- Email: [email protected]
Long-term efficacy of bezafibrate in reduction of small, dense low-density lipoprotein by hypotriglyceridemic action
CURRENT THERAPEUTIC RESEARCH VOL. 61, No.
3, MARCH
2000
Long-Term Efficacy of Bezafibrate in Reduction Small, Dense Low-Density Lipoprotein by Hypotriglyceridemic Action
of
Tsutomu Hirano,’ Tsutomu Kazumi,2 and Gen Yoshino’ ‘First Department of Internal Medicine, Showa University School of Medicine, Tokyo, ‘Department of Medicine, Hyogo Rehabilitation Center General Hospital, Kobe, and .‘Department of Laboratory Medicine, Toho University School of Medicine, Tokyo, Japan
ABSTRACT Objective: We examined both the effects of long-term bezafibrate treatment on low-density lipoprotein (LDL) particle size in hyperlipidemic patients and the relationship between changes in plasma lipids and LDL particle size. Background: It is well recognized that small, dense LDL is a highly atherogenic lipoprotein and is strongly associated with coronary artery disease. Mechanisms for generating small, dense LDL are multifactorial; however, plasma triglyceride concentration is a powerful determinant of LDL size. Methods: We observed changes in LDL particle diameter in 17 hyperlipidemic patients before and during 3 years of bezafibrate treatment (200-mg tablet BID) and examined the relationship between changes in LDL size and plasma lipid levels. Results: Bezafibrate reduced plasma triglyceride concentration by 37% and LDL-C concentration by 13% and increased high-density lipoprotein cholesterol (HDL-C) concentration by 44%. Mean LDL particle diameter determined by 2% to 16% gradient polyacrylamide gel electrophoresis was increased by bezafibrate treatment from 25.2 2 1.4 nm to 26.4 + 0.5 nm (P < 0.0001); thus the prevalence of small, dense LDL (diameter 225.5 nm, pattern B) was markedly reduced from 59% to 12%. The favorable lipoprotein profiles and enlargement of LDL size persisted throughout the study. A significant inverse correlation was observed between LDL size and plasma triglyceride concentration before bezafibrate treatment (r = -.694, P < 0.002), and increased LDL sizes were substantially associated with decreased plasma triglyceride levels after 3 years of bezafibrate treatment (r = -939, P < 0.0001). In contrast, HDL-C plasma concentration was not associated with LDL size at baseline, and changes in LDL size were not associated with changes in HDL-C concentration during the treatment. Conclusions: Results of this study suggest that the increased LDL size that occurs with bezafibrate treatment is strongly attribAddress correspondence to: Tsutomu Hirano, MD, The First Department of Internal Medicine, University School of Medicine, l-5-8 Hatanodai, Shinagawa-ku, Tokyo 142.8666, Japan. Accepted for publication January 11, 2000. Printed in the USA. Reproduction in whole 01‘ part is not permitted. 127
Showa
0011-393x/00/$19.00
CURRENT
THERAPE~ITIC
RESEAIXH@
uted to its hypotriglyceridemic action but not to increased concentrations of HDL-C. Long-term treatment with bezafibrate may have favorable effects on preventing coronary artery disease through the reduction of a potent atherogenic lipoprotein, small, dense LDL. Key words: bezafibrate, small, dense LDL, triglyceride, HDL cholesterol. (Curr Ther Res Clin Exp. 2000;61:127-136)
INTRODUCTION Epidemiologic studies have revealed that dyslipoproteinemia with a low concentration of high-density lipoprotein cholesterol (HDL-C) and an elevated serum triglyceride concentration is associated with a particularly high incidence of coronary artery disease. is2 This atherogenic lipid profile is frequently associated with increased concentrations of small, dense lowdensity lipoprotein (LDL) particles, which are more atherogenic than large, buoyant LDL particles.3 Studies have revealed that substantial correlations of LDL particle size are noted inversely with triglyceride and positively with HDL-C concentrations.4-6 Bezafibrate is a representative fibrate that powerfully decreases plasma triglyceride levels and increases HDL-C levels. Therefore, it is expected that bezafibrate treatment increases LDL particle size by ameliorating the atherogenic lipid profile. Indeed, several studies have already reported that fibrate treatment enlarges LDL particle size.7-10 However, most of the studies have been short term and have not confirmed fully the long-term efficacy of fibrate in reducing small, dense LDL. In addition, it is poorly understood whether the effect of fibrates on LDL particle size is caused by their hypotriglyceridemic action. In the present study, we examined both the effects of long-term bezafibrate treatment on LDL particle size in hyperlipidemic patients and the relationship between changes in plasma lipids and LDL particle size.
