during cholestyramine administration. The fall in cholesterol was mainly due to a decrease in very-low-density lipoprotein (VLDL) and LDL cholesterol; high-density lipoprotein (HDL) cholesterol increased. Apolipoprotein B was reduced dramatically (by 21.7% to 30.6% with cholestyramine and 27.7% to 37.4% with pravastatin). No significant effect on apolipoproteins C-III and E was observed with cholestyramine, while pravastatin reduced these parameters slightly. Apolipoprotein A-l increased during therapy with both drugs, while apolipoprotein A-II was slightly decreased. Although the drugs had nearly the same effects on plasma lipids, their influence on lipoprotein particles defined by their apolipoprotein composition was substantially different. Lp A-II:A-I was increased by both drugs (+8.1% to +41.2% for cholestyramine and +7.2% to +32.6% for pravastatin). Lp A-l was also increased with both drugs, but cholestyramine had a more constant and pronounced effect than pravastatin (+ 15.1% to +21.7% for cholestyramine and + 1.7% to + 13.0% for pravastatin). Lp E:B and Lp C-lll:B were consistently decreased by pravastatin (-10.2% to -36.5% for LP E:B and -7.2% to -20.9% for Lp C-lll:B), while cholestyramine had variable effects on these particles. The latter increased Lp E:B during the first 8 weeks of therapy (about 2%) and thereafter decreased it (-26.2%). In contrast, no significant effect on Lp C-III:B was observed with cholestyramine. These results may be related to the different actions of the drugs on lipoprotein metabolism and suggest that the lipoprotein particle profile may be used to adapt therapy to the individual. @ 1990 by W.B. Saunders Company.
I
T IS NOW CLEARLY ESTABLISHED that reduction of plasma lipoproteins may diminish the risk of atherosclerosis.‘*’ Interruption of the enterohepatic circulation by cholestyramine is widely used for this purpose and has clearly demonstrated its efficacy.’ Pravastatin is a new derivative that inhibits 3-hydroxy-3 methylglutaryl-coenzyme A (HMG CoA) reductase, the key enzyme in the cholesterol synthesis.’ Its action reduces cholesterol synthesis and concomitantly stimulates low-density lipoprotein (LDL) receptor synthesis, leading to an activation of this specific pathway for LDL catabolism.4,5 Lipoproteins are classically defined by their physical properties such as density or electrophoretic mobility. However, the development of immunological methods has clearly established that density classes represent, in fact, a mixture of particles with the same density but different apolipoprotein compositions. Thus, lipoproteins may be distinguished on the basis of their apolipoprotein composition. According to this new concept, developed by Alaupovic,6 lipoproteins may be separated into simple lipoprotein particles, containing one apolipoprotein (Lp B, Lp A-I...) and complex lipoprotein particles, containing two or more apolipoproteins (Lp B:E, Lp B:C-III, Lp B:C-IIII:E, Lp A-II:A-I...).6 We have demonstrated that all the ape A-I-containing lipoproteins may not occupy the same role in promoting reverse cholesterol transport.7.8 In addition, different ape B-containing particles do not behave in the same way in regard to the LDL receptor pathway.9 Thus, studies of lipoproteins defined by their density will not necessarily show changes in different particles with the same density but different protein cOmposition.‘O~” This study examines the effects of pravastatin and cholestyramine in primary hypercholesterolemia with a particular
Metabolism, Vol39, No 3 (March), 1990: pp 269-273
regard on lipoprotein particles defined by their apolipoprotein composition. MATERIALS AND METHODS
Study Design Patients were included in the study if the baseline values remained above 280 mg/dL and 200 mg/dL for total cholesterol and LDL cholesterol, respectively, and below 200 mg/dL for triglycerides. In addition, all subjects showed one of the following criteria: (1) existence of type IIA hyperlipoproteinemia in one first-degree or two second-degree relative(s); (2) existence of a history of coronary heart disease. before 65 years old for women and before 55 years old for men, in at least two first- or second-degree relatives; and (3) tendinous xanthoma, xanthelasma, or cornea1 arcus in patients younger than 40. Although adoption of this protocol favored selection of patients with familial hypercholesterolemia, it did not exclude completely patients with polygenic hypercholesterolemia, familial defective apo B 100, or familial combined hyperlipidemia. The principal exclusion criteria were type I, IIB, III, IV, or V hyperlipoproteinemia; hype- or hyperthyroidism; nephrotic syndrome; biliary obstruction or liver disease; chronic pancreatitis; dysproteinemia; porphyria; lupus erythematosus; diabetes mellitus; severe or unstable angina pectoris; recent myocardial infarction (~3 months); congestive heart failure; hypertension; obesity; alcoholism; malabsorp tion; treatment by corticosteroids, estrogens, androgens, hypolipidemic drugs, quinidine, coumarinic derivatives, theophylline, barbiturates, aluminium salts, laxatives, thiazidic diuretics, and beta blockers unless they were started 8 weeks before the placebo period From the Institut Pasteur, Lille, France; and the Hbpital Purpan. Toulouse, France. Address reprint requests to J.M. Bard, PhD. Institut Pasteur, I rue du Professeur Calmetie, 59019 Lille Cgdex France. CD1990 by W. B. Saunders Company. 0026-0495/90/3903-0009$03.00/0
269
270
BARD ET AL
