Clinical and Experimental Pharmacology and Physiology (2014) 41, 475–481
doi: 10.1111/1440-1681.12243
ORIGINAL ARTICLE
Impact of short-term low-dose atorvastatin on low-density lipoprotein and high-density lipoprotein subfraction phenotype Rui-Xia Xu,† Yuan-Lin Guo,† Xiao-Lin Li, Sha Li and Jian-Jun Li Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
SUMMARY Statins can significantly reduce low-density lipoprotein–cholesterol (LDL-C) and modestly raise or not alter high-density lipoprotein–cholesterol (HDL-C). However, their impact on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) subfractions has been less examined. The aim of the present study was to investigate the short-term impact of lowdose atorvastatin on HDL and LDL subfractions in humans. In this randomized study, data from 52 subjects were analysed. Thirty-seven patients with atherosclerosis were randomized to treatment with atorvastatin 10 mg/day (n = 17) or 20 mg/day (n = 20) for 8 weeks, with 15 healthy subjects without therapy used as a control group. The lipid profile and lipoprotein subfractions were determined using the Lipoprint system at baseline and at 8 weeks. The data suggest that atorvastatin treatment (10 and 20 mg/day) for 8 weeks significantly decreases LDL-C levels and reduces the cholesterol concentration of all LDL subfractions, which is accompanied by an increase of the mean LDL particle size. Although 10 mg/day atorvastatin treatment for 8 weeks had no impact on the HDL subfraction, 20 mg/day atorvastatin for 8 weeks significantly increased the cholesterol concentration of large HDL particles and decreased the cholesterol concentration of small HDL particles without changing serum HDL-C levels in patients with atherosclerosis. Therefore, the results suggest that 20 mg/day atorvastatin treatment for 8 weeks may result in a favourable modification of the HDL subfraction phenotype in addition to its effects on the cholesterol concentration of all LDL subfractions and mean LDL particle size. Key words: atherosclerosis, atorvastatin, high-density lipoprotein subfraction, low-density lipoprotein particle size, low-density lipoprotein subfraction.
INTRODUCTION High low-density lipoprotein–cholesterol (LDL-C) and low highdensity lipoprotein–cholesterol (HDL-C) are the most important predictors for future cardiovascular events.1–3 Therefore, in addition to lowering LDL-C, there is clinical interest in increasing serum HDL-C concentrations in patients because HDL-C, a wellestablished risk factor, has been suggested to be inversely associated with atherosclerosis and coronary heart disease (CHD).4 However, measurement of HDL-C, the cholesterol carried by high-density lipoprotein (HDL) particles, may not fully capture HDL-related risk.5,6 The recent failure of drugs that increase HDL-C without reducing cardiovascular events or atherosclerosis has fuelled interest in alternative indices of HDL quantity, such as HDL particles or apolipoprotein A-I or possibly the quality of HDL (i.e. HDL subfraction or particle size).7,8 Although statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors) have been shown to modestly raise or not alter HDL-C and to significantly reduce LDL-C in patients with coronary artery disease, their effects on lipoprotein subfraction profiles have been less investigated. Moreover, in ‘real world’ clinical practice, many patients with atherosclerotic cardiovascular disease have ‘desirable’ plasma levels of total cholesterol or LDL-C, but low HDL-C levels following statin therapy. Therefore, it is necessary to further explore the potential changes in HDL subfractions following statin treatment due to the lesser impact of these drugs on HDL-C. Atorvastatin is one of the most used statins in many countries, including China, with a common initial dose of 10 or 20 mg.9 Thus, the aim of the present study was to investigate the shortterm effect of conventional doses of atorvastatin (10 and 20 mg/ day) on the HDL subfraction phenotype in patients with atherosclerosis in a randomized manner.
RESULTS
Correspondence: Jian-Jun Li, Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100037, China. Email: [email protected] †
These two authors contributed equally to this study.
Received 28 January 2014; revision 29 March 2014; accepted 4 April 2014. © 2014 Wiley Publishing Asia Pty Ltd
Between July 2011 and September 2013, we consecutively screened and enrolled 41 patients (26 men and 15 women) with atherosclerosis from the Division of Dyslipidemia, Fu Wai Hospital and Cardiovascular Institute. Of these patients, 19 were randomized to the 10 mg/day atorvastatin group and 22 were randomized to the 20 mg/day atorvastatin group. Treatment continued for 8 weeks. During follow-up visits, two patients in the 10 mg/day atorvastatin group and two patients in the 20 mg/day atorvastatin group were excluded from the study because they
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required coronary aortic bypass grafting. Fifteen healthy volunteers who had no drug therapy were recruited to the study as a control group. Thus, data for a total of 52 subjects were analysed. Clinical characteristics The baseline clinical characteristics of the study subjects are summarized in Table 1. Patients in both atorvastatin treatment groups were significantly than the healthy controls (P < 0.05 for both). There were more patients with hypertension in the 20 mg atorvastatin group than in the control group (P < 0.05). Other baseline characteristics were well matched among the three groups. Changes in lipid profile There were no significant differences in baseline serum levels of total cholesterol (TC), triglycerides and LDL-C among the three groups (P > 0.05 for all). However, baseline serum concentrations of HDL-C in patients with atherosclerosis receiving either 10 or 20 mg/day atorvastatin were significantly lower than in the healthy control group (P < 0.05), whereas apolipoprotein (Apo) B levels were higher (P < 0.05). However, following 8 weeks treatment with 10 or 20 mg/day atorvastatin, serum levels of TC, triglycerides, LDL-C and ApoB had decreased significantly compared with baseline (P < 0.05 for all). As indicated in Table 2, LDL-C was decreased by 35.7% and 40.8% in the 10 and
Table 1 Baseline clinical characteristics of the study subjects Control (n = 15)
Demographics Age (years) 39.53 8.78 No. men (%) 8 (53.3) BMI (kg/m2) 22.74 2.03 Clinical profiles (n (%)) Hypertension (%) 2 (13) Diabetes (%) 0 (0) Family history 0 (0) of CHD (%) Smoker 4 (27) Laboratory values WBC count 6.29 1.62 (9109/L) Neutrophils 3.58 1.53 (9109/L) ESR (mm/h) 3.73 3.92 ALT (U/L) 23.61 15.57 AST (U/L) 18.47 5.18 Cr (lmol/L) 69.72 11.18 BUN (mmol/L) 5.29 1.43
Atorvastatin 10 mg/day (n = 17)
Atorvastatin 20 mg/day (n = 20)
53.27 9.65* 10 (58.8) 23.63 2.69
55.63 6.59* 11 (55.0) 27.11 3.32
7 (41) 3 (18) 1 (16)
13 (65)* 5 (25) 1 (8)
7 (41)
8 (40)
6.16 1.03
7.22 1.67
3.65 0.69
4.01 1.43
7.99 27.27 19.73 71.54 5.87
4.21 23.69 10.69 13.32 1.77
10.69 26.25 18.88 73.51 5.68
8.77 8.69 3.91 13.21 1.52
Data are given as either the mean SD or the number of subjects in each group, with percentages in parentheses. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BUN, blood urea nitrogen; CHD, coronary heart disease; Cr, creatinine; ESR, erythrocyte sedimentation rate; WBC, white blood cell. *P < 0.05 compared with control (ANOVA).