PATIENTS
AND METHODS
The study involved men and women with a diagnosis of hyperlipidemia, according to WHO criteria, without coronary artery disease or diabetes mellitus. These patients received bezafibrate 200 mg BID for 3 years. No drugs affecting lipid metabolism were used during the study other than bezafibrate and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors. Blood sampling was performed every 2 to 3 months for determination of serum cholesterol, triglyceride, and HDL-C. LDL particle size was measured using samples taken once a year. Plasma triglyceride and cholesterol levels were measured using enzymatic methods. HDL-C was measured in the plasma after polyanion precipitation of apolipopro128
T. HIRANO
tein (ape) Friedewald Determination
B-containing formula.”
ET AL
lipoproteins.
of Low-Den&g
LDL-C
Lipoprotein
was
estimated
Particle
Size
using
the
The diameter of the major LDL fraction was determined using gradient gel electrophoresis on 2% to 16% polyacrylamide gels according to the method of Nichols et al. i2 Plasma and 3 standards of known diameterapoferritin (12.2 nm), thyroglobulin (17.0 nm), and latex beads (39.0 nm)were simultaneously electrophoresed on the same slab gel for 24 hours. After electrophoresis, LDL was stained with oil red 0 and the standards with Coomassie G-250. The average LDL particle diameter was estimated using interpolation from a plot of the logarithm of the diameter of the standards versus the migration distance of the standards, as described by Coresh et a1.5 Written informed consent was given by each patient, and the study was approved by the ethics committee of our hospital (Showa University Hospital, Tokyo, and Hyogo Rehabilitation Center General Hospital, Kobe, Japan). Statistical significance was estimated using the Student paired t test. The correlation coefficients between 2 variables were determined using the Pearson simple linear regression analysis or polyclonal regression analysis.
RESULTS Ten men and 7 women aged 51 to 71 years participated in the study. There were 6 type IIa, 5 type IIb, 1 type III, and 5 type IV hyperlipidemic patients. All patients had normal liver and kidney function and none had chronic infectious or malignant disease. Three type IIa hyperlipidemic men were already being treated with HMG CoA reductase inhibitors (statins) (pravastatin, 10 mg/d or simvastatin, 5 mg/d); they continued treatment during the study without changing the dose. The effects of 3 years of bezafibrate treatment on plasma lipids are depicted in the table. Bezafibrate treatment for 1 year resulted in a significant (P < 0.001) decrease (mean difference between on-treatment mean and baseline plasma concentrations) in triglyceride concentration (33%) and a significant (P < 0.001) increase in HDL-C concentration (42%). The amelioration of plasma lipid profiles persisted during the 3-year follow-up; 3 years of treatment with bezafibrate significantly (P < 0.001) reduced plasma triglyceride concentration by 37% and significantly (P < 0.001) increased HDL-C concentration by 44%. Total cholesterol (TC) levels were gradually decreased by long-term bezafibrate treatment; the levels were mildly but significantly (P < 0.05) decreased 6% by 2 years and 8% by 3 years of treatment with bezafibrate. Similarly, LDL-C levels were signifi129
CLJI~KENT
Table.
THERAPEUTIC
Changes in plasma lipid concentrations patients (n = 17) and in patients not mean k SD.
RESEAKCH”
after using
3 years statins
of bezafibrate treatment in all (n = 14). Values are given as
Bezafibrate
Treatment
(y)
Baseline
1
2
3
All patients Triglycerides (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL)
195*92 223 t 34 148f36 36 + 6
131 f 82’ 217k 29 14Ok33 51 + 12*
109*46* 210 + 21+ 137 k 20+ 52+10*
123rt59* 205 f 27+ 129*22+ 52 t 13*
Patients not using statins Triglycerides (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL)
200 227 151 36
125*89+ 218 L 32 144*35 49 + 12*
97+35* :;; ;;: f 54 :8*
114?53* 209 + 28+ 132*23+ 53 t 12*
TC = total cholesterol; LDL-C cholesterol. * P < 0.001 versus baseline. + P < 0.05 versus baseline.
f f f f
97 36 38 6
= low-density
lipoprotein
cholesterol;
HDL-C
= high-density
lipoprotem
cantly (P < 0.05) decreased 7% and 13%, respectively, with bezafibrate compared with the levels at baseline. Because 3 men were being treated with statins, we excluded their data from the calculations to explore the sole effect of bezafibrate on changes in
Baseline
Treatment Figure
Duration
1. Changes in the mean low-density lipoprotein of bezafibrate treatment. ‘P < 0.0001 versus
130
(y)
(LDL) particle baseline.
diameter
after
3 years
T. HIWO
ET AL.
