- Email: [email protected]
Effect of simvastatin on apoprotein B—containing lipoproteins in patients with diabetic nephropathy
VOI.UME 63, No. 1.
JANUARY
2002
Effect of Simvastatin on Apoprotein B-Containing Lipoproteins in Patients with Diabetic Nephropathy Tsutomu Hirano, MD,’ Taro Sakaue, MD,’ Cen Yoshino, MD,2 Hiroko Takeuchi, MD,’ and Mitsuru Adachi, MD’ ‘First Department of Internal Medicine, Showa University, School of Medicine, and 2Department of Laboratory Medicine, Toho University School of Medicine, Tokyo, lapan
ABSTRACT Backgrourrd: Diabetic
patients with nephropathy usually have a more atherogenic lipoprotein profile than those without nephropathy, which may be associated with the substantially higher incidence of coronary heart disease (CHD) in this population. Simvastatin has been shown to significantly reduce the incidence of CHD events in diabetic patients. Objective: The purpose of this study was to evaluate the effect of simvastatin (10 mg/d) on atherogenic apoprotein (apo) B-containing lipoproteins in type 2 diabetic patients with nephropathy. Methods: Diabetic patients with nephropathy and a group of healthy control subjects matched for age, sex, and body weight were enrolled. Diabetic patients were administered simvastatin 10 mg/d for 6 months. Apo B-containing lipoproteins were sequentially separated by ultracentrifugation to yield very lowdensity lipoprotein (VLDL) (density cl.006 g/mL), intermediate-density lipoprotein (IDL) (1.006-l .019 g/mL), light low-density lipoprotein (LDL) (1.019-l .044 g/mL), and dense LDL (1.044-1.063 g/mL) fractions. Apo B in lipoproteins was measured by a sensitive enzyme-linked immunosorbent assay at baseline and after 6 months of simvastatin treatment. Results: A total of 18 patients with diabetic nephropathy and 36 matched controls were enrolled. The diabetic patients had significantly higher levels (P < 0.01) of total cholesterol, LDL cholesterol, triglycerides, and apo B compared with age- and weight-matched control subjects at baseline. The diabetic patients also had significantly higher levels (P < 0.05) of cholesterol and apo B in the VLDL, light LDL, and dense LDL fractions. Treatment with simvastatin for 6 months significantly reduced plasma total cholesterol by 21%, LDL cholesterol by 30%, and apo B by 25% (P < O.OOl), but did not affect urinary albumin excretion. Simvastatin significantly decreased both triglyceride and cholesterol levels in VLDL by 18% (P < 0.05), and cholesterol and apo B in IDL by 22% (P < 0.05) and 26% (P < 0.01). Simvastatin decreased both the light and dense Accepted for publication November 13, 200 I, Printed in the USA. Reproduction in whole or part is not permitted.
0011-393X/02/$1
9.00
33
CURRENT THERAPEUTICRESEARCHQ
LDL subfractions to a similar extent, reducing cholesterol and apo B in light LDL by 27% (P < 0.001) and in dense LDL by 28% (P < 0.01) and 18% (P < 0.05) respectively. The light LDL/dense LDL ratio for apo B and for cholesterol were not altered by simvastatin therapy. Conclusions: The results of this study suggest that simvastatin may reduce levels of atherogenic apo B-containing lipoproteins and small dense LDL in diabetic patients with nephropathy. Key words: simvastatin, diabetic nephropathy, low-density lipoprotein fraction, very low-density lipoprotein, intermediate-density lipoprotein (Cur-r Ther Res Clin Exp. 2002;63:33-42)
INTRODUCTION
The incidence of coronary heart disease (CHD) among patients with diabetes mellitus is markedly higher than among people without diabetes. In addition, it has been reported that the risk of death from CHD is substantially increased in diabetic patients with nephropathy compared with diabetic patients without nephropathy.1-3 Elevated levels of very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL), a high proportion of smaller and denser LDL particles (small, dense LDL), and reduced levels of high-density lipoprotein (HDL) have all been frequently reported in patients with diabetes,4 and this atherogenic lipoprotein profile becomes more prominent in patients with diabetic nephropathy.5v6 These abnormal plasma lipoprotein levels may contribute significantly to the increased risk of CHD in patients with diabetic nephropathy. The Scandinavian Simvastatin Survival Study showed that simvastatin-treated diabetic patients experienced significantly fewer CHD events compared with placebo-treated control patients. 7 Therefore, it is reasonable to assume that treatment with simvastatin can improve the atherogenic lipoprotein profile of patients with diabetic nephropathy. It is well known that statins, including simvastatin, can markedly reduce LDL levels by activating the LDL receptor pathway, but it is still not clear how statins affect VLDL, IDL, and the LDL subfractions. Therefore, we investigated the effect of simvastatin on these apo B-containing lipoproteins in patients with diabetic nephropathy.
PATIENTS AND METHODS
Outpatients with diabetic nephropathy as well as a group of healthy control subjects matched for age, body weight, and sex were enrolled. The control subjects were selected from among healthy persons undergoing annual medical examinations at the affiliated hospitals of Showa University. None of the control subjects had a history of hyperglycemia, abnormal hepatic or thyroid function, coronary heart disease, stroke, malignancy, or acute infection. The diabetic
34
T. Hirano et al.
patients fulfilled the World Health Organization criteria for type 2 diabetes mellitus. Diabetic nephropathy was diagnosed from persistent albuminuria in the presence of diabetic retinopathy without clinical or laboratory evidence of other kidney diseases. None of the subjects had clinical or laboratory evidence of abnormal hepatic or thyroid function, malignant tumors, infectious diseases, or renal failure (serum creatinine ~1.2 mg/dL). Each subject gave written informed consent, and the ethics committee of Showa University approved the study. The diabetic patients were treated with simvastatin 10 mg/d for 6 months. No other lipid-lowering drugs were used during the study. Patients were maintained on an isocaloric diet (25-30 kcal/kg ideal body weight) consisting of 17% protein, 23% fat, and 60% carbohydrates under the supervision of dietitians, and their compliance with the diet therapy was monitored. Three patients were being managed with dietary intervention alone (n = 6). Nine were taking sulfonylureas or a-glucosidase inhibitors and 3 were taking insulin; the dosages of these drugs were not changed during simvastatin therapy. Eleven diabetic patients were hypertensive; 5 were being treated with an angiotensin 11receptor antagonist (losartan 25 mg/d) and 6 with a calcium channel blocker (amlodipine 5 mg/d). Blood samples were collected after an overnight fast. Apo B-containing lipoproteins were separated from plasma by ultracentrifugation (Hitachi CP-65G, Hitachi Co, Ltd, Tokyo, Japan) using an RP 55T-708 rotor (Hitachi Co, Ltd) to yield VLDL (density [d] cl.006 g/mL), IDL (d = 1.006-1.019 g/mL), light LDL (d = 1.019-1.044 g/mL), and dense LDL (d = 1.044-1.063 g/mL) fractions according to the method of Have1 et al.” The apo B concentration in each lipoprotein fraction was measured by sensitive enzyme-linked immunosorbent assay (ELISA).’ Glycosylated hemoglobin (HbA,J, fasting plasma glucose, albumin, blood urea nitrogen, creatinine, and lipids in plasma were measured by standard laboratory procedures. Plasma levels of apo AI, AII, B, CII, CIII, and E were determined by immunoturbidimetric assay @aiichi Chemicals Co, Tokyo, Japan). The urinary albumin concentration was measured by the latex turbidimetric immunoassay method using a commercially available kit (LA-system, AIC Co, Tokyo, Japan). Plasma triglyceride and cholesterol levels were measured by enzymatic methods. HDL cholesterol was measured after polyanion precipitation of apo B-containing lipoproteins from the plasma. LDL cholesterol level was estimated using the Friedewald formula.” Statistical
Analysis
The Student paired t test was used to assess differences between plasma parameters before and after simvastatin treatment in the patients with diabetic nephropathy. The unpaired t test was used to evaluate the significance of differences between control subjects and diabetic patients at baseline.