20 mg/day atorvastatin groups, respectively, compared with baseline, although there were no significant changes in serum HDL-C levels. Not surprisingly, there were no changes in any serum lipid parameters at different time points in the healthy control group (Table 2). Changes in cholesterol concentration of HDL subfractions The effects of 8 weeks treatment with 10 or 20 mg/day atorvastatin on the cholesterol mass of HDL particles are summarized in Table 2. As shown in Fig. 1b,e, 10 mg/day atorvastatin had no significant impact on either HDL-C levels or the cholesterol content of each HDL subfraction (P > 0.05 for all). Although serum levels of HDL-C did not change significantly from baseline in the 20 mg/day atorvastatin group (P > 0.05), there was a significant decrease in the cholesterol mass of small HDL particles. Moreover, as indicated in Table 2 and Fig. 1c,f, there was a significant decline in the proportion of small HDL particles in the 20 mg/day atorvastatin group. Meanwhile, there was a significant increase in the cholesterol mass of large HDL particles accompanied by an increase in the proportion of large HDL particles (P < 0.01 for both). There were no significant differences in HDL-C levels or the cholesterol concentration of HDL particles between baseline and 8 weeks in the control group (Table 2; Fig. 1a,d; P > 0.05 for both). Changes in cholesterol concentration of LDL subfractions and mean LDL particle size The changes in the cholesterol mass of LDL particles and mean LDL particle size in patients following atorvastatin treatment and in control subjects without any therapy are given in Table 2. The data suggest that there were no change in the cholesterol mass of LDL particles and mean LDL particle size from baseline to the end of the study (8 weeks) in control subjects. In contrast, there was a significant decrease in the cholesterol concentration of large LDL particles, intermediate LDL particles and small dense LDL particles in patients who received either 10 or 20 mg/day atorvastatin for 8 weeks (P < 0.05 for all; Table 2; Fig. 2a–c). A significant decrease in proportion of each LDL subfraction over the total cholesterol mass was accordingly observed in these patients (Table 2; Fig. 2d–f). However, there was no significant change in the mean LDL particle size at 8 weeks in the control group compared with baseline (from 271 3 to 271 3 A; P > 0.05). In contrast, the mean LDL particle size increased significantly from 267 4 to 272 2 A (P < 0.01) after 8 weeks treatment with 10 mg/day atorvastatin and from 267 4 to 272 3 A after 8 weeks treatment with 20 mg/day atorvastatin (P < 0.01; Table 2). There were no significant differences in the cholesterol concentration, the proportion of each LDL subfraction and mean LDL particle size between the 10 and 20 mg/day atorvastatin groups.
DISCUSSION The impact of statins on subfractons of LDL and HDL has been reported previously,10,11 but the effects of 10 and 20 mg/day atorvastatin have not been reported. In the present study, the data indicate that treatment with 20 but not 10 mg/day atorvastatin for
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Table 2 Dose effect of atorvastatin on lipoprotein subfractions and lipid parameters Healthy controls (n = 15)
Baseline TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) Large HDL-C (mg/dL) Intermediate HDL-C (mg/dL) Small HDL-C (mg/dL) Large HDL (%) Intermediate HDL (%) Small HDL (%) LDL-C (mg/dL) Large LDL-C (mg/dL) Intermediate LDL-C (mg/dL) Small LDL-C (mg/dL) Large LDL (%) Intermediate LDL (%) Small LDL (%) Mean LDL size ( A) IDL-C (mg/dL) IDL (%) VLDL-C (mg/dL) VLDL (%) ApoA-I (g/L) ApoB (g/L) Lp (a) (mg/L) FFA (mmol/L)
195 135 56.73 25.58 24.75 6.92 42.08 44.75 13.25 101 30.50 14.08 2.50 15.84 7.26 1.34 271 49.25 25.84 37.00 19.34 1.79 0.76 219 0.44
25 81 10.84 8.60 5.34 1.93 10.71 6.55 4.41 21 7.65 8.25 2.47 2.93 3.95 1.10 3 10.39 2.29 8.33 3.71 0.36 0.31 251 0.29
Patients with atherosclerosis Atorvastatin 10 mg/day (n = 17)
8 weeks 191 116 55.11 24.45 23.73 6.55 41.91 45.00 13.27 102 30.00 14.09 2.55 15.67 7.30 1.35 271 49.45 27.14 38.67 18.55 1.70 0.75 230 0.40
26 61 9.11 8.56 4.20 1.51 11.21 6.81 4.63 21 7.81 8.65 2.58 3.01 4.14 1.16 3 10.88 1.99 9.09 4.37 0.30 0.15 263 0.12
Baseline 180 191 44.42 14.27 21.45 9.64 31.18 45.73 20.91 105 25.73 20.45 6.36 14.63 11.13 3.41 267 42.27 23.47 35.27 19.69 1.56 1.12 200 0.38
34 113 8.08† 4.45 3.72 2.38 7.39 8.68 5.80 29 7.13 9.26 6.02 4.20 3.96 2.95 4 11.29 3.31 7.07 2.26 0.24 0.20† 259 0.12
8 weeks 123 113 43.61 15.55 20.73 8.18 34.09 46.91 18.73 67.58 19.28 7.00 1.60 12.89 5.65 1.88 272 28.09 22.53 23.64 19.30 1.49 0.65 170 0.40
15*** 45* 10.19† 6.82 4.29 2.14 7.69 3.62 5.14 11.61*** 4.38** 2.72** 0.77* 2.76** 2.01*** 0.61* 2** 4.97*** 2.29 3.59*** 2.26 0.31 0.17* 191 0.28
Atorvastatin 20 mg/day (n = 20)
Baseline 179 188 42.59 11.20 20.35 10.25 26.35 48.85 25.00 114 31.90 19.00 4.50 17.89 10.33 2.34 267 44.50 24.81 33.80 18.96 1.42 1.25 117 0.32
31 112 9.79† 4.38 4.59 3.35 6.92 2.87 5.59 29 8.41 9.52 4.25 3.29 4.16 2.23 4 9.73 2.49 4.98 1.46 0.20 0.28† 125 0.13
8 weeks 123 124 41.74 12.75 19.35 8.65 30.80 47.60 21.50 67.35 18.70 6.80 1.50 14.96 5.26 1.03 272 30.50 24.31 24.70 19.91 1.26 0.67 133 0.33
20*** 45* 8.49† 4.57** 4.57 2.83* 5.99** 3.22 4.91** 20.48*** 4.27*** 4.64** 2.17** 2.52* 2.97** 1.47** 3** 6.79*** 3.89 4.00** 2.65 0.37 0.20* 96 0.26
Data are the mean SD. ApoA-I, apoprotein AI; ApoB, apoprotein B; FFA, free fatty acid; HDL-C, high-density lipoprotein–cholesterol; IDL, intermediate density lipoprotein; LDL-C, low-density lipoprotein–cholesterol; Lp(a), lipoprotein (a); TC, total cholesterol; TG triglycerides; VLDL, very low-density lipoprotein. *P < 0.05, **P < 0.01, ***P < 0.001 compared with baseline (at the same dose; paired samples t-test); †P < 0.05 compared with control (at the same time point; ANOVA).