and their dose was maintained constant during the treatment, other experimental drugs; other illness which may lead to death within 3 years. Probucol, benfluorex, and nicotinic acid had to be stopped for a longer time before the placebo period, 6 months for the first and 3 months for the latter two. After 4 weeks off all lipid-lowering drugs and following administration of a standard lipid-lowering diet, blood was drawn for laboratory measurements (W-2). Although this was an open study, patients received a placebo (two pills at night) for 2 additional weeks with the same regimen, in order to test the compliance. After these 2 weeks, blood was drawn again for laboratory measurements (WO). Baseline values were taken to be the mean between the results obtained at W-2 and WO. At WO, 67 patients (41 women, 26 men), aged 19 to 70 years, were randomized into two groups, one receiving pravastatin, two capsules of 20 mg at night, and the second receiving cholestyramine, 8 g before breakfast and 8 g before supper, with a delay of 30 to 45 minutes between the drug and the meal. The therapy was continued for 12 weeks and blood was drawn for analysis at 4 weeks (W4), 8 weeks (W8) and 12 weeks (W12) of treatment. Adherence to therapy was monitored by capsule and packet count. Diet observance was monitored by inquiry at W-2, W4, and W12. In addition, patients were advised to avoid any change in their life style during the study, and in particular to avoid any new sport activity. Body weight was also checked at each clinical visit. Lipid, Lipoprotein Lipid, and Apolipoprotein Analysis Cholesterol and triglycerides were measured in total serum by enzymatic methods (Boehringer, Mannheim, FRG), adapted to a HITACHI 705 analyzer. Cholesterol was measured in very-lowdensity lipoproteins (VLDL) separated by ultracentrifugation” and in the HDL-containing supernatant after sodium phosphotungstate/ magnesium chloride precipitation (Boehringer). LDL cholesterol was estimated from the following formula: LDL value = serum value - (VLDL value + HDL value). Apolipoproteins AI and B were quantified by laser-immunonephelometry (Behring, Marburg, FRG). Within and between coefficients of variations were below 3% and 5%, respectively. Apolipoproteins AH, C-III, and E were measured by immunoenzymometric assays as previously described, with within and between assay coefficients of variations of less than 5% and 7%. respectively.” Lipoprotein particles containing apo A-II and A-I (Lp A-II:A-I), apo B and E (Lp E:B) and apo B and C-III (Lp C-1II:B) were measured by two-site immunoenzymatic assays as described elsewhere.“‘,‘4 Within and between coefficients of variations were below 6% and lo%, respectively. Particles containing apo A-I but Table 1. Changes in Lipids and Lipoprotein
free of apo A-II were quantified by differential electroimmunoassay on ready-to-use plates marketed by Sebia (Issy les Moulineaux, France),15 with within and between assay coefficients of variations of leas than 3% and 5%, respectively. Statistical Analysis Statistical significance was evaluated by the Wilcoxon paired test (intragroup comparison) and the Mann and Whitney test (intergroup comparison). RESULTS
Since it is known that compliance is difficult to obtain with cholestyramine and the study was designed to assess the efficacy of pravastatin, the randomization was undertaken in order to obtain about two thirds of the patients in the pravastatin group. Thus, 40 subjects (20 women and 20 men, aged 19 to 70 years) were assigned to pravastatin therapy and 27 subjects (21 women and 6 men, aged 16 to 66 years) to cholestyramine therapy. Baseline comparison of the two groups by the Mann and Whitney test showed a significant difference only for apo B (P < .Ol). Capsule and packet count did not show any problems with the adherence to either pravastatin or resin therapy. Body weight was stable throughout the study. Although inquiries revealed good adherence to diet, we cannot completely exclude some alterations in the diet consumed for a short period of time, which could possibly lead to slight modifications in the tested variables, independent of the drug activity. Table 1 shows the changes observed in lipids and lipoprotein lipids. Cholesterol was significantly reduced by 20.6% to 24.3% by pravastatin and by 21.1% to 25.2% by cholestyramine. Triglycerides were reduced by pravastatin, although the difference was statistically significant only at W12 (- 11.8%). In contrast, the increase in this parameter during cholestyramine therapy was not significant. VLDL cholesterol was constantly and significantly reduced by pravastatin (- 17.6 to -26.3%), while cholestyramine resulted in a significant decrease in this parameter only at W 12 (- 18.1%) following slight and insignificant increases at W4 and W8. LDL cholesterol decreased significantly during treatment with the two drugs (-26.2% to -30.7% for pravastatin and -25.1% to -35.0% for cholestyramine).
Lipids on Cholestyramine
or Pravastatin
Chdestyramine BaSS Cholesterol
309
Triglycerides
(48) 100
Prawstatin
W4
WS
w12
-25.2
-21.1
-21.1
l
l
l
+9.7
+ 10.5
+7.7
+6.6
+s.9
(26) VLDL cholesterol
29
LDL cholesterol
(10) 238
HDL cholesterol
(53) 44 (13)
Be80 336 168) 100 (39)
-35.0 . +4.3
-31.0 l
+20.2 $
Therapy
-18.1
33
5 -25.1
263
§ + 10.9
(12) (65) 42
w4
WB
w12
-23.1
-20.6
-24.3
l
l
-4.6
-3.9
-11.8
-20.0
tS -26.3
t - 17.6 $ -27.0 . +8.9
l
-26.2 l
+22.8
(10)
NOTE. Results are in milligrams per deciliter and SD in parentheses at baseline. Percent change given for subsequent visits. Wilcoxon test, treated versus base: lP < .OOl: $P < .Ol; §P -c .05. Mann and Whitney test, cholestyramine versus pravastatin: tP < .05.
l
l
l
- 30.7 l
+22.2 l
PRAVASTATIN, C~~OLES~RAMINE. AND PARTICLES
271
either of the drugs, although it was significantly decreased at W12 for both (-20.2% for pravastatin and -28.5% for cholestyramine).