plasma lipids. As shown in the table, the results remained essentially the same even when data from the statin users were excluded. Changes in mean LDL particle diameter during 3 years of bezafibrate treatment are shown in Figure 1. Before treatment, LDL particle size of the current patients (25.2 f 1.4 nm) was substantially smaller than that of normal control subjects (26.2 + 0.8 nm) reported elsewhere.r3,r4 The prevalence of small, dense LDL (LDL diameter ~25.5 nm, pattern B)12 was markedly increased in the current patients compared with the normal control subjects (59% vs 25%, respectively). Bezafibrate treatment for 1 year resulted in significant enlargement of LDL particle size, and the prevalence of pattern B was markedly reduced. The increase in LDL particle size persisted during the 3 years of bezafibrate treatment; LDL particle size was increased to 26.4 + 0.5 nm, and the prevalence of small, dense LDL (pattern B) was reduced to 12% after 3 years of treatment. Mean LDL particle diameter in the 3 statin users was 26.1 + 0.3 nm before bezafibrate treatment; this increased to 26.5 rt 0.6,26.5 + 0, and 26.7 -t 0.3 nm after 1,2, and 3 years, respectively, of bezafibrate treatment. Changes in LDL particle size after bezafibrate treatment remained the same even when data from the 3 statin users were excluded (data not shown). There was a significant inverse correlation between plasma triglyceride levels and LDL particle diameters at baseline (r = -.694, P < 0.0021, whereas such a correlation was not observed between HDL-C levels and LDL particle diameters (Figure 2). We calculated mean differences of LDL particle size and plasma lipid concentrations between baseline and 3 years of treatment with bezafibrate. A decrease in plasma triglyceride concentration was highly associated with an increase in LDL particle diameter (r = .839, P < 0.0001) (Figure 3). The relationship between changes in 2 variables were curvilinear rather than linear. Thus, the correlation coefficient was greater in polyclonal regression analysis than that in simple regression analysis (Figure 3). Unlike a change in triglyceride levels, an increase in HDL-C was not associated with a change in LDL particle size (Figure 4). Changes in TC and LDL-C concentrations were not associated with changes in LDL particle size (data not shown). A decline in plasma triglyceride concentration was not significantly associated with an increase in HDL after 3 years of treatment with bezafibrate.
DISCUSSION
AND
CONCLUSIONS
We confirmed that bezafibrate treatment significantly (P < 0.001) reduced plasma triglyceride levels and increased HDL-C levels; these favorable effects for the prevention of atherosclerosis persisted throughout the 133
CURRENT
THERAPE~JTIC
RESEARCH@
28 27 26 25 24
P
0 0
Lo 0
m?-
c
i
23 r= -.694 P
22
00
21 50
100
150
Plasma
B
28
VE z %i
27-
E
2625-
00
200
300
Triglyceride
0
0
350
3
o
O
400
(mg/dL)
3 0
0
00
250
0
0
.-0”
1
25
30
35
HDL-C Figure
2. Correlation between low-density ma triglyceride or (B) high-density at baseline.
40
45
50
(mg/dL)
lipoprotein lipoprotein
(LDL) particle cholesterol
diameter (HDL-C)
and (A) plasconcentrations
study. Recently it has been shown that the action of fibrates is exerted by means of a subfamily of the nuclear receptor gene family, the peroxisome proliferator-activated receptor (PPAR)-alpha.i5 PPAR-alpha activation by fibrates leads to both a decreased plasma triglyceride level by increasing lipoprotein lipase expression and decreasing apo C-III expression and an increased HDL-C level by increasing apo A-I and apo A-II expression.‘” Although plasma triglyceride level was decreased and HDL-C level was increased by bezafibrate treatment, we did not observe a significant association between changes in levels of triglyceride and HDL-C with beza132
T. HIRANO
ET AL.
5 + 4
+
y = -0.104x + 4.351 R2 = .643 P < 0.0001
3 i
-250
-150
-50
50
Change in Plasma Triglyceride
150 (mg/dL)
+ -1I---r?-T1T,
-250
I
-150
-50
‘,
‘,
I
50
Change in Plasma Triglyceride Figure
10 (mg/dL)
3. Relationship between change in low-density lipoprotein (LDL) plasma triglyceride level after 3 years of bezafibrate treatment. sion. (B) Curvilinear regression.