35
CURRENTTHERAPEUTIC RESEARCH~
RESULTS
A total of 18 diabetic patients and 36 matched controls were enrolled. Clinical characteristics of all the study subjects are shown in Table I. Sex ratio, age, and body mass index were comparable between the healthy controls and the diabetic patients with nephropathy, whereas the diabetic patients had significantly higher levels of total cholesterol, LDL cholesterol, triglyceride, and apo B (PC 0.01) compared with control subjects. Patients also had significantly higher levels of apo CII and apo E than the control subjects (P < 0.05), but had similar levels of HDL cholesterol, apo AI, and apo CIII. After 6 months of treatment with simvastatin, mean HbA,, decreased slightly but significantly (P < 0.05), with no changes in fasting plasma glucose level. The urinary albumin index was not altered by treatment with simvastatin. Changes in plasma lipids and apoproteins after 6 months’ treatment with simvastatin are shown in Table I. In patients with diabetic nephropathy, simvastatin normalized previously elevated levels of total cholesterol and LDL cholesterol (P < 0.001 vs baseline),
Table I. Clinical characteristics of control subjects and patients with diabetic nephropathy at baseline, and changes in plasma lipids and apoproteins in the diabetic patients after 6 months of treatment with simvastatin 10 mg/d.
Sex Male Female Age, y, mean 2 SD Body mass index, kg/m2 Urinary albumin index, mg/g creatinine Plasma glucose, mg/dL HbA,., % Total cholesterol, mg/dL LDL cholesterol, mg/dL HDL cholesterol, mg/dL Triglyceride, mg/dL Apoprotein Al, mg/dL Apoprotein B, mg/dL Apoprotein CII, mg/dL Apoprotein CIII, mg/dL Apoprotein E, mg/dL
Diabetic Nephropathy (n = 18)
Control (n = 36)
Baseline
After Treatment
18 18 59 * 12 23.4 !I 3.7 NA
8 10 60 ? 12 23.5 ? 3.0 831 ? 1945
1067 ? 2627
97 * NA 184 ? 111 t 55 + 96 ? 127 k 74 + 3.9 +10.0 * 3.9 *
177 7.4 257 169 54 168 140 140 4.7 11.2 5.3
12 30 24 16 32 24 15 0.7 2.5 0.9
-+ f -c ? ? ? f 2 ? 2 *
60* 1.2 46” 37* 16 65* 31 38* 2.09 5.6 1.59
181 7.2 204 118 57 145 146 104 4.5 10.0 4.3
? * + + * 2 -c ? ? t r
76 1.3+ 52’ 38* 17 50 36 28* 1.7 3.0 0.8+
HbA,, = glycosylated hemoglobin; LDL = low-density lipoprotein; HDL = high-density lipoprotein. *P < 0.01 (control vs patient at baseline, by unpaired t test). +P < 0.05 (baseline vs after treatment, by paired t test). *P < 0.001 (baseline vs after treatment, by paired t test). §P < 0.05 (control vs patient at baseline, by unpaired t test).
36
T. /-/iron0 et ol.
but plasma triglyceride and HDL cholesterol levels were not significantly altered. Plasma apo B was significantly decreased after simvastatin treatment (P < 0.001) and apo E levels decreased slightly (P < 0.05) whereas apo AI, apo CII, and apo CIII levels were not altered significantly. Compliance with simvastatin therapy was excellent (100%) for all patients, and no adverse reactions to simvastatin treatment were reported. Concentrations of cholesterol, triglyceride, and apo B in VLDL, IDL, light LDL, and dense LDL fractions from the control subjects and diabetic patients with nephropathy (at baseline and after treatment) are shown in Table II. Diabetic patients initially had significantly higher levels of cholesterol and apo B in VLDL, light LDL, and dense LDL fractions (P < 0.05). VLDL cholesterol and triglyceride levels decreased significantly after simvastatin treatment (P < 0.05). VLDL apo B decreased but this result was not statistically significant (P = 0.078). IDL levels did not increase, but IDL cholesterol and IDL apo B levels decreased significantly after simvastatin treatment (P < 0.05 and P < 0.01, respectively). Cholesterol and apo B levels decreased significantly after treatment in both the light LDL (P < 0.001) and dense LDL fractions (P < 0.05). There was also a slight significant decrease in triglyceride level in the light LDL fraction (P< O.Ol), but the decrease was not significant in the dense LDL subfraction.
Table II. Concentrations of apoprotein B-containing lipoproteins control subjects and patients with diabetic nephropathy, after 6 months of treatment with simvastatin 10 mg/d. Control (n = 36) VLDL cholesterol VLDL triglyceride VLDL apoprotein B IDL cholesterol IDL triglyceride IDL apoprotein B Light LDL cholesterol Light LDL triglyceride Light LDL apoprotein B Dense LDL cholesterol Dense LDL triglyceride Dense LDL apoprotein B
13 k 45 k 14 * 5+-2 4kl 5k4 55 t 11 2 45 k 23 k 5kl 23 k
7 27 8
21 3 17 8 8
(mean +- SD mg/dL) in and changes in diabetic patients
Diabetic Nephropathy Baseline 22 k 98 2 20 k 6?4 4+-3 6t3 73 2 12 2 86 t 39 ? 624 56 k
After Treatment
11* 45* II’
20* 5 27* 17* 23*
VLDL = very low-density lipoprotein; IDL = intermediate-density lipoprotein. *P < 0.01 (control vs patient at baseline, by unpaired t test). +P < 0.05 (baseline vs after treatment, by paired t test). *P < 0.05 (control vs patient at baseline, by unpaired t test). §P < 0.01 (baseline vs after treatment, by unpaired t test). "P < 0.001 (baseline vs after treatment, by paired t test).