HDL cholesterol increased significantly during the WS and W12 of pravastatin phase (+22.8% and +22.2%, respectively) and at W8 of cholestyramine (+20.2%). The results obtained on apolipoproteins are presented in Table 2. Apolipoprotein AI was slightly increased by both therapies, but these variations were statistically significant only at W12 for pravastatin and W8 for cholestyramine. A slight reduction of apo AI1 was observed with both drugs. However, this effect was statistically significant only at W4 in both groups. A significant decrease in apo B was observed with both drugs. Pravastatin decreased this parameter by 27.7% to 37.4% and cholestyramine by 21.7% to 30.5%. Apolipoprotein C-III was slightly but nonsignificantly increased by choiestyramine treatment, while pravastatin reduced this apolipoprotein significantly after 4 weeks of therapy. This parameter responded differently to both therapies at W4, according to the Mann and Whitney test. Cholestyramine had inconstant and insignificant effects on apo E, while pravastatin decreased this parameter significantly (-3.7% to -15.6%). The effects of both treatments on lipoprotein particles are presented in Table 3. Lp A-II:A-I was increased by both therapies. This effect was significant at W12 for cholestyramine (+41.2%) and WS and W12 for pravastatin (+24.9% and + 32.6%). Cholestyramine increased Lp A-I significantly (+ 15.1% to +21.7%) at each sampling time, while the incremental action of pravastatin was significant only at W8 (+ 13.0%). However, comparison of the changes in Lp A-I with both therapies by the Mann and Whitney test showed a significant difference between the two groups at W4. Study of the two apo B-containing particles, Lp E:B and Lp C-III:B, showed important differences between the two drugs. Cholestyramine had variable effects on Lp E:B (+2.3% [NS] at W4, +2.1% [P < .05] at W8, and -26.2% [P < .OS] at W 12), while pravastatin decreased this parameter very significantly at each sampling time (by 10.2% to 36.5%). Lp C-III:B was slightly but insignificantly increased by cholestyramine, while pravastatin decreased this parameter significantly (-7.2% to -20.9%). The Mann and Whitney test showed a significant difference between the effects of the two drugs on this parameter at W4 (P < .05). The ratio Lp E:B/Lp C-1II:B did not change at W4 and W8 with Table 2. Changes in Apolipoproteins
DISCUSSION
Both cholestyramine and pravastatin were effective in reducing cholesterol and LDL cholesterol. The effect on these two parameters may be explained by the depletion of cellular cholesterol that activates the LDL receptor specific pathway.4*5 However, pravastatin may also inhibit production of LDL, as has already been shown for an other HMG CoA reductase inhibitor.16 Direct inhibition of cholesterol synthesis by pravastatin may lead to less VLDL secretion from the liver. This may explain the effect of this drug on triglycerides and VLDL cholesterol. In contrast, it is now well known that cholestyramine stimulates triglyceride synthesis.” However, the drug may consequently enhance VLDL catabolism.” These phenomena may explain its tendency to increased triglyceride and VLDL cholesterol. It is most probable that the decrease in VLDL cholesterol at W 12 of cholestyramine reflects enhanced VLDL catabolism following activation of synthesis. This phenomenon may prelude a complete triglyceride normalisation. HDL cholesterol values were increased by both therapies. This may potentiate the beneficial cardiovascular effect of LDL cholesterol reduction.* Such an action is consistent with the results of other studies.‘8.‘9 It should be kept in mind that the response in HDL is extremely variable from study to study. Such variability may be due either to different methodologies used for HDL cholesterol determination or to variations in patient recruitment. However, the discrete increase in apo A-I, associated with a slight decrease in apo A-II, does not argue in favor of an increase in HDL particle number. The results are more consistent with structural modifications in HDL and show some differences between the two drugs tested. Cholestyramine increased significantly the plasma mass of particles containing apo A-I but free of apo A-II (Lp A-I), and had less constant effect on Lp A-II:A-I, ie, on particles containing both apolipoproteins. Pravastatin had a less pronounced incremental action on Lp A-I and seems to be more potent in raising Lp A-II:A-I. Since it has been suggested that Lp A-I may represent the on Cholestyramine
or Pravastatin
Therapy Pravastatin
Cholestyramine BaSe
w4
WB
w12
Apo A-l
120
+3.3
+8.9
-12.7
116
Apo A-II
(21) 34
Apo B
173
(8) (30) Apo C-III Apo E
l
BasS
-13.6
+11.0
-3.5
(19) 36
t -21.7
-22.5
-30.5
(9) 199
5.4
$ +2.7
$ +4.3
$ -29.5
(1.81 6.8
-0.4
+9.7
- 13.0
(2.3)
(44)§
w4
W8
w12
+3.5
+5.9
+4.5
- 10.0 .
+1.8
-2.0
l
-27.7
-28.8
6.1
$ +6.6
$ +16.6
(2.7) 7.6
tll -15.6
-3.7
-12.0
(10)
$
NOTE. Results are in milligrams per deciliter and SD in parentheses at baseline. Percent change given for subsequent visits. Wilcoxon test, treated versus base: lP -z .05; tP < .Ol; $P < .OOl. Mann and Whitney test, cholestyramine versus pravastatin: BP i .Ol; 11P < .05.
-37.4
$ -13.4
t
272
BARD ET AL
Table 3. Changes in Lipoprotein Particles on Cholastyramine or Pravartatin Therapy Chdestyramine Base Lp A-II:A-I
93
w4
+B.l
Pravastatin W8
+ll.B
(24) Lp A-l
45 (10)
Lp E:B
52
Lp E:B/Lp C-lll:B
24 (11) 2.27
+41.2 l
f21.4 $ +2.3
(31) Lp C-lll:B
w12
-0.8
+21.7 .