size and change in (A) Linear regres-
fibrate treatment, suggesting that the effects of bezafibrate on metabolism of triglyceride-rich lipoproteins and HDL-C are in part dissociated. Compared with the relationship between triglyceride and HDL-C, substantial close association was observed between changes in the plasma 133
CIJKRENT
TIIERAPEIJTIC
RESEAKCH~~
0
ii
-10
6 Figure
4. Relationship between high-density lipoprotein brate treatment.
0
10
Change
20
in HDL-C
change in low-density cholesterol (HDL-C)
30
40
50
(mg/dL) lipoprotein concentration
(LDL) size and change in after 3 years of bezafi-
level and LDL particle diameter with bezafibrate treatment. A significant correlation between LDL particle size and HDL-C has been because a substantial intercorrelation usually exists reported 4-6. however between H’DL-C anh triglyceride levels, it remains to be determined whether the association of LDL particle size with HDL-C level is direct. The present results strongly suggest that LDL particle size is influenced primarily by the plasma triglyceride level; however, the association of LDL particle size with HDL-C level is at most indirect. It has been postulated that high plasma triglyceride levels can influence the LDL composition through a cycle of lipid exchange, so that LDL becomes enriched in triglycerides and is then acted on by hepatic triglyceride lipase to produce small, lipid-poor LDL particles.16 Therefore, activities of lipid transfer proteins and hepatic triglyceride lipase may also be involved in the mechanisms for generating small, dense LDL. Nevertheless, it has been recognized that the most powerful determinant of LDL particle size is plasma triglyceride concentration itself.4~“*1”,17 Our present results may support this recognition. Plasma triglycerides do not maintain a fasting value, but postprandial increase occurs several times per day as a result of fat intake in connection with meals. Thus fluctuation of postprandial triglyceride levels becomes a more important determinant of LDL particle size than fasting levels, especially in individuals who have a marked postprandial lipidemia.14~1s Fibrates efficiently ameliorate postprandial lipidemia as well as fasting hypertriglyceridemia.” Therefore, the enlargement effect of bezafibrate on triglyceride
134
T. HIRANO
ET AL.
LDL particle size would be attributable to the overall amelioration of both fasting and postprandial hypertriglyceridemia. Recently, Ruotolo et ali’ reported that in dyslipoproteinemic men who survived a first myocardial infarction before age 45 years were assigned randomly to treatment for 5 years with bezafibrate, LDL particle size became larger with bezafibrate treatment; however, this increase was unrelated to disease progression assessed using coronary angiography. In that study, however, the degree of enlargement of the LDL-C particle diameter was very small (0.3 nm), whereas a marked increase in LDL diameter was observed in the present study (1.2 nm). Thus, it remains to be elucidated whether a substantial increase in LDL particle size by bezafibrate could prevent the development of coronary artery disease. References: 1. Grundy SM, Balady GJ, Criqui Guidance from Framingham: Task Force on Risk Reduction. 1887.
MH, et al. Primary prevention of coronary A statement for healthcare professionals American Heart Association. Circulation.
2. Lamarche B, Despres JP, Moorjani factors for ischemic heart disease. erosclerosis. 1996;119:235-245.
heart disease: from the AHA 1998;97:1876-
S, et al. Triglycerides and HDL-cholesterol Results from the Quebec Cardiovascular
Study.
3. Lamarche B, Tchernof A, Moorjani S, et al. Small, dense low-density lipoprotein as a predictor of the risk of ischemic heart disease in men. Prospective results Quebec Cardiovascular Study. Circulation. 1997;95:69-75. 4. McNamara JR, Jenner JL, Li Z, et al. Change in LDL change in plasma triglyceride concentration. Arterioscler
as risk Ath-
particles from the
particle size is associated Thromb. 1992;12:1284-1290.
with
5. Coresh J, Kwiterovich PO, Smith HH, Bachorik PS. Association of plasma triglyceride concentration and LDL particle diameter, density, and chemical composition with premature coronary artery disease in men and women. J Lipid Res. 1993;34:1687-1697. 6. Katzel LI, Krauss to body composition ticles. Arterioscler
RM, Goldberg AI’. Relations and plasma insulin levels Thromb. 1994;14:1121-1128.
of plasma TG and HDL-C are altered in men with
7. Yoshida H, Ishikawa T, Ayaori M, et al. Beneficial effect of gemfibrozil composition and oxidative susceptibility of low density lipoprotein: double-blind, placebo-controlled study. Atherosclerosis. 1998;139:179-187. 8. Guerin M, Bruckert transfer from HDL bined hyperlipidemia. 9. Toyota affinity 10.
Y, Yamamura for the LDL
concentrations small LDL paron the chemical A randomized,
E, Dolphin PJ, et al. Fenofibrate reduces plasma cholesteryl to VLDL and normalizes the atherogenic, dense LDL profile Arterioscler Thromb Vast Biol. 1996;16:763-772. T, Miyake Y, Yamamoto A. Low receptor in hyperlipoproteinemia.
density lipoprotein Atherosclerosis.
ester in com-
(LDL) binding 1999;147:77-86.