8 weeks can significantly change the HDL subfraction phenotype. However, similar to a previous study,12 our data also demonstrated that atorvastatin treatment for 8 weeks with either 10 or 20 mg/day can effectively lower serum concentrations of LDL-C, accompanied by a decrease in the cholesterol concentration of all LDL subfractions. These results suggest that a dose of 20 mg/day atorvastatin is likely to have better outcomes with regard to HDL subfraction phenotypes than a dose of 10 mg/day atorvastatin. The emerging opinion is that the quality of HDL particles may be more important than their quantity. Although the effect of statins on HDL-C has been studied extensively, only a few studies have investigated the effect of statins on HDL subfractions and the results were not consistent. For example, a recent study by Kostapanos et al.10 demonstrated that 12 weeks treatment with either 10 or 20 mg/day rosuvastatin in a population without evidence of cardiovascular disease increased HDL-C in a dosedependent manner, accompanied by an increase in only the cholesterol mass of the large HDL particles. Another study in patients with mixed dyslipidaemia and metabolic syndrome showed that high-dose rosuvastatin (40 mg/day) combined with fenofibrate treatment for 12 weeks significantly raised HDL-C levels, accompanied by increases in the cholesterol concentration of small HDL particles.11 However, Toth et al.13 reported that treatment with 40 mg/day simvastatin in combination with niacin
resulted in a significant reduction in the number of small HDL particles and an increase in the number of large HDL particles as well as a marked increase in HDL particle size. Another study found that 10 mg/day atorvastatin for 6 weeks increased HDL-C by 10% in patients with type 2 diabetes mellitus (T2DM) and mixed hyperlipoproteinaemia,14 whereas another study used the same drug at the same dose for 8 weeks and reported an increase in HDL-C of 3% in patients with combined hyperlipidaemia and dense LDL.15 In the present study, the serum HDL-C level was not altered by 10 or 20 mg/day atorvastatin treatment for 8 weeks in patients with atherosclerosis, although 20 mg/day atorvastatin treatment significantly increased the cholesterol concentration of large HDL particles and decreased that of small HDL particles, accompanied by an increase in the proportion of the cholesterol concentration of large HDL particles and a reduction in the proportion of small HDL particles. However, 10 mg/day atorvastatin treatment had no effect on the HDL subfraction phenotype. These results indicate that atorvastatin can change small HDL particles towards large HDL particles with less atherogenic potential and that the effect of atorvastatin on HDL subfractions was somewhat dose dependent in patients with atherosclerosis. The most frequently administered doses of atorvastatin in clinical practice are low or medium doses of 10 or 20 mg/day, especially in China. Thus, the present study addressed the issue of the effects on
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(d)
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Fig. 1 Effects of atorvastatin on high-density lipoprotein (HDL) subfractions analysed by ANOVA or paired samples t-test in (a,d) the control group, (b,d) patients receiving 10 mg/day atorvastatin and (c,f) patients receiving 20 mg/day atorvastatin. (□), large high-density lipoprotein–cholesterol (HDL-C); ( ), intermediate HDL-C; (■), small HDL-C. (a,d) There were no significant differences in HDL subfractions between baseline and at the 8 week follow up in the control group (P > 0.05 for all). (b,e) At 10 mg/day, atorvastatin had no significant effect on any HDL subfraction (P > 0.05 for all). (c,f) After 8 weeks treatment with 20 mg/day atorvastatin, there was a significant increase in the cholesterol mass of the large HDL-C subfraction and a decrease in the cholesterol mass of the small HDL-C subfraction (P = 0.021 and P = 0.004, respectively), accompanied by an increase in the proportion of large HDL particles and a decrease in the proportion of small HDL particles (P = 0.002 and P = 0.001, respectively). Data are the mean SD. *P < 0.05, **P < 0.01 compared with baseline (at the same dose).
lipoprotein subfractions, particularly HDL, following the shortterm (8 weeks) use of low-dose (10 and 20 mg/day) atorvastatin. One of the principle targets in preventive cardiology is lowering LDL-C.16 Nevertheless, recent studies have demonstrated that the content of LDL particles in LDL-C exhibits considerable interindividual variation.17,18 Consequently, patients with the same LDL-C levels but different amounts of LDL particles differ in terms of absolute risk of coronary artery disease (CAD). Therefore, assessment of LDL subfractions and LDL particle size has been proposed as a more reliable method to quantify atherogenicity of the lipoprotein fraction.19 In the present study we demonstrated that 10 and 20 mg/day atorvastatin treatment effectively reduces serum LDL-C levels by decreasing the cholesterol concentration of all LDL subfractions. It should be noted that the effect was not significantly different between these two doses of atorvastatin, indicating that low-dose atorvastatin is robust in reducing LDL-C and LDL subfarctions. In particular, the present results are similar to those reported in several earlier studies, which found that atorvastatin decreased all LDL subfractions in patients with T2DM and mixed hyperlipoproteinaemia, patients with hypercholesterolaemia and healthy controls.12,14 More importantly, a significant increase in mean LDL particle size after 10 and 20 mg/day atorvastatin treatment was observed in the present study. Furthermore, we found a significant decrease in three LDL subfractions (i.e. large, intermediate and small LDL particles). It has been reported that atorvastatin reduces the risk of CAD not only by decreasing cholesterol concentration in all LDL
subfractions, but also by increasing the mean LDL particle size. In another study, neither conventional (10 mg/day) nor highdose (80 mg/day) atorvastatin affected LDL mean peak diameter measured by polyacrylamide gradient gel electrophoresis, although the two doses of atorvastatin decreased cholesterol concentrations in all LDL subfractions in patients with T2DM.20 A slight, albeit non-significant increase, in LDL size, measured by nuclear magnetic resonance (NMR) spectroscopy, was found in response to atorvastatin in T2DM.21 It was reported that atorvastatin markedly reduced all LDL subclasses, resulting in a significant reduction in the number of LDL particles, as well as an increase in LDL particle size, as evaluated by NMR,22,23 findings that are very similar to those of the present study. Moreover, the mechanisms underlying the changes in LDL and HDL subfractions in response to statin treatment are still not fully understood. It has been reported that cholesteryl ester transfer protein (CETP) may contribute to the formation of small dense LDL particles via an indirect mechanism involving an enhanced cholesteryl ester transfer from HDL to large VLDL1, which are regarded as precursors of small dense LDL.20 Moreover, it has been shown that both plasma CETP mass and cholesteryl ester transfer out of HDL decrease in response to statin treatment.6 Inhibition of this process would favour an increase in LDL diameter and a density shift towards more buoyant LDL in response to statin therapy, together with a decrease in triglyceride-rich lipoproteins, an important substrate for CETP.20 There are several limitations to the present study. First, the study had a relatively small sample size from a single centre.