f15.1
+2.1
t -26.2
t + 19.2
t -12.7
-4.3
-28.5
Base
96
(2.3)
l
W8
wt2
i7.2
+24.9
+32.6
(29) 43 (9) 56 (44) 27 (12)
+13.4
w4
1.99
t +1.7
f13.0
t +4.8
l
0 - 19.6
- 10.2
$ - 20.9
t -7.2
$ - 14.8
-3.0
-20.2
l
II
- 1.4
-36.5
t
(1.3)
$
NOTE. Results are in milligrams per deciliter and SD in parentheses at baseline. Percent change given for subsequent visits. Wilcoxon test, treated versus base: lP < .Ol; tP < .05; $P < ,001. Mann and Whitney test, cholestyramine versus pravastatin: $P < .Ol; IIP -c .05. “antiatherogenic” apo A-Ixontaining particle,‘.8 one may speculate that the action of pravastatin on HDL may be of less substantial benefit. Metabolic studies need to be done to determine if the increase in plasma Lp A-I is due to overproduction or underutilization. Since it has been suggested that 1ecithin:cholesterol:acyltransferase ( LCAT) may determine the composition of HDL in men,*’ further basic studies should be performed to determine if these variations in apo A-I-containing particles are related in changes in LCAT mass and/or activity. The decrease in apo B may relate to the fall in LDL cholesterol obtained with both therapies. The significant decrease in apo E on pravastatin therapy could be related to a reduction in intermediate density lipoproteins due to the activation of the receptor-specific pathway. Apolipoprotein C-III fell significantly after 4 weeks of pravastatin therapy. This may be related to the effect of the drug on VLDL synthesis. The lack of effect of pravastatin on this parameter after 4 weeks of therapy remains unclear. However, it should be pointed out that apo C-III comes back to baseline values concomitantly with a major increase in HDL cholesterol. This may suggest that the apo C-III remains associated with lipoprotein particles from the HDL density range. In contrast with the results obtained with pravastatin, cholestyramine did not lead to any change in apo C-III and E. Nevertheless, although the effect was not statistically significant, apo C-III increased slightly. Activation of the apo B, E receptor could lead to a decrease in ape E. However, the apo E receptor, whose existence has been suggested earlier, is apparently refractory to cholestyramine treatment.” Moreover, activation of VLDL synthesis should lead to an increase in both apo C-III and apo E. The combination of these phenomena probably explains the absence of effect of cholestyramine on these apolipoproteins. Apolipoprotein B-containing particles may be divided into Lp B:E, containing apo B and apo E, Lp B:C-III, containing apo C-III and apo B, and Lp B:C-III:E, containing all these three apolipoproteins. It should be kept in mind that our methodology measures the pool of particles containing both apo E and apo B (Lp E:B) or both apo C-III and apo B (Lp C-1II:B). Activation of the receptor-specific pathway as a result of depletion of cellular cholesterol may lead to an accelerated clearance of ape B-containing particles and
true
explains probably the decrease in Lp E:B and Lp C-1II:B. Moreover, at least for pravastatin, this phenomenon could be potentiated by inhibition of direct synthesis of these particles, due to reduction in the cholesterol synthesis. However, the results obtained with cholestyramine reflect the complexity mechanism of action of the drug. Since Lp E:B and Lp C-1II:B levels are highly correlated with triglycerides at the baseline (data not shown), activation of triglyceride synthesis may lead to increased values for these parameters. In the first period of time, the accelerated clearance of these complex particles is balanced by their increased synthesis related to the activation of triglyceride synthesis. This probably explains the increase in Lp C-1II:B and Lp E:B at W4 and W8. In the second time period, activation of the catabolism of these apo B-containing particles supplants the activation of synthesis and results in a decrease in Lp E:B. As suggested by the decrease in Lp E:B/Lp C-1II:B ratio at W 12, both drugs could be more effective in reducing Lp E:B than Lp C-1II:B. This could reflect a better affinity of Lp B:E for the receptor.’ Finally, a correlation between the baseline level of Lp E:B and the mean triglyceride reduction (r = .31, P -c.05, data not shown) is found in the pravastatin group, but not in the cholestyramine group. This means that, on pravastatin therapy, the higher the level of Lp E:B, the greater is the triglyceride reduction. Apparently, clearance of Lp E:B particles through the receptor pathway leads to triglyceride reduction. Thus, the baseline level of Lp E:B may help to understand the variability in response to pravastatin therapy. CONCLUSION
This study demonstrates that the apolipoprotein composition of lipoprotein particles may be important in studying lipoprotein metabolism in clinical pharmacology. The results presented here show that the two drugs have similar effects on the classic lipid parameters, while the different lipoprotein particles change in different ways. Further investigation are necessary in this field to determine how these parameters may be used to adapt therapy to the individual. ACKNOWLEDGMENT The authors thank Professor J. Shepherd help in preparation of the manuscript.
(Glasgow,
Scotland)
for
PRAVASTATIN,
CHOLESTYRAMINE,
273
AND PARTICLES
REFERENCES
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11. Alaupovic P, McConathy WJ, Fesmire J, et al: Profiles of apolipoproteins and apolipoprotein B containing lipoprotein particles indyslipoproteinemias. Clin. Chem. 34/8B:B13-B27,1988 12. Alaupovic P, Lee DM, McConathy WJ: Studies of the composition and structure of plasma lipoproteins. Distribution of lipoprotein families in major density classes of normal human plasma lipoproteins. B&hem Biophys Acta 260:689-707,1972 13. Fruchart JC, Fievet C, Puchois P: Ape-lipoproteins. Metabolite-s III: Lipids, amino acids and related compounds, in Bergmeyer HU (ed): Methods of Enzymatic Analysis. New York, NY, VCH, 1985, pp 126-138 14. Fruchart JC, Kandoussi A, Parsy D, et al: Measurement of lipoprotein particles defined by their apolipoprotein composition using immunosorbent assay. Annual Meeting on Clinical Chemistry, Mannheim, September 25-28, 1985. J Clin Chem Clin Biochem 23:619, 1985 (abstr) 15. Parra HJ, Mezdour H, Ghalim N, et al: Differential electroimmunoassay on ready-to-use plates for human Lp A-I lipoprotein particles. Clin Chem (in press) 16. Huff MW, Telford DE, Woodkrofdt K, et al: Mevinolin