Ruotolo G, Ericsson CG, Tettamanti C, et al. Treatment effects on serum lipoprotein lipids, apolipoproteins and low density lipoprotein particle size and relationships of lipoprotein variables to progression of coronary artery disease in the Bezafibrate Coronary Atherosclerosis Intervention Trial (BECAIT). J Am Co11 Cardiol. 1998;32:1648-1656.
11. Freidewald
WT, Levy
RI, Fredrickson
DS. Estimation 135
of the concentration
of low-density
CURRENT
lipoprotein cholesterol Chem. 1972;18:499-502. 12. Nichols AV, Krauss trophoresis. Methods 13. Hirano T, Naito insulin-dependent
THERAPEUTIC
in plasma,
without
RESEARCH@
use of the preparative
RM, Musliner TA. Nondenaturing Enzymol. 1986;128:417431.
H, Kurokawa M, et al. High prevalence diabetic patients with nephropathy.
14. Hirano T, Oi K, Sakai S, et al. High athy is not directly associated with mia. Atherosclerosis. 1998;141:77-85. 15. Fruchart JC, Duriez P, Staels vators regulate genes governing erosclerosis. Curr Opin Lipidol.
Clin
polyacrylamide
gradient
of small LDL Atherosclerosis.
particles in non1996;123:57-72.
prevalence of small dense kidney damage: A possible
Y, et al. Plasma triglyceride lipoproteins. Arteriosclerosis.
gel elec-
LDL in diabetic nephroprole of postprandial lipe-
B. Peroxisome proliferator-activated lipoprotein metabolism, vascular 1999;10:245-257.
RJ, Grannot E, Oschry in low and high density
ultracentrifuge.
receptor-alpha actiinflammation and ath-
16.
Deckelbaum composition
17.
Griffin BA, Freeman DJ, Tait GW, et al. Role of plasma triglyceride in the regulation of plasma low density lipoprotein (LDL) subfractions: Relative contribution of small, dense LDL to coronary heart disease risk. Atherosclerosis. 1994;106:241-253.
18. Karpe F, Tornvall P, Olivecrona T, et al. Composition Effects of postprandial triglyceride-rich lipoproteins, and cholesteryl ester transfer protein. Atherosclerosis. 19. Weintraub MS, on postprandial
Charach lipemia.
G, Grosskopf Atherosclerosis.
I. Effects of fibric 1998;141:S71-575.
136
determines structure1984;4:225-231.
of human low density lipoprotein: lipoprotein lipase, hepatic lipase 1993;98:3349. acid
derivatives
and metformin
3, MARCH
2000
Long-Term Efficacy of Bezafibrate in Reduction Small, Dense Low-Density Lipoprotein by Hypotriglyceridemic Action
of
Tsutomu Hirano,’ Tsutomu Kazumi,2 and Gen Yoshino’ ‘First Department of Internal Medicine, Showa University School of Medicine, Tokyo, ‘Department of Medicine, Hyogo Rehabilitation Center General Hospital, Kobe, and .‘Department of Laboratory Medicine, Toho University School of Medicine, Tokyo, Japan
ABSTRACT Objective: We examined both the effects of long-term bezafibrate treatment on low-density lipoprotein (LDL) particle size in hyperlipidemic patients and the relationship between changes in plasma lipids and LDL particle size. Background: It is well recognized that small, dense LDL is a highly atherogenic lipoprotein and is strongly associated with coronary artery disease. Mechanisms for generating small, dense LDL are multifactorial; however, plasma triglyceride concentration is a powerful determinant of LDL size. Methods: We observed changes in LDL particle diameter in 17 hyperlipidemic patients before and during 3 years of bezafibrate treatment (200-mg tablet BID) and examined the relationship between changes in LDL size and plasma lipid levels. Results: Bezafibrate reduced plasma triglyceride concentration by 37% and LDL-C concentration by 13% and increased high-density lipoprotein cholesterol (HDL-C) concentration by 44%. Mean LDL particle diameter determined by 2% to 16% gradient polyacrylamide gel electrophoresis was increased by bezafibrate treatment from 25.2 2 1.4 nm to 26.4 + 0.5 nm (P < 0.0001); thus the prevalence of small, dense LDL (diameter 225.5 nm, pattern B) was markedly reduced from 59% to 12%. The favorable lipoprotein profiles and enlargement of LDL size persisted throughout the study. A significant inverse correlation was observed between LDL size and plasma triglyceride concentration before bezafibrate treatment (r = -.694, P < 0.002), and increased LDL sizes were substantially associated with decreased plasma triglyceride levels after 3 years of bezafibrate treatment (r = -939, P < 0.0001). In contrast, HDL-C plasma concentration was not associated with LDL size at baseline, and changes in LDL size were not associated with changes in HDL-C concentration during the treatment. Conclusions: Results of this study suggest that the increased LDL size that occurs with bezafibrate treatment is strongly attribAddress correspondence to: Tsutomu Hirano, MD, The First Department of Internal Medicine, University School of Medicine, l-5-8 Hatanodai, Shinagawa-ku, Tokyo 142.8666, Japan. Accepted for publication January 11, 2000. Printed in the USA. Reproduction in whole 01‘ part is not permitted. 127
Showa
0011-393x/00/$19.00
CURRENT
THERAPE~ITIC
RESEAIXH@