(n = 18)
lipoprotein;
19 k 75 2 15 k 4 k 3k3 4 + 52 +10 k 63 2 26 2 421 41 k
lo+ 40+ 7 3+ 29 15” 4§ 28” IO” 14+
LDL = low-density
37
CURRENT THERAPEUTICRESEARCH@
The percent reduction of cholesterol, triglyceride, and apo B in various lipoproteins after 6 months of treatment with simvastatin is illustrated in the figure. Simvastatin significantly decreased VLDL triglycerides by -18% (f’ < 0.05). Simvastatin treatment decreased plasma apo B by 25.0%. Simvastatin decreased VLDL cholesterol and IDL cholesterol by 17.5% and 22.1%, respectively (P < 0.05) and had a more profound effect on LDL cholesterol. The percent reduction of LDL cholesterol calculated by the Friedewald formula was 29.8%, which was similar to the decrease in dense LDL cholesterol and light LDL cholesterol separated by ultracentrifugation. There was no statistical difference between the percent reduction in dense LDL cholesterol and in light LDL cholesterol after simvastatin treatment (-27.3% and -28.1%, respectively). The percent reduction in light LDL apo B (-27.2%) was greater than the reduction in dense LDL apo B (-17.5%) but this difference did not attain statistical significance. The light LDL/dense LDL apo B ratio (w/w) was not altered by treatment with simvastatin (1.68 f 0.49 at baseline vs 1.71 k 1.15 after treatment). Similarly, the light LDL/dense LDL cholesterol ratio and triglyceride ratio (w/w) were not altered by simvastatin therapy (cholesterol: 2.15 f 1.10 at baseline vs 2.67 + 2.25 after treatment; triglycerides: 3.49 f 3.29 at baseline vs 3.73 f 4.25 after treatment). DISCUSSION
Several studies, including the present one, have demonstrated that patients with diabetic nephropathy have a more atherogenic lipoprotein profile than diabetic patients without nephropathy,5*6*11~12 which may explain the high incihypercholesteroldence of CHD in this population.‘3s’4 Hypertriglyceridemia, emia, and low HDL cholesterolemia are typical plasma lipid abnormalities in patients with diabetic nephropathy. 5,6~12LDL, as well as VLDL and IDL, can be taken up by the liver via LDL receptors.15 Since statins enhance the expression of hepatic LDL receptors, the VLDL and IDL concentrations could also theoretically be decreased by treatment with simvastatin. Gaw et all6 have reported that simvastatin significantly decreased small VLDL fraction (VLDL-II) and IDL concentrations by stimulating their catabolism in hypercholesterolemic subjects. Similarly, we found that simvastatin significantly reduced the VLDL and IDL concentrations in patients with diabetic nephropathy. Steiner and colleagues”~‘* have demonstrated that the number of VLDL particles (estimated from the VLDL apo B concentration) is closely associated with the severity of coronary atherosclerosis in diabetic patients with CHD, independent of other coronary risk factors, suggesting that VLDL is essentially as atherogenic as IDL or LDL. It is well known that IDL, a remnant of VLDL, is a potently atherogenic lipoprotein. lg Although we did not observe a significantly increased IDL level in our diabetic patients at baseline, several other studies have shown that the IDL levels are elevated in patients with diabetes5,” or renal failure.” The present study demonstrated that simvastatin could reduce IDL concentration even when it was not markedly high before treatment. The reduction in plasma apo E
38
T. Hirono et al.
Cholesterol
Plasma VLDL
IDL
Light LDL
Dense LDL
HDL
Triglyceride
-40
1
J
-22.3
Plasma
Apoprotein
n
VLDL
IDL
Li ht L8 L
Dense LDL
Light LDL
Dense LDL
B
-401
-t -26.4
Plasma
VLDL
IDL
Figure. Percent changes in cholesterol, triglyceride, and apoprotein B in very low-density lipoprotein (VLDL) (density cl.006 g/mL), intermediate-density lipoprotein (IDL) (d = 1.006-l .019 g/mL), light low-density lipoprotein (LDL) (d = 1 .019-l .044 g/mL), and dense LDL (d = 1.044-l .063 g/mL) in patients with diabetic nephropathy after treatment with simvastatin for 6 months. Continuous variables are expressed as mean +- SD. HDL = highdensity lipoprotein. *P-c0.001 versus baseline; +P < 0.05 versus baseline; *P < 0.01 versus baseline.
39
CURRENT THERAPEUTICRESEARCH~
levels may have reflected a reduction in the remnant lipoproteins by simvastatin, suggesting that this drug may be effective in reducing levels of potentially atherogenic lipoproteins such as VLDL and its remnants. As expected, simvastatin substantially reduced LDL cholesterol levels in patients with diabetic nephropathy. Because the plasma apo B level approximates the LDL apo B concentration, it can also be expected that simvastatin would substantially reduce LDL apo B levels. Considerable evidence has been accumulated to show that small dense LDL particles are more atherogenic than large buoyant LDL particles. 21-23Therefore, it was of interest to examine which LDL subfraction is more strongly affected by treatment with simvastatin. We found that simvastatin therapy reduced the cholesterol level in light LDL (large and buoyant) and dense LDL (small and dense) to a similar extent, and that it also reduced the apo B level in light LDL and dense LDL to a similar extent, Thus, the light LDL/dense LDL ratio for apo B and cholesterol were not affected by simvastatin treatment. These results suggest that simvastatin therapy reduces the total LDL mass without affecting the distribution of LDL subfractions. Previous studies have shown that simvastatin and pravastatin do not remarkably alter LDL size as determined by gradient gel electrophoresis 24S25a finding that appears to be in good agreement with the present data. LDL size is strictly regulated by the plasma triglyceride level,21S23Z26 and fibrates, which are potent hypotriglyceridemic agents, cause marked enlargement of LDL particles. 27 Because statins cause a less marked reduction in plasma triglyceride levels than do fibrates, there is less increase in LDL particle size during statin therapy than during fibrate therapy. In contrast, fibrates do not cause a marked reduction in LDL concentration, whereas statins do. Even if LDL particle size is enlarged (as is the case during treatment with fibrates), atherogenicity will remain unless the total LDL mass is also reduced significantly. Simvastatin markedly reduces both light LDL and dense LDL mass, indicating that the quantity of small, dense LDL particles is reduced even though LDL size is not significantly altered by this drug. Griffin et al23 have reported that the incidence of CHD substantially increases as the concentration of small, dense LDL (LDL-III fraction, d = 1.044-1.060 g/mL) increases. Therefore, the marked reduction in small, dense LDL mass induced by simvastatin should help reduce the high risk of CHD in patients with diabetic nephropathy, in whom LDL particle size is usually smaller.26 Finally, we failed to observe any effect of simvastatin on the urinary albumin excretion rate in our patients with overt diabetic nephropathy, although there have been reports that simvastatin can significantly reduce the urinary albumin excretion rate in diabetic patients with microalbuminuria.28~2g The present study was small and uncontrolled and therefore may lack the power to truly estimate the efficacy of simvastatin in this population. Further studies will be needed in a large number of subjects to determine whether simvastatin treatment can reduce urinary albumin excretion rate at different stages of diabetic nephropathy.