and cholestyramine inhibit the direct synthesis of low density lipoprotein apoprotein B in miniature pigs. J Lipid Res 26: 1175- 1186, 1985 17. Angelin B, Einarsson K, Hellstrom K, et al: Effects of cholestyramine and chenodeoxycholic acid on the metabolism of endogenous triglyceride in hyperlipoproteinemia. J Lipid Res 19: 1017-1024,1978 18. Mabuchi H, Kamon N, Fujita H, et al: Effects of CS-514 on serum lipoprotein lipid and apolipoprotein levels in patients with familial hypercholesterolemia. Metabolism 36:475-479, 1987 19. Mabuchi H, Sakai T, Sakai Y, et al: Reduction of serum cholesterol in heterozygous patients with familial hypercholesterolemia. N Engl J Med 308:609-613, 1983 20. Miller NE, Rajput-Williams J, Nanjee MN, et al: Relationship of high density lipoprotein composition to plasma lecithin: Cholesterol acyltransferase concentration in men. Atherosclerosis 69:123-129,1988 21. Hui DY, Innerarity TL, Mahley RW: Lipoprotein binding to canine hepatic membranes. J Biol Chem 256:5646-5655, 198 1
Bard, H.J. Parra, P. Douste-Blazy,
and J.C. Fruchart
This study compares the effects of cholestyramine (16 g/d) and pravastatin (40 mg/d) on lipoprotein particles defined by their apolipoprotein composition (Lp A-l, Lp A-II:A-I, Lp E:B, and Lp C-lll:Bl. Analysis was performed after 4,B. and 12 weeks of therapy. Low-density lipoprotein (LDL) cholesterol decreased by 26.1% to 35.0% with cholestyramine and 26.2% to 30.7% with pravastatin, while triglycerides decreased slightly with pravastatin therapy and increased slightly during cholestyramine administration. The fall in cholesterol was mainly due to a decrease in very-low-density lipoprotein (VLDL) and LDL cholesterol; high-density lipoprotein (HDL) cholesterol increased. Apolipoprotein B was reduced dramatically (by 21.7% to 30.6% with cholestyramine and 27.7% to 37.4% with pravastatin). No significant effect on apolipoproteins C-III and E was observed with cholestyramine, while pravastatin reduced these parameters slightly. Apolipoprotein A-l increased during therapy with both drugs, while apolipoprotein A-II was slightly decreased. Although the drugs had nearly the same effects on plasma lipids, their influence on lipoprotein particles defined by their apolipoprotein composition was substantially different. Lp A-II:A-I was increased by both drugs (+8.1% to +41.2% for cholestyramine and +7.2% to +32.6% for pravastatin). Lp A-l was also increased with both drugs, but cholestyramine had a more constant and pronounced effect than pravastatin (+ 15.1% to +21.7% for cholestyramine and + 1.7% to + 13.0% for pravastatin). Lp E:B and Lp C-lll:B were consistently decreased by pravastatin (-10.2% to -36.5% for LP E:B and -7.2% to -20.9% for Lp C-lll:B), while cholestyramine had variable effects on these particles. The latter increased Lp E:B during the first 8 weeks of therapy (about 2%) and thereafter decreased it (-26.2%). In contrast, no significant effect on Lp C-III:B was observed with cholestyramine. These results may be related to the different actions of the drugs on lipoprotein metabolism and suggest that the lipoprotein particle profile may be used to adapt therapy to the individual. @ 1990 by W.B. Saunders Company.
I
T IS NOW CLEARLY ESTABLISHED that reduction of plasma lipoproteins may diminish the risk of atherosclerosis.‘*’ Interruption of the enterohepatic circulation by cholestyramine is widely used for this purpose and has clearly demonstrated its efficacy.’ Pravastatin is a new derivative that inhibits 3-hydroxy-3 methylglutaryl-coenzyme A (HMG CoA) reductase, the key enzyme in the cholesterol synthesis.’ Its action reduces cholesterol synthesis and concomitantly stimulates low-density lipoprotein (LDL) receptor synthesis, leading to an activation of this specific pathway for LDL catabolism.4,5 Lipoproteins are classically defined by their physical properties such as density or electrophoretic mobility. However, the development of immunological methods has clearly established that density classes represent, in fact, a mixture of particles with the same density but different apolipoprotein compositions. Thus, lipoproteins may be distinguished on the basis of their apolipoprotein composition. According to this new concept, developed by Alaupovic,6 lipoproteins may be separated into simple lipoprotein particles, containing one apolipoprotein (Lp B, Lp A-I...) and complex lipoprotein particles, containing two or more apolipoproteins (Lp B:E, Lp B:C-III, Lp B:C-IIII:E, Lp A-II:A-I...).6 We have demonstrated that all the ape A-I-containing lipoproteins may not occupy the same role in promoting reverse cholesterol transport.7.8 In addition, different ape B-containing particles do not behave in the same way in regard to the LDL receptor pathway.9 Thus, studies of lipoproteins defined by their density will not necessarily show changes in different particles with the same density but different protein cOmposition.‘O~” This study examines the effects of pravastatin and cholestyramine in primary hypercholesterolemia with a particular
Metabolism, Vol39, No 3 (March), 1990: pp 269-273
regard on lipoprotein particles defined by their apolipoprotein composition. MATERIALS AND METHODS
Study Design Patients were included in the study if the baseline values remained above 280 mg/dL and 200 mg/dL for total cholesterol and LDL cholesterol, respectively, and below 200 mg/dL for triglycerides. In addition, all subjects showed one of the following criteria: (1) existence of type IIA hyperlipoproteinemia in one first-degree or two second-degree relative(s); (2) existence of a history of coronary heart disease. before 65 years old for women and before 55 years old for men, in at least two first- or second-degree relatives; and (3) tendinous xanthoma, xanthelasma, or cornea1 arcus in patients younger than 40. Although adoption of this protocol favored selection of patients with familial hypercholesterolemia, it did not exclude completely patients with polygenic hypercholesterolemia, familial defective apo B 100, or familial combined hyperlipidemia. The principal exclusion criteria were type I, IIB, III, IV, or V hyperlipoproteinemia; hype- or hyperthyroidism; nephrotic syndrome; biliary obstruction or liver disease; chronic pancreatitis; dysproteinemia; porphyria; lupus erythematosus; diabetes mellitus; severe or unstable angina pectoris; recent myocardial infarction (~3 months); congestive heart failure; hypertension; obesity; alcoholism; malabsorp tion; treatment by corticosteroids, estrogens, androgens, hypolipidemic drugs, quinidine, coumarinic derivatives, theophylline, barbiturates, aluminium salts, laxatives, thiazidic diuretics, and beta blockers unless they were started 8 weeks before the placebo period From the Institut Pasteur, Lille, France; and the Hbpital Purpan. Toulouse, France. Address reprint requests to J.M. Bard, PhD. Institut Pasteur, I rue du Professeur Calmetie, 59019 Lille Cgdex France. CD1990 by W. B. Saunders Company. 0026-0495/90/3903-0009$03.00/0