uted to its hypotriglyceridemic action but not to increased concentrations of HDL-C. Long-term treatment with bezafibrate may have favorable effects on preventing coronary artery disease through the reduction of a potent atherogenic lipoprotein, small, dense LDL. Key words: bezafibrate, small, dense LDL, triglyceride, HDL cholesterol. (Curr Ther Res Clin Exp. 2000;61:127-136)
INTRODUCTION Epidemiologic studies have revealed that dyslipoproteinemia with a low concentration of high-density lipoprotein cholesterol (HDL-C) and an elevated serum triglyceride concentration is associated with a particularly high incidence of coronary artery disease. is2 This atherogenic lipid profile is frequently associated with increased concentrations of small, dense lowdensity lipoprotein (LDL) particles, which are more atherogenic than large, buoyant LDL particles.3 Studies have revealed that substantial correlations of LDL particle size are noted inversely with triglyceride and positively with HDL-C concentrations.4-6 Bezafibrate is a representative fibrate that powerfully decreases plasma triglyceride levels and increases HDL-C levels. Therefore, it is expected that bezafibrate treatment increases LDL particle size by ameliorating the atherogenic lipid profile. Indeed, several studies have already reported that fibrate treatment enlarges LDL particle size.7-10 However, most of the studies have been short term and have not confirmed fully the long-term efficacy of fibrate in reducing small, dense LDL. In addition, it is poorly understood whether the effect of fibrates on LDL particle size is caused by their hypotriglyceridemic action. In the present study, we examined both the effects of long-term bezafibrate treatment on LDL particle size in hyperlipidemic patients and the relationship between changes in plasma lipids and LDL particle size.
PATIENTS
AND METHODS
The study involved men and women with a diagnosis of hyperlipidemia, according to WHO criteria, without coronary artery disease or diabetes mellitus. These patients received bezafibrate 200 mg BID for 3 years. No drugs affecting lipid metabolism were used during the study other than bezafibrate and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors. Blood sampling was performed every 2 to 3 months for determination of serum cholesterol, triglyceride, and HDL-C. LDL particle size was measured using samples taken once a year. Plasma triglyceride and cholesterol levels were measured using enzymatic methods. HDL-C was measured in the plasma after polyanion precipitation of apolipopro128
T. HIRANO
tein (ape) Friedewald Determination
B-containing formula.”
ET AL
lipoproteins.
of Low-Den&g
LDL-C
Lipoprotein
was
estimated
Particle
Size
using
the
The diameter of the major LDL fraction was determined using gradient gel electrophoresis on 2% to 16% polyacrylamide gels according to the method of Nichols et al. i2 Plasma and 3 standards of known diameterapoferritin (12.2 nm), thyroglobulin (17.0 nm), and latex beads (39.0 nm)were simultaneously electrophoresed on the same slab gel for 24 hours. After electrophoresis, LDL was stained with oil red 0 and the standards with Coomassie G-250. The average LDL particle diameter was estimated using interpolation from a plot of the logarithm of the diameter of the standards versus the migration distance of the standards, as described by Coresh et a1.5 Written informed consent was given by each patient, and the study was approved by the ethics committee of our hospital (Showa University Hospital, Tokyo, and Hyogo Rehabilitation Center General Hospital, Kobe, Japan). Statistical significance was estimated using the Student paired t test. The correlation coefficients between 2 variables were determined using the Pearson simple linear regression analysis or polyclonal regression analysis.
RESULTS Ten men and 7 women aged 51 to 71 years participated in the study. There were 6 type IIa, 5 type IIb, 1 type III, and 5 type IV hyperlipidemic patients. All patients had normal liver and kidney function and none had chronic infectious or malignant disease. Three type IIa hyperlipidemic men were already being treated with HMG CoA reductase inhibitors (statins) (pravastatin, 10 mg/d or simvastatin, 5 mg/d); they continued treatment during the study without changing the dose. The effects of 3 years of bezafibrate treatment on plasma lipids are depicted in the table. Bezafibrate treatment for 1 year resulted in a significant (P < 0.001) decrease (mean difference between on-treatment mean and baseline plasma concentrations) in triglyceride concentration (33%) and a significant (P < 0.001) increase in HDL-C concentration (42%). The amelioration of plasma lipid profiles persisted during the 3-year follow-up; 3 years of treatment with bezafibrate significantly (P < 0.001) reduced plasma triglyceride concentration by 37% and significantly (P < 0.001) increased HDL-C concentration by 44%. Total cholesterol (TC) levels were gradually decreased by long-term bezafibrate treatment; the levels were mildly but significantly (P < 0.05) decreased 6% by 2 years and 8% by 3 years of treatment with bezafibrate. Similarly, LDL-C levels were signifi129
CLJI~KENT
Table.