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Fig. 2 Effects of atorvastatin on low-density lipoprotein (LDL) subfractions analysed by ANOVA or paired samples t-test in (a,d) the control group, (b,d) patients receiving 10 mg/day atorvastatin and (c,f) patients receiving 20 mg/day atorvastatin. (□), large low-density lipoprotein–cholesterol (LDL-C); ( ), intermediate LDL-C; (■), small LDL-C. (a,d) There were no significant differences in LDL subfractions between baseline and at the 8 week follow up in the control group (P > 0.05 for all). (b,e) Afer 8 weeks treatment, 10 mg/day atorvastatin significantly decreased the cholesterol concentration of all LDL subfractions (P = 0.007, P < 0.001 and P = 0.012 for large, intermediate and small LDL-C, respectively), as well as the proportion of each LDL subfraction (P = 0.004, P < 0.001 and P = 0.013, respectively). (c,f) After 8 weeks treatment with 20 mg/day atorvastatin, there was a significant decrease in the cholesterol concentration of all LDL subfractions (P < 0.001, P = 0.001 and P = 0.006 for large, intermediate and small LDL-C, respectively), as well as the proportion of each LDL subfraction (P = 0.010, P = 0.002 and P = 0.003, respectively). Data are the mean SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with baseline (at the same dose).
The study protocol was reviewed and approved by the Ethics Committee of Fu Wai Hospital and Cardiovascular Institute, Beijing, China. All patients and control subjects provided informed consent.
receive either 10 mg/day (n = 17) or 20 mg/day (n = 20) atorvastatin for 8 weeks to evaluate the short-term and dose-dependent effects of low–moderate doses atorvastatin on HDL and LDL subfractions. Healthy subjects who had not received any drug treatment previously were enrolled as the control group (n = 15). Data regarding serum lipid profile and HDL and LDL subfractions were collected at baseline and at Week 8. The inclusion criteria for the present study were as follows: (i) definite diagnostic imaging evidence of atherosclerotic lesions detected by arterial ultrasound, coronary chest tomography or coronary angiography; (ii) no treatment history with statins or other drugs known to affect blood lipids within the previous 4 weeks; and (iii) age 18–70 years. Patients with triglycerides ≥ 500 mg/dL (5.6 mmol/L), previous acute coronary syndrome within 1 month, serious heart failure or arrhythmia, infectious disease within 1 month, serious liver or renal dysfunction, autoimmune disease, malignant disease, those who were pregnant or lactating or those with a psychiatric disorder were excluded from the study. In addition, patients with laboratory values more than threefold the upper limit of normal (ULN) for aspartate aminotransferase or alanine aminotransferase, or more than fivefold the ULN for creatine phosphokinase were also excluded from the study.
Study design and population
Laboratory examinations
In the present randomized controlled open-label prospective study, 37 patients with atherosclerosis were randomly assigned to
Blood samples were obtained from the cubital vein at baseline and after 8 weeks treatment in all subjects after an overnight fast.
Second, the study duration was short, which may have enabled us to fully uncover the effects of atorvastatin on lipoprotein subfractions. Moreover, we did not use age-matched healthy volunteers as the control group. Furthermore, the impact of high-does atorvastatin on lipoprotein was not evaluated in the present study. Finally, the impact of atrovastatin-induced changes in lipoprotein subfractions on clinical outcome was not investigated because of the small sample size. In conclusion, the findings of the present randomized controlled open-label prospective study indicate that 8 weeks therapy with a clinically conventional dose of 20 mg/day atorvastatin results in a favourable modification of the HDL subfraction phenotype in addition to effects on the cholesterol concentration of all LDL subfractions and mean LDL particle size.
METHODS Ethics approval
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All samples were subsequently stored at –80°C and analysed immediately after thawing. Serum concentrations of TC, triglycerides, HDL-C, LDL-C, ApoA-I, ApoB, Lp(a) and free fatty acids (FFA) were measured using an automatic biochemistry analyser (Model 7150; Hitachi, Tokyo, Japan). Total cholesterol, triglycerides, HDL-C, LDL-C and FFA levels were measured by enzymatic assay, whereas ApoA-I, ApoB and Lp(a) were determined by a turbidimetric immunoassay. Analysis of HDL subfractions Blood samples were also used for subfraction analysis. The HDL subclass analysis was performed electrophoretically using highresolution 3% polyacrylamide gel tubes and the Lipoprint HDL System (Quantimetrix, Redondo Beach, CA, USA) according to the manufacturer’s instructions and as described previously.24,25 By this analysis, HDL was divided into 10 subfractions: Subfractions 1–3 represent large HDL particles, Subfractions 4–7 indicate intermediate HDL particles and Subfractions 8–10 indicate small HDL particles. The cholesterol concentration (mg/dL) of each HDL subfraction and the proportion of the cholesterol concentration of each HDL subfraction as a percentage of the HDLC concentration were subsequently determined. Analysis of LDL subfractions The cholesterol content of LDL subfractions was also determined electrophoretically using high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL System, as previously described.25,26 Using this assay, LDL was divided into seven subfractions: Subfraction 1 represents large LDL particles, Subfraction 2 indicates intermediate LDL particles and Subfractions 3–7 refer to small dense LDL particles. The intermediate density lipoprotein can also be detected and the cholesterol mass (mg/dL) of each lipoprotein subfraction, the mean LDL particle size ( A) and the proportion of the cholesterol mass of each lipoprotein subfraction as a percentage of the total cholesterol mass were determined by this assay. Statistical analysis Data are expressed as the mean SD. To compare the dose effect at the same time point, Student’s t-test and one-way ANOVA were used. The significance of differences between baseline and after treatment were evaluated by paired samples t-test. Statistical significance was defined as P < 0.05. All statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).
ACKNOWLEDGEMENTS This work was supported, in part, by grants from the National Natural Scientific Foundation (81070171, 81241121, 81100118), Specialized Research Fund for the Doctoral Program of Higher Education of China (20111106110013, 20101106120007), Capital Special Foundation of Clinical Application Research (Z121107001012015), Capital Health Development Fund (2011400302), Beijing Natural Science Foundation (7131014) and Specialized Research Personnel Fund of Fu Wai Hospital (2012-FWXX02).