40
T. Hirana et al.
CONCLUSIONS
The results of this study suggest that simvastatin may reduce levels of atherogenie apo B-containing lipoproteins and small, dense LDL in diabetic patients with diabetic nephropathy. ACKNOWLEDGMENT
Financial support for the publication Pharmaceutical, Tokyo, Japan.
of this paper
was provided
by Banyu
REFERENCES 1. Valmadrid CT, Klein R, Moss SE, Klein BE. The risk of cardiovascular disease mortality associated with microalbuminuria and gross proteinuria in persons with olderonset diabetes mellitus. Arch Intern Med. 2000;160:1093-1100. 2. Jensen T, Borch-Johnsen K, Kofoed-Enevoldsen A, Deckert T. Coronary heart disease in young type 1 (insulin-dependent) diabetic patients with and without diabetic nephropathy: Incidence and risk factors. Diabetologia. 1987;30:144-148. 3. De Cosmo S, Bacci S, Piras GP, et al. High prevalence of risk factors for cardiovascular disease in parents of IDDM patients with albuminuria. Diabetologia. 1997;40: 1191-l 196. 4. Steiner G. Diabetes and atherosclerosis-a lipoprotein perspective. Diabetic Med. 1997;14(Suppl 3):S38-s44. 5. Attman PO, Knight-Gibson C, Tavella M, et al. The compositional abnormalities of lipoproteins in diabetic renal failure. Nephrol Dial Transplant. 1998;13:2833-2841. 6. Hirano T. Lipoprotein 71:S22-S24.
abnormalities
in diabetic nephropathy.
Kidney Int Suppl. 1999;
7. Pyorala K, Pedersen TR, Kjekshus J, et al. Cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease. A subgroup analysis of the Scandinavian Simvastatin Survival Study (4s). Diabetes Care. 1997;20:614-620. 8. Have1 RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345-1353. 9. Young SG, Smith RS, Hogle DM, et al. Two new monoclonal antibody-based enzymelinked assays of apolipoprotein B. Clin Chem. 1986;32:1484-1490. 10. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of lowdensity lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499-502. 11. Kashiwazaki K, Hirano T, Yoshino G, et al. Decreased release of lipoprotein lipase is associated with vascular endothelial damage in NIDDM patients with microalbuminuria. Diabetes Care. 1998;21:2016-2020. 12. Sakaue T, Hirano T, Yoshino G, et al. The influence of diabetic nephropathy on remnant lipoproteins. J Jpn Diabetes Sot. 1999;42:439-446. 13. Hahn R, Oette K, Mondorf H, et al. Analysis of cardiovascular risk factors in chronic hemodialysis patients with special attention to the hyperlipoproteinemias. Atherosclerosis. 1983;48:279-288. 14. Cheung AK, Sarnak MJ, Yan G, et al. Atherosclerotic cardiovascular disease risks in chronic hemodialysis patients. Kidney Int. 2000;58:353-362.
41
CURRENTTHERAPEUTIC RESEARCH@
ME, Garcia Z, et al. Uptake of cholesterol-rich remnant 15. Koo C, Wernette-Hammond lipoproteins by human monocyte-derived macrophages is mediated by low-density lipoprotein receptors. J Clin fnoest. 1988;81:1332-1340. 16. Gaw A, Packard CJ, Murray EF, et al. Effects of simvastatin on apoB metabolism and LDL subfraction distribution. Arterioscler Thromb. 1993;13:170-189. 17. Steiner G, Schwartz L, Shumak S, Poapst M. The association of increased levels of intermediate-density lipoproteins with smoking and with coronary artery disease. Circulation. 1987;75:124-130. in type 18. Tkac I, Kimball BP, Lewis G, et al. The severity of coronary atherosclerosis 2 diabetes mellitus is related to the number of circulating triglyceride-rich lipoprotein particles. Arterioscler Thromb Vast Biol. 1997;17:3633-3638. 19. Krauss RM, Lindgren FT, Williams PT, et al. Intermediate-density lipoproteins and progression of coronary artery disease in hypercholesterolaemic men. Lancet. 1987;2:62-66. 20. Attman PO, Alaupovic P, Tavella M, Knight-Gibson C. Abnormal lipid and apolipoprotein composition of major lipoprotein density classes in patients with chronic renal failure. Nephrol Dial Transplant. 1996;11:63-69. 21. Austin MA, Breslow JL, Hennekens CH, et al. Low-density lipoprotein subclass patterns and risk of myocardial infarction. JAMA. 1988;260:1917-1921. 22. Stampfer MJ, Krauss RM, Ma J, et al. A prospective study of triglyceride level, lowdensity lipoprotein particle diameter, and risk of myocardial infarction. JAMA. 1996; 276:882-888. 23. 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. 24. Zhao SP, Hollaar L, van’t Hooft FM, et al. Effect of simvastatin on the apparent size of LDL particles in patients with type IIB hyperlipoproteinemia. C/in Chim Acta. 1991;203:109-117. 25. Zambon S, Cortella A, Sartore G, et al. Pravastatin treatment in combined hyperlipidae mia. Effect on plasma lipoprotein levels and size. Eur J Clin Pharmacol. 1994;46:221-224. 26. Hirano T, Oi K, Sakai S, et al. High prevalence of small dense LDL in diabetic nephropathy is not directly associated with kidney damage: A possible role of postprandial lipemia. Atherosclerosis. 1998;141:77-85. 27. Hirano T, Kazumi T, Yoshino G. Long-term efficacy of bezafibrate in reduction of small, dense low-density lipoprotein by hypotriglyceridemic action. Curr Ther Res Clin Exp. 2000;61:127-136. 28. Tonolo G, Ciccarese M, Brizzi P, et al. Reduction of albumin excretion rate in normotensive microalbuminuric type 2 diabetic patients during long-term simvastatin treatment. Diabetes Care. 1997;20:1891-1895. diabetes 29. Fried LF, Forrest KY, Ellis D, et al. Lipid modulation in insulin-dependent mellitus: Effect on microvascular outcomes. J Diabetes Complications. 2001;15:113-119. Address correspondence to: Tsutomu Hirano, MD
First Department of Internal Medicine Showa University School of Medicine l-5-8 Hatanodai, Shinagawa-ku Tokyo 142-8666 Japan E-mail: [email protected]
42
JANUARY
2002
Effect of Simvastatin on Apoprotein B-Containing Lipoproteins in Patients with Diabetic Nephropathy Tsutomu Hirano, MD,’ Taro Sakaue, MD,’ Cen Yoshino, MD,2 Hiroko Takeuchi, MD,’ and Mitsuru Adachi, MD’ ‘First Department of Internal Medicine, Showa University, School of Medicine, and 2Department of Laboratory Medicine, Toho University School of Medicine, Tokyo, lapan
ABSTRACT Backgrourrd: Diabetic