THERAPEUTIC
Changes in plasma lipid concentrations patients (n = 17) and in patients not mean k SD.
RESEAKCH”
after using
3 years statins
of bezafibrate treatment in all (n = 14). Values are given as
Bezafibrate
Treatment
(y)
Baseline
1
2
3
All patients Triglycerides (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL)
195*92 223 t 34 148f36 36 + 6
131 f 82’ 217k 29 14Ok33 51 + 12*
109*46* 210 + 21+ 137 k 20+ 52+10*
123rt59* 205 f 27+ 129*22+ 52 t 13*
Patients not using statins Triglycerides (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL)
200 227 151 36
125*89+ 218 L 32 144*35 49 + 12*
97+35* :;; ;;: f 54 :8*
114?53* 209 + 28+ 132*23+ 53 t 12*
TC = total cholesterol; LDL-C cholesterol. * P < 0.001 versus baseline. + P < 0.05 versus baseline.
f f f f
97 36 38 6
= low-density
lipoprotein
cholesterol;
HDL-C
= high-density
lipoprotem
cantly (P < 0.05) decreased 7% and 13%, respectively, with bezafibrate compared with the levels at baseline. Because 3 men were being treated with statins, we excluded their data from the calculations to explore the sole effect of bezafibrate on changes in
Baseline
Treatment Figure
Duration
1. Changes in the mean low-density lipoprotein of bezafibrate treatment. ‘P < 0.0001 versus
130
(y)
(LDL) particle baseline.
diameter
after
3 years
T. HIWO
ET AL.
plasma lipids. As shown in the table, the results remained essentially the same even when data from the statin users were excluded. Changes in mean LDL particle diameter during 3 years of bezafibrate treatment are shown in Figure 1. Before treatment, LDL particle size of the current patients (25.2 f 1.4 nm) was substantially smaller than that of normal control subjects (26.2 + 0.8 nm) reported elsewhere.r3,r4 The prevalence of small, dense LDL (LDL diameter ~25.5 nm, pattern B)12 was markedly increased in the current patients compared with the normal control subjects (59% vs 25%, respectively). Bezafibrate treatment for 1 year resulted in significant enlargement of LDL particle size, and the prevalence of pattern B was markedly reduced. The increase in LDL particle size persisted during the 3 years of bezafibrate treatment; LDL particle size was increased to 26.4 + 0.5 nm, and the prevalence of small, dense LDL (pattern B) was reduced to 12% after 3 years of treatment. Mean LDL particle diameter in the 3 statin users was 26.1 + 0.3 nm before bezafibrate treatment; this increased to 26.5 rt 0.6,26.5 + 0, and 26.7 -t 0.3 nm after 1,2, and 3 years, respectively, of bezafibrate treatment. Changes in LDL particle size after bezafibrate treatment remained the same even when data from the 3 statin users were excluded (data not shown). There was a significant inverse correlation between plasma triglyceride levels and LDL particle diameters at baseline (r = -.694, P < 0.0021, whereas such a correlation was not observed between HDL-C levels and LDL particle diameters (Figure 2). We calculated mean differences of LDL particle size and plasma lipid concentrations between baseline and 3 years of treatment with bezafibrate. A decrease in plasma triglyceride concentration was highly associated with an increase in LDL particle diameter (r = .839, P < 0.0001) (Figure 3). The relationship between changes in 2 variables were curvilinear rather than linear. Thus, the correlation coefficient was greater in polyclonal regression analysis than that in simple regression analysis (Figure 3). Unlike a change in triglyceride levels, an increase in HDL-C was not associated with a change in LDL particle size (Figure 4). Changes in TC and LDL-C concentrations were not associated with changes in LDL particle size (data not shown). A decline in plasma triglyceride concentration was not significantly associated with an increase in HDL after 3 years of treatment with bezafibrate.