DISCLOSURE The authors declare no conflict of interests.
REFERENCES 1. Martin SS, Blaha MJ, Blankstein R et al. Dyslipidemia, coronary artery calcium, and incident atherosclerotic cardiovascular disease: Implications for statin therapy from the multi-ethnic study of atherosclerosis. Circulation 2014; 129: 77–86. 2. Li JJ, Yang P, Liu J et al. Impact of 10 mg rosuvastatin daily or alternate-day on lipid profile and inflammatory markers. Clin. Chim. Acta 2012; 413: 139–42. 3. Gotto AM Jr, Brinton EA. Assessing low levels of high-density lipoprotein cholesterol as a risk factor in coronary heart disease: A Working Group report and update. J. Am. Coll. Cardiol. 2004; 43: 717–24. 4. Vergeer M, Holleboom AG, Kastelein JJ et al. The HDL hypothesis: does high-density lipoprotein protect from atherosclerosis? J. Lipid Res. 2010; 51: 2058–73. 5. deGoma EM, deGoma RL, Rader DJ. Beyond high-density lipoprotein cholesterol levels evaluating high-density lipoprotein function as influenced by novel therapeutic approaches. J. Am. Coll. Cardiol. 2008; 51: 2199–211. 6. Mackey RH, Greenland P, Goff DC Jr et al. High-density lipoprotein cholesterol and particle concentrations, carotid atherosclerosis, and coronary events: MESA (Multi-Ethnic Study of Atherosclerosis). J. Am. Coll. Cardiol. 2012; 60: 508–16. 7. Rosenson RS, Brewer HB, Chapman JM et al. HDL measures, particle heterogeneity, proposed nomenclature, and relation to atherosclerotic cardiovascular events. Clin. Chem. 2011; 57: 392–410. 8. Rizzo JA1, Mallow PJ, Waters HC et al. Managing to low-density lipoprotein particles compared with low-density lipoprotein cholesterol: a cost-effectiveness analysis. J. Clin. Lipidol. 2013; 7: 642–52. 9. Guo YL, Liu J, Li JJ et al. A multi-center survey of achieving recommended lipid goals in Chinese patients with coronary artery disease in real world cardiovascular practice. Int. J. Cardiol. 2011; 153: 211–12. 10. Kostapanos MS, Milionis HJ, Filippatos TD et al. Dose-dependent effect of rosuvastatin treatment on HDL-subfraction phenotype in patients with primary hyperlipidemia. J. Cardiovasc. Pharmacol. Ther. 2009; 14: 5–13. 11. Agouridis AP, Kostapanos MS, Tsimihodimos V et al. Effect of rosuvastatin monotherapy or in combination with fenofibrate or x-3 fatty acids on lipoprotein subfraction profile in patients with mixed dyslipidaemia and metabolic syndrome. Int. J. Clin. Pract. 2012; 66: 843–53. 12. Geiss HC, Otto C, Schwandt P et al. Effect of atorvastatin on lowdensity lipoprotein subtypes in patients with different forms of hyperlipoproteinemia and control subjects. Metabolism 2001; 50: 983–8. 13. Toth PP, Thakker KM, Jiang P et al. Niacin extended-release/simvastatin combination therapy produces larger favorable changes in high-density lipoprotein particles than atorvastatin monotherapy. Vasc. Health Risk Manag. 2012; 8: 39–44. 14. Frost RJ, Otto C, Geiss HC et al. Effects of atorvastatin versus fenofibrate on lipoprotein profiles, low-density lipoprotein subfraction distribution, and hemorheologic parameters in type 2 diabetes mellitus with mixed hyperlipoproteinemia. Am. J. Cardiol. 2001; 87: 44–8. 15. Winkler K, Weltzien P, Friedrich I et al. Qualitative effect of fenofibrate and quantitative effect of atorvastatin on LDL profile in combined hyperlipidemia with dense LDL. Exp. Clin. Endocrinol. Diabetes 2004; 112: 241–7. 16. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Third Report of the National Cholesterol Education Program NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Final report. Circulation 2002; 106: 3143–421.
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Atorvastatin and LDL and HDL subfractions 17. Superko HR, Gadesam RR. Is it LDL particle size or number that correlates with risk for cardiovascular disease? Curr. Atheroscler. Rep. 2008; 10: 377–85. 18. Irving BA, Nair KS, Srinivasan M. Effects of insulin sensitivity, body composition, and fitness on lipoprotein particle sizes and concentrations determined by nuclear magnetic resonance. J. Clin. Endocrinol. Metab. 2011; 96: E713–18. 19. Harchaoui KE, van der Steeg WA, Stroes ES et al. Value of lowdensity lipoprotein particle number and size as predictors of coronary artery disease in apparently healthy men and women: the EPIC-Norfolk Prospective Population Study. J. Am. Coll. Cardiol. 2007; 49: 547–53. 20. Kappelle PJ, Dallinga-Thie GM, Dullaart RP et al. Atorvastatin treatment lowers fasting remnant-like particle cholesterol and LDL subfraction cholesterol without affecting LDL size in type 2 diabetes mellitus: Relevance for non-HDL cholesterol and apolipoprotein B guideline targets. Biochim. Biophys. Acta 2010; 1801: 89– 94. 21. Soedamah-Muthu SS, Colhoun HM, Thomason MJ et al. The effect of atorvastatin on serum lipids, lipoproteins and NMR spectroscopy
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defined lipoprotein subclasses in type 2 diabetic patients with ischaemic heart disease. Atherosclerosis 2003; 167: 243–55. Ikewaki K, Terao Y, Ozasa H et al. Effects of atorvastatin on nuclear magnetic resonance-defined lipoprotein subclasses and inflammatory markers in patients with hypercholesterolemia. J. Atheroscler. Thromb. 2009; 16: 51–6. Chung M, Lichtenstein AH, Ip S et al. Comparability of methods for LDL subfraction determination: A systematic review. Atherosclerosis 2009; 205: 342–8. Goliasch G, Oravec S, Blessberger H et al. Relative importance of different lipid risk factors for the development of myocardial infarction at a very young age (≤40 years of age). Eur. J. Clin. Invest. 2012; 42: 631–6. Christogiannis LG, Kostapanos MS, Tellis CC et al. Distinct effects of fixed combinations of valsartan with either amlodipine or hydrochlorothiazide on lipoprotein subfraction profile in patients with hypertension. J. Hum. Hypertens. 2013; 27: 44–50. Banuls C, Bellod L, Jover A et al. Comparability of two different polyacrylamide gel electrophoresis methods for the classification of LDL pattern type. Clin. Chim. Acta 2012; 413: 251–7.