269
270
BARD ET AL
and their dose was maintained constant during the treatment, other experimental drugs; other illness which may lead to death within 3 years. Probucol, benfluorex, and nicotinic acid had to be stopped for a longer time before the placebo period, 6 months for the first and 3 months for the latter two. After 4 weeks off all lipid-lowering drugs and following administration of a standard lipid-lowering diet, blood was drawn for laboratory measurements (W-2). Although this was an open study, patients received a placebo (two pills at night) for 2 additional weeks with the same regimen, in order to test the compliance. After these 2 weeks, blood was drawn again for laboratory measurements (WO). Baseline values were taken to be the mean between the results obtained at W-2 and WO. At WO, 67 patients (41 women, 26 men), aged 19 to 70 years, were randomized into two groups, one receiving pravastatin, two capsules of 20 mg at night, and the second receiving cholestyramine, 8 g before breakfast and 8 g before supper, with a delay of 30 to 45 minutes between the drug and the meal. The therapy was continued for 12 weeks and blood was drawn for analysis at 4 weeks (W4), 8 weeks (W8) and 12 weeks (W12) of treatment. Adherence to therapy was monitored by capsule and packet count. Diet observance was monitored by inquiry at W-2, W4, and W12. In addition, patients were advised to avoid any change in their life style during the study, and in particular to avoid any new sport activity. Body weight was also checked at each clinical visit. Lipid, Lipoprotein Lipid, and Apolipoprotein Analysis Cholesterol and triglycerides were measured in total serum by enzymatic methods (Boehringer, Mannheim, FRG), adapted to a HITACHI 705 analyzer. Cholesterol was measured in very-lowdensity lipoproteins (VLDL) separated by ultracentrifugation” and in the HDL-containing supernatant after sodium phosphotungstate/ magnesium chloride precipitation (Boehringer). LDL cholesterol was estimated from the following formula: LDL value = serum value - (VLDL value + HDL value). Apolipoproteins AI and B were quantified by laser-immunonephelometry (Behring, Marburg, FRG). Within and between coefficients of variations were below 3% and 5%, respectively. Apolipoproteins AH, C-III, and E were measured by immunoenzymometric assays as previously described, with within and between assay coefficients of variations of less than 5% and 7%. respectively.” Lipoprotein particles containing apo A-II and A-I (Lp A-II:A-I), apo B and E (Lp E:B) and apo B and C-III (Lp C-1II:B) were measured by two-site immunoenzymatic assays as described elsewhere.“‘,‘4 Within and between coefficients of variations were below 6% and lo%, respectively. Particles containing apo A-I but Table 1. Changes in Lipids and Lipoprotein
free of apo A-II were quantified by differential electroimmunoassay on ready-to-use plates marketed by Sebia (Issy les Moulineaux, France),15 with within and between assay coefficients of variations of leas than 3% and 5%, respectively. Statistical Analysis Statistical significance was evaluated by the Wilcoxon paired test (intragroup comparison) and the Mann and Whitney test (intergroup comparison). RESULTS
Since it is known that compliance is difficult to obtain with cholestyramine and the study was designed to assess the efficacy of pravastatin, the randomization was undertaken in order to obtain about two thirds of the patients in the pravastatin group. Thus, 40 subjects (20 women and 20 men, aged 19 to 70 years) were assigned to pravastatin therapy and 27 subjects (21 women and 6 men, aged 16 to 66 years) to cholestyramine therapy. Baseline comparison of the two groups by the Mann and Whitney test showed a significant difference only for apo B (P < .Ol). Capsule and packet count did not show any problems with the adherence to either pravastatin or resin therapy. Body weight was stable throughout the study. Although inquiries revealed good adherence to diet, we cannot completely exclude some alterations in the diet consumed for a short period of time, which could possibly lead to slight modifications in the tested variables, independent of the drug activity. Table 1 shows the changes observed in lipids and lipoprotein lipids. Cholesterol was significantly reduced by 20.6% to 24.3% by pravastatin and by 21.1% to 25.2% by cholestyramine. Triglycerides were reduced by pravastatin, although the difference was statistically significant only at W12 (- 11.8%). In contrast, the increase in this parameter during cholestyramine therapy was not significant. VLDL cholesterol was constantly and significantly reduced by pravastatin (- 17.6 to -26.3%), while cholestyramine resulted in a significant decrease in this parameter only at W 12 (- 18.1%) following slight and insignificant increases at W4 and W8. LDL cholesterol decreased significantly during treatment with the two drugs (-26.2% to -30.7% for pravastatin and -25.1% to -35.0% for cholestyramine).
Lipids on Cholestyramine
or Pravastatin
Chdestyramine BaSS Cholesterol
309
Triglycerides
(48) 100
Prawstatin
W4
WS
w12
-25.2
-21.1
-21.1
l
l
l
+9.7
+ 10.5
+7.7
+6.6
+s.9
(26) VLDL cholesterol
29
LDL cholesterol
(10) 238
HDL cholesterol
(53) 44 (13)
Be80 336 168) 100 (39)
-35.0 . +4.3
-31.0 l
+20.2 $
Therapy
-18.1
33
5 -25.1
263
§ + 10.9
(12) (65) 42
w4
WB
w12
-23.1
-20.6
-24.3
l
l
-4.6
-3.9
-11.8
-20.0
tS -26.3
t - 17.6 $ -27.0 . +8.9
l
-26.2 l
+22.8
(10)
NOTE. Results are in milligrams per deciliter and SD in parentheses at baseline. Percent change given for subsequent visits. Wilcoxon test, treated versus base: lP < .OOl: $P < .Ol; §P -c .05. Mann and Whitney test, cholestyramine versus pravastatin: tP < .05.
l
l
l
- 30.7 l
+22.2 l
PRAVASTATIN, C~~OLES~RAMINE. AND PARTICLES
271
either of the drugs, although it was significantly decreased at W12 for both (-20.2% for pravastatin and -28.5% for cholestyramine).