DISCUSSION
AND
CONCLUSIONS
We confirmed that bezafibrate treatment significantly (P < 0.001) reduced plasma triglyceride levels and increased HDL-C levels; these favorable effects for the prevention of atherosclerosis persisted throughout the 133
CURRENT
THERAPE~JTIC
RESEARCH@
28 27 26 25 24
P
0 0
Lo 0
m?-
c
i
23 r= -.694 P
22
00
21 50
100
150
Plasma
B
28
VE z %i
27-
E
2625-
00
200
300
Triglyceride
0
0
350
3
o
O
400
(mg/dL)
3 0
0
00
250
0
0
.-0”
1
25
30
35
HDL-C Figure
2. Correlation between low-density ma triglyceride or (B) high-density at baseline.
40
45
50
(mg/dL)
lipoprotein lipoprotein
(LDL) particle cholesterol
diameter (HDL-C)
and (A) plasconcentrations
study. Recently it has been shown that the action of fibrates is exerted by means of a subfamily of the nuclear receptor gene family, the peroxisome proliferator-activated receptor (PPAR)-alpha.i5 PPAR-alpha activation by fibrates leads to both a decreased plasma triglyceride level by increasing lipoprotein lipase expression and decreasing apo C-III expression and an increased HDL-C level by increasing apo A-I and apo A-II expression.‘” Although plasma triglyceride level was decreased and HDL-C level was increased by bezafibrate treatment, we did not observe a significant association between changes in levels of triglyceride and HDL-C with beza132
T. HIRANO
ET AL.
5 + 4
+
y = -0.104x + 4.351 R2 = .643 P < 0.0001
3 i
-250
-150
-50
50
Change in Plasma Triglyceride
150 (mg/dL)
+ -1I---r?-T1T,
-250
I
-150
-50
‘,
‘,
I
50
Change in Plasma Triglyceride Figure
10 (mg/dL)
3. Relationship between change in low-density lipoprotein (LDL) plasma triglyceride level after 3 years of bezafibrate treatment. sion. (B) Curvilinear regression.
size and change in (A) Linear regres-
fibrate treatment, suggesting that the effects of bezafibrate on metabolism of triglyceride-rich lipoproteins and HDL-C are in part dissociated. Compared with the relationship between triglyceride and HDL-C, substantial close association was observed between changes in the plasma 133
CIJKRENT
TIIERAPEIJTIC
RESEAKCH~~
0
ii
-10
6 Figure
4. Relationship between high-density lipoprotein brate treatment.
0
10
Change
20
in HDL-C
change in low-density cholesterol (HDL-C)
30
40
50
(mg/dL) lipoprotein concentration
(LDL) size and change in after 3 years of bezafi-
level and LDL particle diameter with bezafibrate treatment. A significant correlation between LDL particle size and HDL-C has been because a substantial intercorrelation usually exists reported 4-6. however between H’DL-C anh triglyceride levels, it remains to be determined whether the association of LDL particle size with HDL-C level is direct. The present results strongly suggest that LDL particle size is influenced primarily by the plasma triglyceride level; however, the association of LDL particle size with HDL-C level is at most indirect. It has been postulated that high plasma triglyceride levels can influence the LDL composition through a cycle of lipid exchange, so that LDL becomes enriched in triglycerides and is then acted on by hepatic triglyceride lipase to produce small, lipid-poor LDL particles.16 Therefore, activities of lipid transfer proteins and hepatic triglyceride lipase may also be involved in the mechanisms for generating small, dense LDL. Nevertheless, it has been recognized that the most powerful determinant of LDL particle size is plasma triglyceride concentration itself.4~“*1”,17 Our present results may support this recognition. Plasma triglycerides do not maintain a fasting value, but postprandial increase occurs several times per day as a result of fat intake in connection with meals. Thus fluctuation of postprandial triglyceride levels becomes a more important determinant of LDL particle size than fasting levels, especially in individuals who have a marked postprandial lipidemia.14~1s Fibrates efficiently ameliorate postprandial lipidemia as well as fasting hypertriglyceridemia.” Therefore, the enlargement effect of bezafibrate on triglyceride
134
T. HIRANO
ET AL.
LDL particle size would be attributable to the overall amelioration of both fasting and postprandial hypertriglyceridemia. Recently, Ruotolo et ali’ reported that in dyslipoproteinemic men who survived a first myocardial infarction before age 45 years were assigned randomly to treatment for 5 years with bezafibrate, LDL particle size became larger with bezafibrate treatment; however, this increase was unrelated to disease progression assessed using coronary angiography. In that study, however, the degree of enlargement of the LDL-C particle diameter was very small (0.3 nm), whereas a marked increase in LDL diameter was observed in the present study (1.2 nm). Thus, it remains to be elucidated whether a substantial increase in LDL particle size by bezafibrate could prevent the development of coronary artery disease. References: 1. Grundy SM, Balady GJ, Criqui Guidance from Framingham: Task Force on Risk Reduction. 1887.
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