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doi: 10.1111/1440-1681.12243
ORIGINAL ARTICLE
Impact of short-term low-dose atorvastatin on low-density lipoprotein and high-density lipoprotein subfraction phenotype Rui-Xia Xu,† Yuan-Lin Guo,† Xiao-Lin Li, Sha Li and Jian-Jun Li Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
SUMMARY Statins can significantly reduce low-density lipoprotein–cholesterol (LDL-C) and modestly raise or not alter high-density lipoprotein–cholesterol (HDL-C). However, their impact on high-density lipoprotein (HDL) and low-density lipoprotein (LDL) subfractions has been less examined. The aim of the present study was to investigate the short-term impact of lowdose atorvastatin on HDL and LDL subfractions in humans. In this randomized study, data from 52 subjects were analysed. Thirty-seven patients with atherosclerosis were randomized to treatment with atorvastatin 10 mg/day (n = 17) or 20 mg/day (n = 20) for 8 weeks, with 15 healthy subjects without therapy used as a control group. The lipid profile and lipoprotein subfractions were determined using the Lipoprint system at baseline and at 8 weeks. The data suggest that atorvastatin treatment (10 and 20 mg/day) for 8 weeks significantly decreases LDL-C levels and reduces the cholesterol concentration of all LDL subfractions, which is accompanied by an increase of the mean LDL particle size. Although 10 mg/day atorvastatin treatment for 8 weeks had no impact on the HDL subfraction, 20 mg/day atorvastatin for 8 weeks significantly increased the cholesterol concentration of large HDL particles and decreased the cholesterol concentration of small HDL particles without changing serum HDL-C levels in patients with atherosclerosis. Therefore, the results suggest that 20 mg/day atorvastatin treatment for 8 weeks may result in a favourable modification of the HDL subfraction phenotype in addition to its effects on the cholesterol concentration of all LDL subfractions and mean LDL particle size. Key words: atherosclerosis, atorvastatin, high-density lipoprotein subfraction, low-density lipoprotein particle size, low-density lipoprotein subfraction.
INTRODUCTION High low-density lipoprotein–cholesterol (LDL-C) and low highdensity lipoprotein–cholesterol (HDL-C) are the most important predictors for future cardiovascular events.1–3 Therefore, in addition to lowering LDL-C, there is clinical interest in increasing serum HDL-C concentrations in patients because HDL-C, a wellestablished risk factor, has been suggested to be inversely associated with atherosclerosis and coronary heart disease (CHD).4 However, measurement of HDL-C, the cholesterol carried by high-density lipoprotein (HDL) particles, may not fully capture HDL-related risk.5,6 The recent failure of drugs that increase HDL-C without reducing cardiovascular events or atherosclerosis has fuelled interest in alternative indices of HDL quantity, such as HDL particles or apolipoprotein A-I or possibly the quality of HDL (i.e. HDL subfraction or particle size).7,8 Although statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors) have been shown to modestly raise or not alter HDL-C and to significantly reduce LDL-C in patients with coronary artery disease, their effects on lipoprotein subfraction profiles have been less investigated. Moreover, in ‘real world’ clinical practice, many patients with atherosclerotic cardiovascular disease have ‘desirable’ plasma levels of total cholesterol or LDL-C, but low HDL-C levels following statin therapy. Therefore, it is necessary to further explore the potential changes in HDL subfractions following statin treatment due to the lesser impact of these drugs on HDL-C. Atorvastatin is one of the most used statins in many countries, including China, with a common initial dose of 10 or 20 mg.9 Thus, the aim of the present study was to investigate the shortterm effect of conventional doses of atorvastatin (10 and 20 mg/ day) on the HDL subfraction phenotype in patients with atherosclerosis in a randomized manner.
RESULTS
Correspondence: Jian-Jun Li, Division of Dyslipidemia, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100037, China. Email: [email protected] †
These two authors contributed equally to this study.
Received 28 January 2014; revision 29 March 2014; accepted 4 April 2014. © 2014 Wiley Publishing Asia Pty Ltd
Between July 2011 and September 2013, we consecutively screened and enrolled 41 patients (26 men and 15 women) with atherosclerosis from the Division of Dyslipidemia, Fu Wai Hospital and Cardiovascular Institute. Of these patients, 19 were randomized to the 10 mg/day atorvastatin group and 22 were randomized to the 20 mg/day atorvastatin group. Treatment continued for 8 weeks. During follow-up visits, two patients in the 10 mg/day atorvastatin group and two patients in the 20 mg/day atorvastatin group were excluded from the study because they
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required coronary aortic bypass grafting. Fifteen healthy volunteers who had no drug therapy were recruited to the study as a control group. Thus, data for a total of 52 subjects were analysed. Clinical characteristics The baseline clinical characteristics of the study subjects are summarized in Table 1. Patients in both atorvastatin treatment groups were significantly than the healthy controls (P < 0.05 for both). There were more patients with hypertension in the 20 mg atorvastatin group than in the control group (P < 0.05). Other baseline characteristics were well matched among the three groups. Changes in lipid profile There were no significant differences in baseline serum levels of total cholesterol (TC), triglycerides and LDL-C among the three groups (P > 0.05 for all). However, baseline serum concentrations of HDL-C in patients with atherosclerosis receiving either 10 or 20 mg/day atorvastatin were significantly lower than in the healthy control group (P < 0.05), whereas apolipoprotein (Apo) B levels were higher (P < 0.05). However, following 8 weeks treatment with 10 or 20 mg/day atorvastatin, serum levels of TC, triglycerides, LDL-C and ApoB had decreased significantly compared with baseline (P < 0.05 for all). As indicated in Table 2, LDL-C was decreased by 35.7% and 40.8% in the 10 and
Table 1 Baseline clinical characteristics of the study subjects Control (n = 15)
Demographics Age (years) 39.53 8.78 No. men (%) 8 (53.3) BMI (kg/m2) 22.74 2.03 Clinical profiles (n (%)) Hypertension (%) 2 (13) Diabetes (%) 0 (0) Family history 0 (0) of CHD (%) Smoker 4 (27) Laboratory values WBC count 6.29 1.62 (9109/L) Neutrophils 3.58 1.53 (9109/L) ESR (mm/h) 3.73 3.92 ALT (U/L) 23.61 15.57 AST (U/L) 18.47 5.18 Cr (lmol/L) 69.72 11.18 BUN (mmol/L) 5.29 1.43
Atorvastatin 10 mg/day (n = 17)
Atorvastatin 20 mg/day (n = 20)
53.27 9.65* 10 (58.8) 23.63 2.69
55.63 6.59* 11 (55.0) 27.11 3.32
7 (41) 3 (18) 1 (16)
13 (65)* 5 (25) 1 (8)
7 (41)
8 (40)
6.16 1.03
7.22 1.67
3.65 0.69
4.01 1.43
7.99 27.27 19.73 71.54 5.87
4.21 23.69 10.69 13.32 1.77
10.69 26.25 18.88 73.51 5.68
8.77 8.69 3.91 13.21 1.52
Data are given as either the mean SD or the number of subjects in each group, with percentages in parentheses. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BUN, blood urea nitrogen; CHD, coronary heart disease; Cr, creatinine; ESR, erythrocyte sedimentation rate; WBC, white blood cell. *P < 0.05 compared with control (ANOVA).