HDL cholesterol increased significantly during the WS and W12 of pravastatin phase (+22.8% and +22.2%, respectively) and at W8 of cholestyramine (+20.2%). The results obtained on apolipoproteins are presented in Table 2. Apolipoprotein AI was slightly increased by both therapies, but these variations were statistically significant only at W12 for pravastatin and W8 for cholestyramine. A slight reduction of apo AI1 was observed with both drugs. However, this effect was statistically significant only at W4 in both groups. A significant decrease in apo B was observed with both drugs. Pravastatin decreased this parameter by 27.7% to 37.4% and cholestyramine by 21.7% to 30.5%. Apolipoprotein C-III was slightly but nonsignificantly increased by choiestyramine treatment, while pravastatin reduced this apolipoprotein significantly after 4 weeks of therapy. This parameter responded differently to both therapies at W4, according to the Mann and Whitney test. Cholestyramine had inconstant and insignificant effects on apo E, while pravastatin decreased this parameter significantly (-3.7% to -15.6%). The effects of both treatments on lipoprotein particles are presented in Table 3. Lp A-II:A-I was increased by both therapies. This effect was significant at W12 for cholestyramine (+41.2%) and WS and W12 for pravastatin (+24.9% and + 32.6%). Cholestyramine increased Lp A-I significantly (+ 15.1% to +21.7%) at each sampling time, while the incremental action of pravastatin was significant only at W8 (+ 13.0%). However, comparison of the changes in Lp A-I with both therapies by the Mann and Whitney test showed a significant difference between the two groups at W4. Study of the two apo B-containing particles, Lp E:B and Lp C-III:B, showed important differences between the two drugs. Cholestyramine had variable effects on Lp E:B (+2.3% [NS] at W4, +2.1% [P < .05] at W8, and -26.2% [P < .OS] at W 12), while pravastatin decreased this parameter very significantly at each sampling time (by 10.2% to 36.5%). Lp C-III:B was slightly but insignificantly increased by cholestyramine, while pravastatin decreased this parameter significantly (-7.2% to -20.9%). The Mann and Whitney test showed a significant difference between the effects of the two drugs on this parameter at W4 (P < .05). The ratio Lp E:B/Lp C-1II:B did not change at W4 and W8 with Table 2. Changes in Apolipoproteins
DISCUSSION
Both cholestyramine and pravastatin were effective in reducing cholesterol and LDL cholesterol. The effect on these two parameters may be explained by the depletion of cellular cholesterol that activates the LDL receptor specific pathway.4*5 However, pravastatin may also inhibit production of LDL, as has already been shown for an other HMG CoA reductase inhibitor.16 Direct inhibition of cholesterol synthesis by pravastatin may lead to less VLDL secretion from the liver. This may explain the effect of this drug on triglycerides and VLDL cholesterol. In contrast, it is now well known that cholestyramine stimulates triglyceride synthesis.” However, the drug may consequently enhance VLDL catabolism.” These phenomena may explain its tendency to increased triglyceride and VLDL cholesterol. It is most probable that the decrease in VLDL cholesterol at W 12 of cholestyramine reflects enhanced VLDL catabolism following activation of synthesis. This phenomenon may prelude a complete triglyceride normalisation. HDL cholesterol values were increased by both therapies. This may potentiate the beneficial cardiovascular effect of LDL cholesterol reduction.* Such an action is consistent with the results of other studies.‘8.‘9 It should be kept in mind that the response in HDL is extremely variable from study to study. Such variability may be due either to different methodologies used for HDL cholesterol determination or to variations in patient recruitment. However, the discrete increase in apo A-I, associated with a slight decrease in apo A-II, does not argue in favor of an increase in HDL particle number. The results are more consistent with structural modifications in HDL and show some differences between the two drugs tested. Cholestyramine increased significantly the plasma mass of particles containing apo A-I but free of apo A-II (Lp A-I), and had less constant effect on Lp A-II:A-I, ie, on particles containing both apolipoproteins. Pravastatin had a less pronounced incremental action on Lp A-I and seems to be more potent in raising Lp A-II:A-I. Since it has been suggested that Lp A-I may represent the on Cholestyramine
or Pravastatin
Therapy Pravastatin
Cholestyramine BaSe
w4
WB
w12
Apo A-l
120
+3.3
+8.9
-12.7
116
Apo A-II
(21) 34
Apo B
173
(8) (30) Apo C-III Apo E
l
BasS
-13.6
+11.0
-3.5
(19) 36
t -21.7
-22.5
-30.5
(9) 199
5.4
$ +2.7
$ +4.3
$ -29.5
(1.81 6.8
-0.4
+9.7
- 13.0
(2.3)
(44)§
w4
W8
w12
+3.5
+5.9
+4.5
- 10.0 .
+1.8
-2.0
l
-27.7
-28.8
6.1
$ +6.6
$ +16.6
(2.7) 7.6
tll -15.6
-3.7
-12.0
(10)
$
NOTE. Results are in milligrams per deciliter and SD in parentheses at baseline. Percent change given for subsequent visits. Wilcoxon test, treated versus base: lP -z .05; tP < .Ol; $P < .OOl. Mann and Whitney test, cholestyramine versus pravastatin: BP i .Ol; 11P < .05.
-37.4
$ -13.4
t
272
BARD ET AL
Table 3. Changes in Lipoprotein Particles on Cholastyramine or Pravartatin Therapy Chdestyramine Base Lp A-II:A-I
93
w4
+B.l
Pravastatin W8
+ll.B
(24) Lp A-l
45 (10)
Lp E:B
52
Lp E:B/Lp C-lll:B
24 (11) 2.27
+41.2 l
f21.4 $ +2.3
(31) Lp C-lll:B
w12
-0.8
+21.7 .