patients with nephropathy usually have a more atherogenic lipoprotein profile than those without nephropathy, which may be associated with the substantially higher incidence of coronary heart disease (CHD) in this population. Simvastatin has been shown to significantly reduce the incidence of CHD events in diabetic patients. Objective: The purpose of this study was to evaluate the effect of simvastatin (10 mg/d) on atherogenic apoprotein (apo) B-containing lipoproteins in type 2 diabetic patients with nephropathy. Methods: Diabetic patients with nephropathy and a group of healthy control subjects matched for age, sex, and body weight were enrolled. Diabetic patients were administered simvastatin 10 mg/d for 6 months. Apo B-containing lipoproteins were sequentially separated by ultracentrifugation to yield very lowdensity lipoprotein (VLDL) (density cl.006 g/mL), intermediate-density lipoprotein (IDL) (1.006-l .019 g/mL), light low-density lipoprotein (LDL) (1.019-l .044 g/mL), and dense LDL (1.044-1.063 g/mL) fractions. Apo B in lipoproteins was measured by a sensitive enzyme-linked immunosorbent assay at baseline and after 6 months of simvastatin treatment. Results: A total of 18 patients with diabetic nephropathy and 36 matched controls were enrolled. The diabetic patients had significantly higher levels (P < 0.01) of total cholesterol, LDL cholesterol, triglycerides, and apo B compared with age- and weight-matched control subjects at baseline. The diabetic patients also had significantly higher levels (P < 0.05) of cholesterol and apo B in the VLDL, light LDL, and dense LDL fractions. Treatment with simvastatin for 6 months significantly reduced plasma total cholesterol by 21%, LDL cholesterol by 30%, and apo B by 25% (P < O.OOl), but did not affect urinary albumin excretion. Simvastatin significantly decreased both triglyceride and cholesterol levels in VLDL by 18% (P < 0.05), and cholesterol and apo B in IDL by 22% (P < 0.05) and 26% (P < 0.01). Simvastatin decreased both the light and dense Accepted for publication November 13, 200 I, Printed in the USA. Reproduction in whole or part is not permitted.
0011-393X/02/$1
9.00
33
CURRENT THERAPEUTICRESEARCHQ
LDL subfractions to a similar extent, reducing cholesterol and apo B in light LDL by 27% (P < 0.001) and in dense LDL by 28% (P < 0.01) and 18% (P < 0.05) respectively. The light LDL/dense LDL ratio for apo B and for cholesterol were not altered by simvastatin therapy. Conclusions: The results of this study suggest that simvastatin may reduce levels of atherogenic apo B-containing lipoproteins and small dense LDL in diabetic patients with nephropathy. Key words: simvastatin, diabetic nephropathy, low-density lipoprotein fraction, very low-density lipoprotein, intermediate-density lipoprotein (Cur-r Ther Res Clin Exp. 2002;63:33-42)
INTRODUCTION
The incidence of coronary heart disease (CHD) among patients with diabetes mellitus is markedly higher than among people without diabetes. In addition, it has been reported that the risk of death from CHD is substantially increased in diabetic patients with nephropathy compared with diabetic patients without nephropathy.1-3 Elevated levels of very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL), a high proportion of smaller and denser LDL particles (small, dense LDL), and reduced levels of high-density lipoprotein (HDL) have all been frequently reported in patients with diabetes,4 and this atherogenic lipoprotein profile becomes more prominent in patients with diabetic nephropathy.5v6 These abnormal plasma lipoprotein levels may contribute significantly to the increased risk of CHD in patients with diabetic nephropathy. The Scandinavian Simvastatin Survival Study showed that simvastatin-treated diabetic patients experienced significantly fewer CHD events compared with placebo-treated control patients. 7 Therefore, it is reasonable to assume that treatment with simvastatin can improve the atherogenic lipoprotein profile of patients with diabetic nephropathy. It is well known that statins, including simvastatin, can markedly reduce LDL levels by activating the LDL receptor pathway, but it is still not clear how statins affect VLDL, IDL, and the LDL subfractions. Therefore, we investigated the effect of simvastatin on these apo B-containing lipoproteins in patients with diabetic nephropathy.
PATIENTS AND METHODS
Outpatients with diabetic nephropathy as well as a group of healthy control subjects matched for age, body weight, and sex were enrolled. The control subjects were selected from among healthy persons undergoing annual medical examinations at the affiliated hospitals of Showa University. None of the control subjects had a history of hyperglycemia, abnormal hepatic or thyroid function, coronary heart disease, stroke, malignancy, or acute infection. The diabetic
34
T. Hirano et al.
patients fulfilled the World Health Organization criteria for type 2 diabetes mellitus. Diabetic nephropathy was diagnosed from persistent albuminuria in the presence of diabetic retinopathy without clinical or laboratory evidence of other kidney diseases. None of the subjects had clinical or laboratory evidence of abnormal hepatic or thyroid function, malignant tumors, infectious diseases, or renal failure (serum creatinine ~1.2 mg/dL). Each subject gave written informed consent, and the ethics committee of Showa University approved the study. The diabetic patients were treated with simvastatin 10 mg/d for 6 months. No other lipid-lowering drugs were used during the study. Patients were maintained on an isocaloric diet (25-30 kcal/kg ideal body weight) consisting of 17% protein, 23% fat, and 60% carbohydrates under the supervision of dietitians, and their compliance with the diet therapy was monitored. Three patients were being managed with dietary intervention alone (n = 6). Nine were taking sulfonylureas or a-glucosidase inhibitors and 3 were taking insulin; the dosages of these drugs were not changed during simvastatin therapy. Eleven diabetic patients were hypertensive; 5 were being treated with an angiotensin 11receptor antagonist (losartan 25 mg/d) and 6 with a calcium channel blocker (amlodipine 5 mg/d). Blood samples were collected after an overnight fast. Apo B-containing lipoproteins were separated from plasma by ultracentrifugation (Hitachi CP-65G, Hitachi Co, Ltd, Tokyo, Japan) using an RP 55T-708 rotor (Hitachi Co, Ltd) to yield VLDL (density [d] cl.006 g/mL), IDL (d = 1.006-1.019 g/mL), light LDL (d = 1.019-1.044 g/mL), and dense LDL (d = 1.044-1.063 g/mL) fractions according to the method of Have1 et al.” The apo B concentration in each lipoprotein fraction was measured by sensitive enzyme-linked immunosorbent assay (ELISA).’ Glycosylated hemoglobin (HbA,J, fasting plasma glucose, albumin, blood urea nitrogen, creatinine, and lipids in plasma were measured by standard laboratory procedures. Plasma levels of apo AI, AII, B, CII, CIII, and E were determined by immunoturbidimetric assay @aiichi Chemicals Co, Tokyo, Japan). The urinary albumin concentration was measured by the latex turbidimetric immunoassay method using a commercially available kit (LA-system, AIC Co, Tokyo, Japan). Plasma triglyceride and cholesterol levels were measured by enzymatic methods. HDL cholesterol was measured after polyanion precipitation of apo B-containing lipoproteins from the plasma. LDL cholesterol level was estimated using the Friedewald formula.” Statistical
Analysis
The Student paired t test was used to assess differences between plasma parameters before and after simvastatin treatment in the patients with diabetic nephropathy. The unpaired t test was used to evaluate the significance of differences between control subjects and diabetic patients at baseline.