20 mg/day atorvastatin groups, respectively, compared with baseline, although there were no significant changes in serum HDL-C levels. Not surprisingly, there were no changes in any serum lipid parameters at different time points in the healthy control group (Table 2). Changes in cholesterol concentration of HDL subfractions The effects of 8 weeks treatment with 10 or 20 mg/day atorvastatin on the cholesterol mass of HDL particles are summarized in Table 2. As shown in Fig. 1b,e, 10 mg/day atorvastatin had no significant impact on either HDL-C levels or the cholesterol content of each HDL subfraction (P > 0.05 for all). Although serum levels of HDL-C did not change significantly from baseline in the 20 mg/day atorvastatin group (P > 0.05), there was a significant decrease in the cholesterol mass of small HDL particles. Moreover, as indicated in Table 2 and Fig. 1c,f, there was a significant decline in the proportion of small HDL particles in the 20 mg/day atorvastatin group. Meanwhile, there was a significant increase in the cholesterol mass of large HDL particles accompanied by an increase in the proportion of large HDL particles (P < 0.01 for both). There were no significant differences in HDL-C levels or the cholesterol concentration of HDL particles between baseline and 8 weeks in the control group (Table 2; Fig. 1a,d; P > 0.05 for both). Changes in cholesterol concentration of LDL subfractions and mean LDL particle size The changes in the cholesterol mass of LDL particles and mean LDL particle size in patients following atorvastatin treatment and in control subjects without any therapy are given in Table 2. The data suggest that there were no change in the cholesterol mass of LDL particles and mean LDL particle size from baseline to the end of the study (8 weeks) in control subjects. In contrast, there was a significant decrease in the cholesterol concentration of large LDL particles, intermediate LDL particles and small dense LDL particles in patients who received either 10 or 20 mg/day atorvastatin for 8 weeks (P < 0.05 for all; Table 2; Fig. 2a–c). A significant decrease in proportion of each LDL subfraction over the total cholesterol mass was accordingly observed in these patients (Table 2; Fig. 2d–f). However, there was no significant change in the mean LDL particle size at 8 weeks in the control group compared with baseline (from 271 3 to 271 3 A; P > 0.05). In contrast, the mean LDL particle size increased significantly from 267 4 to 272 2 A (P < 0.01) after 8 weeks treatment with 10 mg/day atorvastatin and from 267 4 to 272 3 A after 8 weeks treatment with 20 mg/day atorvastatin (P < 0.01; Table 2). There were no significant differences in the cholesterol concentration, the proportion of each LDL subfraction and mean LDL particle size between the 10 and 20 mg/day atorvastatin groups.
DISCUSSION The impact of statins on subfractons of LDL and HDL has been reported previously,10,11 but the effects of 10 and 20 mg/day atorvastatin have not been reported. In the present study, the data indicate that treatment with 20 but not 10 mg/day atorvastatin for
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Table 2 Dose effect of atorvastatin on lipoprotein subfractions and lipid parameters Healthy controls (n = 15)
Baseline TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) Large HDL-C (mg/dL) Intermediate HDL-C (mg/dL) Small HDL-C (mg/dL) Large HDL (%) Intermediate HDL (%) Small HDL (%) LDL-C (mg/dL) Large LDL-C (mg/dL) Intermediate LDL-C (mg/dL) Small LDL-C (mg/dL) Large LDL (%) Intermediate LDL (%) Small LDL (%) Mean LDL size ( A) IDL-C (mg/dL) IDL (%) VLDL-C (mg/dL) VLDL (%) ApoA-I (g/L) ApoB (g/L) Lp (a) (mg/L) FFA (mmol/L)
195 135 56.73 25.58 24.75 6.92 42.08 44.75 13.25 101 30.50 14.08 2.50 15.84 7.26 1.34 271 49.25 25.84 37.00 19.34 1.79 0.76 219 0.44
25 81 10.84 8.60 5.34 1.93 10.71 6.55 4.41 21 7.65 8.25 2.47 2.93 3.95 1.10 3 10.39 2.29 8.33 3.71 0.36 0.31 251 0.29
Patients with atherosclerosis Atorvastatin 10 mg/day (n = 17)
8 weeks 191 116 55.11 24.45 23.73 6.55 41.91 45.00 13.27 102 30.00 14.09 2.55 15.67 7.30 1.35 271 49.45 27.14 38.67 18.55 1.70 0.75 230 0.40
26 61 9.11 8.56 4.20 1.51 11.21 6.81 4.63 21 7.81 8.65 2.58 3.01 4.14 1.16 3 10.88 1.99 9.09 4.37 0.30 0.15 263 0.12
Baseline 180 191 44.42 14.27 21.45 9.64 31.18 45.73 20.91 105 25.73 20.45 6.36 14.63 11.13 3.41 267 42.27 23.47 35.27 19.69 1.56 1.12 200 0.38
34 113 8.08† 4.45 3.72 2.38 7.39 8.68 5.80 29 7.13 9.26 6.02 4.20 3.96 2.95 4 11.29 3.31 7.07 2.26 0.24 0.20† 259 0.12
8 weeks 123 113 43.61 15.55 20.73 8.18 34.09 46.91 18.73 67.58 19.28 7.00 1.60 12.89 5.65 1.88 272 28.09 22.53 23.64 19.30 1.49 0.65 170 0.40
15*** 45* 10.19† 6.82 4.29 2.14 7.69 3.62 5.14 11.61*** 4.38** 2.72** 0.77* 2.76** 2.01*** 0.61* 2** 4.97*** 2.29 3.59*** 2.26 0.31 0.17* 191 0.28
Atorvastatin 20 mg/day (n = 20)
Baseline 179 188 42.59 11.20 20.35 10.25 26.35 48.85 25.00 114 31.90 19.00 4.50 17.89 10.33 2.34 267 44.50 24.81 33.80 18.96 1.42 1.25 117 0.32
31 112 9.79† 4.38 4.59 3.35 6.92 2.87 5.59 29 8.41 9.52 4.25 3.29 4.16 2.23 4 9.73 2.49 4.98 1.46 0.20 0.28† 125 0.13
8 weeks 123 124 41.74 12.75 19.35 8.65 30.80 47.60 21.50 67.35 18.70 6.80 1.50 14.96 5.26 1.03 272 30.50 24.31 24.70 19.91 1.26 0.67 133 0.33
20*** 45* 8.49† 4.57** 4.57 2.83* 5.99** 3.22 4.91** 20.48*** 4.27*** 4.64** 2.17** 2.52* 2.97** 1.47** 3** 6.79*** 3.89 4.00** 2.65 0.37 0.20* 96 0.26
Data are the mean SD. ApoA-I, apoprotein AI; ApoB, apoprotein B; FFA, free fatty acid; HDL-C, high-density lipoprotein–cholesterol; IDL, intermediate density lipoprotein; LDL-C, low-density lipoprotein–cholesterol; Lp(a), lipoprotein (a); TC, total cholesterol; TG triglycerides; VLDL, very low-density lipoprotein. *P < 0.05, **P < 0.01, ***P < 0.001 compared with baseline (at the same dose; paired samples t-test); †P < 0.05 compared with control (at the same time point; ANOVA).