f15.1
+2.1
t -26.2
t + 19.2
t -12.7
-4.3
-28.5
Base
96
(2.3)
l
W8
wt2
i7.2
+24.9
+32.6
(29) 43 (9) 56 (44) 27 (12)
+13.4
w4
1.99
t +1.7
f13.0
t +4.8
l
0 - 19.6
- 10.2
$ - 20.9
t -7.2
$ - 14.8
-3.0
-20.2
l
II
- 1.4
-36.5
t
(1.3)
$
NOTE. Results are in milligrams per deciliter and SD in parentheses at baseline. Percent change given for subsequent visits. Wilcoxon test, treated versus base: lP < .Ol; tP < .05; $P < ,001. Mann and Whitney test, cholestyramine versus pravastatin: $P < .Ol; IIP -c .05. “antiatherogenic” apo A-Ixontaining particle,‘.8 one may speculate that the action of pravastatin on HDL may be of less substantial benefit. Metabolic studies need to be done to determine if the increase in plasma Lp A-I is due to overproduction or underutilization. Since it has been suggested that 1ecithin:cholesterol:acyltransferase ( LCAT) may determine the composition of HDL in men,*’ further basic studies should be performed to determine if these variations in apo A-I-containing particles are related in changes in LCAT mass and/or activity. The decrease in apo B may relate to the fall in LDL cholesterol obtained with both therapies. The significant decrease in apo E on pravastatin therapy could be related to a reduction in intermediate density lipoproteins due to the activation of the receptor-specific pathway. Apolipoprotein C-III fell significantly after 4 weeks of pravastatin therapy. This may be related to the effect of the drug on VLDL synthesis. The lack of effect of pravastatin on this parameter after 4 weeks of therapy remains unclear. However, it should be pointed out that apo C-III comes back to baseline values concomitantly with a major increase in HDL cholesterol. This may suggest that the apo C-III remains associated with lipoprotein particles from the HDL density range. In contrast with the results obtained with pravastatin, cholestyramine did not lead to any change in apo C-III and E. Nevertheless, although the effect was not statistically significant, apo C-III increased slightly. Activation of the apo B, E receptor could lead to a decrease in ape E. However, the apo E receptor, whose existence has been suggested earlier, is apparently refractory to cholestyramine treatment.” Moreover, activation of VLDL synthesis should lead to an increase in both apo C-III and apo E. The combination of these phenomena probably explains the absence of effect of cholestyramine on these apolipoproteins. Apolipoprotein B-containing particles may be divided into Lp B:E, containing apo B and apo E, Lp B:C-III, containing apo C-III and apo B, and Lp B:C-III:E, containing all these three apolipoproteins. It should be kept in mind that our methodology measures the pool of particles containing both apo E and apo B (Lp E:B) or both apo C-III and apo B (Lp C-1II:B). Activation of the receptor-specific pathway as a result of depletion of cellular cholesterol may lead to an accelerated clearance of ape B-containing particles and
true
explains probably the decrease in Lp E:B and Lp C-1II:B. Moreover, at least for pravastatin, this phenomenon could be potentiated by inhibition of direct synthesis of these particles, due to reduction in the cholesterol synthesis. However, the results obtained with cholestyramine reflect the complexity mechanism of action of the drug. Since Lp E:B and Lp C-1II:B levels are highly correlated with triglycerides at the baseline (data not shown), activation of triglyceride synthesis may lead to increased values for these parameters. In the first period of time, the accelerated clearance of these complex particles is balanced by their increased synthesis related to the activation of triglyceride synthesis. This probably explains the increase in Lp C-1II:B and Lp E:B at W4 and W8. In the second time period, activation of the catabolism of these apo B-containing particles supplants the activation of synthesis and results in a decrease in Lp E:B. As suggested by the decrease in Lp E:B/Lp C-1II:B ratio at W 12, both drugs could be more effective in reducing Lp E:B than Lp C-1II:B. This could reflect a better affinity of Lp B:E for the receptor.’ Finally, a correlation between the baseline level of Lp E:B and the mean triglyceride reduction (r = .31, P -c.05, data not shown) is found in the pravastatin group, but not in the cholestyramine group. This means that, on pravastatin therapy, the higher the level of Lp E:B, the greater is the triglyceride reduction. Apparently, clearance of Lp E:B particles through the receptor pathway leads to triglyceride reduction. Thus, the baseline level of Lp E:B may help to understand the variability in response to pravastatin therapy. CONCLUSION
This study demonstrates that the apolipoprotein composition of lipoprotein particles may be important in studying lipoprotein metabolism in clinical pharmacology. The results presented here show that the two drugs have similar effects on the classic lipid parameters, while the different lipoprotein particles change in different ways. Further investigation are necessary in this field to determine how these parameters may be used to adapt therapy to the individual. ACKNOWLEDGMENT The authors thank Professor J. Shepherd help in preparation of the manuscript.
(Glasgow,
Scotland)
for
PRAVASTATIN,
CHOLESTYRAMINE,
273
AND PARTICLES
REFERENCES
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11. Alaupovic P, McConathy WJ, Fesmire J, et al: Profiles of apolipoproteins and apolipoprotein B containing lipoprotein particles indyslipoproteinemias. Clin. Chem. 34/8B:B13-B27,1988 12. Alaupovic P, Lee DM, McConathy WJ: Studies of the composition and structure of plasma lipoproteins. Distribution of lipoprotein families in major density classes of normal human plasma lipoproteins. B&hem Biophys Acta 260:689-707,1972 13. Fruchart JC, Fievet C, Puchois P: Ape-lipoproteins. Metabolite-s III: Lipids, amino acids and related compounds, in Bergmeyer HU (ed): Methods of Enzymatic Analysis. New York, NY, VCH, 1985, pp 126-138 14. Fruchart JC, Kandoussi A, Parsy D, et al: Measurement of lipoprotein particles defined by their apolipoprotein composition using immunosorbent assay. Annual Meeting on Clinical Chemistry, Mannheim, September 25-28, 1985. J Clin Chem Clin Biochem 23:619, 1985 (abstr) 15. Parra HJ, Mezdour H, Ghalim N, et al: Differential electroimmunoassay on ready-to-use plates for human Lp A-I lipoprotein particles. Clin Chem (in press) 16. Huff MW, Telford DE, Woodkrofdt K, et al: Mevinolin and cholestyramine inhibit the direct synthesis of low density lipoprotein apoprotein B in miniature pigs. J Lipid Res 26: 1175- 1186, 1985 17. Angelin B, Einarsson K, Hellstrom K, et al: Effects of cholestyramine and chenodeoxycholic acid on the metabolism of endogenous triglyceride in hyperlipoproteinemia. J Lipid Res 19: 1017-1024,1978 18. Mabuchi H, Kamon N, Fujita H, et al: Effects of CS-514 on serum lipoprotein lipid and apolipoprotein levels in patients with familial hypercholesterolemia. Metabolism 36:475-479, 1987 19. Mabuchi H, Sakai T, Sakai Y, et al: Reduction of serum cholesterol in heterozygous patients with familial hypercholesterolemia. N Engl J Med 308:609-613, 1983 20. Miller NE, Rajput-Williams J, Nanjee MN, et al: Relationship of high density lipoprotein composition to plasma lecithin: Cholesterol acyltransferase concentration in men. Atherosclerosis 69:123-129,1988 21. Hui DY, Innerarity TL, Mahley RW: Lipoprotein binding to canine hepatic membranes. J Biol Chem 256:5646-5655, 198 1