35
CURRENTTHERAPEUTIC RESEARCH~
RESULTS
A total of 18 diabetic patients and 36 matched controls were enrolled. Clinical characteristics of all the study subjects are shown in Table I. Sex ratio, age, and body mass index were comparable between the healthy controls and the diabetic patients with nephropathy, whereas the diabetic patients had significantly higher levels of total cholesterol, LDL cholesterol, triglyceride, and apo B (PC 0.01) compared with control subjects. Patients also had significantly higher levels of apo CII and apo E than the control subjects (P < 0.05), but had similar levels of HDL cholesterol, apo AI, and apo CIII. After 6 months of treatment with simvastatin, mean HbA,, decreased slightly but significantly (P < 0.05), with no changes in fasting plasma glucose level. The urinary albumin index was not altered by treatment with simvastatin. Changes in plasma lipids and apoproteins after 6 months’ treatment with simvastatin are shown in Table I. In patients with diabetic nephropathy, simvastatin normalized previously elevated levels of total cholesterol and LDL cholesterol (P < 0.001 vs baseline),
Table I. Clinical characteristics of control subjects and patients with diabetic nephropathy at baseline, and changes in plasma lipids and apoproteins in the diabetic patients after 6 months of treatment with simvastatin 10 mg/d.
Sex Male Female Age, y, mean 2 SD Body mass index, kg/m2 Urinary albumin index, mg/g creatinine Plasma glucose, mg/dL HbA,., % Total cholesterol, mg/dL LDL cholesterol, mg/dL HDL cholesterol, mg/dL Triglyceride, mg/dL Apoprotein Al, mg/dL Apoprotein B, mg/dL Apoprotein CII, mg/dL Apoprotein CIII, mg/dL Apoprotein E, mg/dL
Diabetic Nephropathy (n = 18)
Control (n = 36)
Baseline
After Treatment
18 18 59 * 12 23.4 !I 3.7 NA
8 10 60 ? 12 23.5 ? 3.0 831 ? 1945
1067 ? 2627
97 * NA 184 ? 111 t 55 + 96 ? 127 k 74 + 3.9 +10.0 * 3.9 *
177 7.4 257 169 54 168 140 140 4.7 11.2 5.3
12 30 24 16 32 24 15 0.7 2.5 0.9
-+ f -c ? ? ? f 2 ? 2 *
60* 1.2 46” 37* 16 65* 31 38* 2.09 5.6 1.59
181 7.2 204 118 57 145 146 104 4.5 10.0 4.3
? * + + * 2 -c ? ? t r
76 1.3+ 52’ 38* 17 50 36 28* 1.7 3.0 0.8+
HbA,, = glycosylated hemoglobin; LDL = low-density lipoprotein; HDL = high-density lipoprotein. *P < 0.01 (control vs patient at baseline, by unpaired t test). +P < 0.05 (baseline vs after treatment, by paired t test). *P < 0.001 (baseline vs after treatment, by paired t test). §P < 0.05 (control vs patient at baseline, by unpaired t test).
36
T. /-/iron0 et ol.
but plasma triglyceride and HDL cholesterol levels were not significantly altered. Plasma apo B was significantly decreased after simvastatin treatment (P < 0.001) and apo E levels decreased slightly (P < 0.05) whereas apo AI, apo CII, and apo CIII levels were not altered significantly. Compliance with simvastatin therapy was excellent (100%) for all patients, and no adverse reactions to simvastatin treatment were reported. Concentrations of cholesterol, triglyceride, and apo B in VLDL, IDL, light LDL, and dense LDL fractions from the control subjects and diabetic patients with nephropathy (at baseline and after treatment) are shown in Table II. Diabetic patients initially had significantly higher levels of cholesterol and apo B in VLDL, light LDL, and dense LDL fractions (P < 0.05). VLDL cholesterol and triglyceride levels decreased significantly after simvastatin treatment (P < 0.05). VLDL apo B decreased but this result was not statistically significant (P = 0.078). IDL levels did not increase, but IDL cholesterol and IDL apo B levels decreased significantly after simvastatin treatment (P < 0.05 and P < 0.01, respectively). Cholesterol and apo B levels decreased significantly after treatment in both the light LDL (P < 0.001) and dense LDL fractions (P < 0.05). There was also a slight significant decrease in triglyceride level in the light LDL fraction (P< O.Ol), but the decrease was not significant in the dense LDL subfraction.
Table II. Concentrations of apoprotein B-containing lipoproteins control subjects and patients with diabetic nephropathy, after 6 months of treatment with simvastatin 10 mg/d. Control (n = 36) VLDL cholesterol VLDL triglyceride VLDL apoprotein B IDL cholesterol IDL triglyceride IDL apoprotein B Light LDL cholesterol Light LDL triglyceride Light LDL apoprotein B Dense LDL cholesterol Dense LDL triglyceride Dense LDL apoprotein B
13 k 45 k 14 * 5+-2 4kl 5k4 55 t 11 2 45 k 23 k 5kl 23 k
7 27 8
21 3 17 8 8
(mean +- SD mg/dL) in and changes in diabetic patients
Diabetic Nephropathy Baseline 22 k 98 2 20 k 6?4 4+-3 6t3 73 2 12 2 86 t 39 ? 624 56 k
After Treatment
11* 45* II’
20* 5 27* 17* 23*
VLDL = very low-density lipoprotein; IDL = intermediate-density lipoprotein. *P < 0.01 (control vs patient at baseline, by unpaired t test). +P < 0.05 (baseline vs after treatment, by paired t test). *P < 0.05 (control vs patient at baseline, by unpaired t test). §P < 0.01 (baseline vs after treatment, by unpaired t test). "P < 0.001 (baseline vs after treatment, by paired t test).
(n = 18)
lipoprotein;
19 k 75 2 15 k 4 k 3k3 4 + 52 +10 k 63 2 26 2 421 41 k
lo+ 40+ 7 3+ 29 15” 4§ 28” IO” 14+
LDL = low-density
37
CURRENT THERAPEUTICRESEARCH@
The percent reduction of cholesterol, triglyceride, and apo B in various lipoproteins after 6 months of treatment with simvastatin is illustrated in the figure. Simvastatin significantly decreased VLDL triglycerides by -18% (f’ < 0.05). Simvastatin treatment decreased plasma apo B by 25.0%. Simvastatin decreased VLDL cholesterol and IDL cholesterol by 17.5% and 22.1%, respectively (P < 0.05) and had a more profound effect on LDL cholesterol. The percent reduction of LDL cholesterol calculated by the Friedewald formula was 29.8%, which was similar to the decrease in dense LDL cholesterol and light LDL cholesterol separated by ultracentrifugation. There was no statistical difference between the percent reduction in dense LDL cholesterol and in light LDL cholesterol after simvastatin treatment (-27.3% and -28.1%, respectively). The percent reduction in light LDL apo B (-27.2%) was greater than the reduction in dense LDL apo B (-17.5%) but this difference did not attain statistical significance. The light LDL/dense LDL apo B ratio (w/w) was not altered by treatment with simvastatin (1.68 f 0.49 at baseline vs 1.71 k 1.15 after treatment). Similarly, the light LDL/dense LDL cholesterol ratio and triglyceride ratio (w/w) were not altered by simvastatin therapy (cholesterol: 2.15 f 1.10 at baseline vs 2.67 + 2.25 after treatment; triglycerides: 3.49 f 3.29 at baseline vs 3.73 f 4.25 after treatment). DISCUSSION
Several studies, including the present one, have demonstrated that patients with diabetic nephropathy have a more atherogenic lipoprotein profile than diabetic patients without nephropathy,5*6*11~12 which may explain the high incihypercholesteroldence of CHD in this population.‘3s’4 Hypertriglyceridemia, emia, and low HDL cholesterolemia are typical plasma lipid abnormalities in patients with diabetic nephropathy. 5,6~12LDL, as well as VLDL and IDL, can be taken up by the liver via LDL receptors.15 Since statins enhance the expression of hepatic LDL receptors, the VLDL and IDL concentrations could also theoretically be decreased by treatment with simvastatin. Gaw et all6 have reported that simvastatin significantly decreased small VLDL fraction (VLDL-II) and IDL concentrations by stimulating their catabolism in hypercholesterolemic subjects. Similarly, we found that simvastatin significantly reduced the VLDL and IDL concentrations in patients with diabetic nephropathy. Steiner and colleagues”~‘* have demonstrated that the number of VLDL particles (estimated from the VLDL apo B concentration) is closely associated with the severity of coronary atherosclerosis in diabetic patients with CHD, independent of other coronary risk factors, suggesting that VLDL is essentially as atherogenic as IDL or LDL. It is well known that IDL, a remnant of VLDL, is a potently atherogenic lipoprotein. lg Although we did not observe a significantly increased IDL level in our diabetic patients at baseline, several other studies have shown that the IDL levels are elevated in patients with diabetes5,” or renal failure.” The present study demonstrated that simvastatin could reduce IDL concentration even when it was not markedly high before treatment. The reduction in plasma apo E
38
T. Hirono et al.
Cholesterol
Plasma VLDL
IDL
Light LDL
Dense LDL
HDL
Triglyceride
-40
1
J
-22.3
Plasma
Apoprotein
n
VLDL
IDL
Li ht L8 L
Dense LDL
Light LDL
Dense LDL
B
-401
-t -26.4
Plasma
VLDL
IDL
Figure. Percent changes in cholesterol, triglyceride, and apoprotein B in very low-density lipoprotein (VLDL) (density cl.006 g/mL), intermediate-density lipoprotein (IDL) (d = 1.006-l .019 g/mL), light low-density lipoprotein (LDL) (d = 1 .019-l .044 g/mL), and dense LDL (d = 1.044-l .063 g/mL) in patients with diabetic nephropathy after treatment with simvastatin for 6 months. Continuous variables are expressed as mean +- SD. HDL = highdensity lipoprotein. *P-c0.001 versus baseline; +P < 0.05 versus baseline; *P < 0.01 versus baseline.
39
CURRENT THERAPEUTICRESEARCH~
levels may have reflected a reduction in the remnant lipoproteins by simvastatin, suggesting that this drug may be effective in reducing levels of potentially atherogenic lipoproteins such as VLDL and its remnants. As expected, simvastatin substantially reduced LDL cholesterol levels in patients with diabetic nephropathy. Because the plasma apo B level approximates the LDL apo B concentration, it can also be expected that simvastatin would substantially reduce LDL apo B levels. Considerable evidence has been accumulated to show that small dense LDL particles are more atherogenic than large buoyant LDL particles. 21-23Therefore, it was of interest to examine which LDL subfraction is more strongly affected by treatment with simvastatin. We found that simvastatin therapy reduced the cholesterol level in light LDL (large and buoyant) and dense LDL (small and dense) to a similar extent, and that it also reduced the apo B level in light LDL and dense LDL to a similar extent, Thus, the light LDL/dense LDL ratio for apo B and cholesterol were not affected by simvastatin treatment. These results suggest that simvastatin therapy reduces the total LDL mass without affecting the distribution of LDL subfractions. Previous studies have shown that simvastatin and pravastatin do not remarkably alter LDL size as determined by gradient gel electrophoresis 24S25a finding that appears to be in good agreement with the present data. LDL size is strictly regulated by the plasma triglyceride level,21S23Z26 and fibrates, which are potent hypotriglyceridemic agents, cause marked enlargement of LDL particles. 27 Because statins cause a less marked reduction in plasma triglyceride levels than do fibrates, there is less increase in LDL particle size during statin therapy than during fibrate therapy. In contrast, fibrates do not cause a marked reduction in LDL concentration, whereas statins do. Even if LDL particle size is enlarged (as is the case during treatment with fibrates), atherogenicity will remain unless the total LDL mass is also reduced significantly. Simvastatin markedly reduces both light LDL and dense LDL mass, indicating that the quantity of small, dense LDL particles is reduced even though LDL size is not significantly altered by this drug. Griffin et al23 have reported that the incidence of CHD substantially increases as the concentration of small, dense LDL (LDL-III fraction, d = 1.044-1.060 g/mL) increases. Therefore, the marked reduction in small, dense LDL mass induced by simvastatin should help reduce the high risk of CHD in patients with diabetic nephropathy, in whom LDL particle size is usually smaller.26 Finally, we failed to observe any effect of simvastatin on the urinary albumin excretion rate in our patients with overt diabetic nephropathy, although there have been reports that simvastatin can significantly reduce the urinary albumin excretion rate in diabetic patients with microalbuminuria.28~2g The present study was small and uncontrolled and therefore may lack the power to truly estimate the efficacy of simvastatin in this population. Further studies will be needed in a large number of subjects to determine whether simvastatin treatment can reduce urinary albumin excretion rate at different stages of diabetic nephropathy.
40
T. Hirana et al.
CONCLUSIONS
The results of this study suggest that simvastatin may reduce levels of atherogenie apo B-containing lipoproteins and small, dense LDL in diabetic patients with diabetic nephropathy. ACKNOWLEDGMENT
Financial support for the publication Pharmaceutical, Tokyo, Japan.
of this paper
was provided
by Banyu
REFERENCES 1. Valmadrid CT, Klein R, Moss SE, Klein BE. The risk of cardiovascular disease mortality associated with microalbuminuria and gross proteinuria in persons with olderonset diabetes mellitus. Arch Intern Med. 2000;160:1093-1100. 2. Jensen T, Borch-Johnsen K, Kofoed-Enevoldsen A, Deckert T. Coronary heart disease in young type 1 (insulin-dependent) diabetic patients with and without diabetic nephropathy: Incidence and risk factors. Diabetologia. 1987;30:144-148. 3. De Cosmo S, Bacci S, Piras GP, et al. High prevalence of risk factors for cardiovascular disease in parents of IDDM patients with albuminuria. Diabetologia. 1997;40: 1191-l 196. 4. Steiner G. Diabetes and atherosclerosis-a lipoprotein perspective. Diabetic Med. 1997;14(Suppl 3):S38-s44. 5. Attman PO, Knight-Gibson C, Tavella M, et al. The compositional abnormalities of lipoproteins in diabetic renal failure. Nephrol Dial Transplant. 1998;13:2833-2841. 6. Hirano T. Lipoprotein 71:S22-S24.
abnormalities
in diabetic nephropathy.
Kidney Int Suppl. 1999;
7. Pyorala K, Pedersen TR, Kjekshus J, et al. Cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease. A subgroup analysis of the Scandinavian Simvastatin Survival Study (4s). Diabetes Care. 1997;20:614-620. 8. Have1 RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345-1353. 9. Young SG, Smith RS, Hogle DM, et al. Two new monoclonal antibody-based enzymelinked assays of apolipoprotein B. Clin Chem. 1986;32:1484-1490. 10. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of lowdensity lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499-502. 11. Kashiwazaki K, Hirano T, Yoshino G, et al. Decreased release of lipoprotein lipase is associated with vascular endothelial damage in NIDDM patients with microalbuminuria. Diabetes Care. 1998;21:2016-2020. 12. Sakaue T, Hirano T, Yoshino G, et al. The influence of diabetic nephropathy on remnant lipoproteins. J Jpn Diabetes Sot. 1999;42:439-446. 13. Hahn R, Oette K, Mondorf H, et al. Analysis of cardiovascular risk factors in chronic hemodialysis patients with special attention to the hyperlipoproteinemias. Atherosclerosis. 1983;48:279-288. 14. Cheung AK, Sarnak MJ, Yan G, et al. Atherosclerotic cardiovascular disease risks in chronic hemodialysis patients. Kidney Int. 2000;58:353-362.
41
CURRENTTHERAPEUTIC RESEARCH@
ME, Garcia Z, et al. Uptake of cholesterol-rich remnant 15. Koo C, Wernette-Hammond lipoproteins by human monocyte-derived macrophages is mediated by low-density lipoprotein receptors. J Clin fnoest. 1988;81:1332-1340. 16. Gaw A, Packard CJ, Murray EF, et al. Effects of simvastatin on apoB metabolism and LDL subfraction distribution. Arterioscler Thromb. 1993;13:170-189. 17. Steiner G, Schwartz L, Shumak S, Poapst M. The association of increased levels of intermediate-density lipoproteins with smoking and with coronary artery disease. Circulation. 1987;75:124-130. in type 18. Tkac I, Kimball BP, Lewis G, et al. The severity of coronary atherosclerosis 2 diabetes mellitus is related to the number of circulating triglyceride-rich lipoprotein particles. Arterioscler Thromb Vast Biol. 1997;17:3633-3638. 19. Krauss RM, Lindgren FT, Williams PT, et al. Intermediate-density lipoproteins and progression of coronary artery disease in hypercholesterolaemic men. Lancet. 1987;2:62-66. 20. Attman PO, Alaupovic P, Tavella M, Knight-Gibson C. Abnormal lipid and apolipoprotein composition of major lipoprotein density classes in patients with chronic renal failure. Nephrol Dial Transplant. 1996;11:63-69. 21. Austin MA, Breslow JL, Hennekens CH, et al. Low-density lipoprotein subclass patterns and risk of myocardial infarction. JAMA. 1988;260:1917-1921. 22. Stampfer MJ, Krauss RM, Ma J, et al. A prospective study of triglyceride level, lowdensity lipoprotein particle diameter, and risk of myocardial infarction. JAMA. 1996; 276:882-888. 23. 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. 24. Zhao SP, Hollaar L, van’t Hooft FM, et al. Effect of simvastatin on the apparent size of LDL particles in patients with type IIB hyperlipoproteinemia. C/in Chim Acta. 1991;203:109-117. 25. Zambon S, Cortella A, Sartore G, et al. Pravastatin treatment in combined hyperlipidae mia. Effect on plasma lipoprotein levels and size. Eur J Clin Pharmacol. 1994;46:221-224. 26. Hirano T, Oi K, Sakai S, et al. High prevalence of small dense LDL in diabetic nephropathy is not directly associated with kidney damage: A possible role of postprandial lipemia. Atherosclerosis. 1998;141:77-85. 27. Hirano T, Kazumi T, Yoshino G. Long-term efficacy of bezafibrate in reduction of small, dense low-density lipoprotein by hypotriglyceridemic action. Curr Ther Res Clin Exp. 2000;61:127-136. 28. Tonolo G, Ciccarese M, Brizzi P, et al. Reduction of albumin excretion rate in normotensive microalbuminuric type 2 diabetic patients during long-term simvastatin treatment. Diabetes Care. 1997;20:1891-1895. diabetes 29. Fried LF, Forrest KY, Ellis D, et al. Lipid modulation in insulin-dependent mellitus: Effect on microvascular outcomes. J Diabetes Complications. 2001;15:113-119. Address correspondence to: Tsutomu Hirano, MD
First Department of Internal Medicine Showa University School of Medicine l-5-8 Hatanodai, Shinagawa-ku Tokyo 142-8666 Japan E-mail: [email protected]
42