8 weeks can significantly change the HDL subfraction phenotype. However, similar to a previous study,12 our data also demonstrated that atorvastatin treatment for 8 weeks with either 10 or 20 mg/day can effectively lower serum concentrations of LDL-C, accompanied by a decrease in the cholesterol concentration of all LDL subfractions. These results suggest that a dose of 20 mg/day atorvastatin is likely to have better outcomes with regard to HDL subfraction phenotypes than a dose of 10 mg/day atorvastatin. The emerging opinion is that the quality of HDL particles may be more important than their quantity. Although the effect of statins on HDL-C has been studied extensively, only a few studies have investigated the effect of statins on HDL subfractions and the results were not consistent. For example, a recent study by Kostapanos et al.10 demonstrated that 12 weeks treatment with either 10 or 20 mg/day rosuvastatin in a population without evidence of cardiovascular disease increased HDL-C in a dosedependent manner, accompanied by an increase in only the cholesterol mass of the large HDL particles. Another study in patients with mixed dyslipidaemia and metabolic syndrome showed that high-dose rosuvastatin (40 mg/day) combined with fenofibrate treatment for 12 weeks significantly raised HDL-C levels, accompanied by increases in the cholesterol concentration of small HDL particles.11 However, Toth et al.13 reported that treatment with 40 mg/day simvastatin in combination with niacin
resulted in a significant reduction in the number of small HDL particles and an increase in the number of large HDL particles as well as a marked increase in HDL particle size. Another study found that 10 mg/day atorvastatin for 6 weeks increased HDL-C by 10% in patients with type 2 diabetes mellitus (T2DM) and mixed hyperlipoproteinaemia,14 whereas another study used the same drug at the same dose for 8 weeks and reported an increase in HDL-C of 3% in patients with combined hyperlipidaemia and dense LDL.15 In the present study, the serum HDL-C level was not altered by 10 or 20 mg/day atorvastatin treatment for 8 weeks in patients with atherosclerosis, although 20 mg/day atorvastatin treatment significantly increased the cholesterol concentration of large HDL particles and decreased that of small HDL particles, accompanied by an increase in the proportion of the cholesterol concentration of large HDL particles and a reduction in the proportion of small HDL particles. However, 10 mg/day atorvastatin treatment had no effect on the HDL subfraction phenotype. These results indicate that atorvastatin can change small HDL particles towards large HDL particles with less atherogenic potential and that the effect of atorvastatin on HDL subfractions was somewhat dose dependent in patients with atherosclerosis. The most frequently administered doses of atorvastatin in clinical practice are low or medium doses of 10 or 20 mg/day, especially in China. Thus, the present study addressed the issue of the effects on
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Fig. 1 Effects of atorvastatin on high-density lipoprotein (HDL) subfractions analysed by ANOVA or paired samples t-test in (a,d) the control group, (b,d) patients receiving 10 mg/day atorvastatin and (c,f) patients receiving 20 mg/day atorvastatin. (□), large high-density lipoprotein–cholesterol (HDL-C); ( ), intermediate HDL-C; (■), small HDL-C. (a,d) There were no significant differences in HDL subfractions between baseline and at the 8 week follow up in the control group (P > 0.05 for all). (b,e) At 10 mg/day, atorvastatin had no significant effect on any HDL subfraction (P > 0.05 for all). (c,f) After 8 weeks treatment with 20 mg/day atorvastatin, there was a significant increase in the cholesterol mass of the large HDL-C subfraction and a decrease in the cholesterol mass of the small HDL-C subfraction (P = 0.021 and P = 0.004, respectively), accompanied by an increase in the proportion of large HDL particles and a decrease in the proportion of small HDL particles (P = 0.002 and P = 0.001, respectively). Data are the mean SD. *P < 0.05, **P < 0.01 compared with baseline (at the same dose).
lipoprotein subfractions, particularly HDL, following the shortterm (8 weeks) use of low-dose (10 and 20 mg/day) atorvastatin. One of the principle targets in preventive cardiology is lowering LDL-C.16 Nevertheless, recent studies have demonstrated that the content of LDL particles in LDL-C exhibits considerable interindividual variation.17,18 Consequently, patients with the same LDL-C levels but different amounts of LDL particles differ in terms of absolute risk of coronary artery disease (CAD). Therefore, assessment of LDL subfractions and LDL particle size has been proposed as a more reliable method to quantify atherogenicity of the lipoprotein fraction.19 In the present study we demonstrated that 10 and 20 mg/day atorvastatin treatment effectively reduces serum LDL-C levels by decreasing the cholesterol concentration of all LDL subfractions. It should be noted that the effect was not significantly different between these two doses of atorvastatin, indicating that low-dose atorvastatin is robust in reducing LDL-C and LDL subfarctions. In particular, the present results are similar to those reported in several earlier studies, which found that atorvastatin decreased all LDL subfractions in patients with T2DM and mixed hyperlipoproteinaemia, patients with hypercholesterolaemia and healthy controls.12,14 More importantly, a significant increase in mean LDL particle size after 10 and 20 mg/day atorvastatin treatment was observed in the present study. Furthermore, we found a significant decrease in three LDL subfractions (i.e. large, intermediate and small LDL particles). It has been reported that atorvastatin reduces the risk of CAD not only by decreasing cholesterol concentration in all LDL
subfractions, but also by increasing the mean LDL particle size. In another study, neither conventional (10 mg/day) nor highdose (80 mg/day) atorvastatin affected LDL mean peak diameter measured by polyacrylamide gradient gel electrophoresis, although the two doses of atorvastatin decreased cholesterol concentrations in all LDL subfractions in patients with T2DM.20 A slight, albeit non-significant increase, in LDL size, measured by nuclear magnetic resonance (NMR) spectroscopy, was found in response to atorvastatin in T2DM.21 It was reported that atorvastatin markedly reduced all LDL subclasses, resulting in a significant reduction in the number of LDL particles, as well as an increase in LDL particle size, as evaluated by NMR,22,23 findings that are very similar to those of the present study. Moreover, the mechanisms underlying the changes in LDL and HDL subfractions in response to statin treatment are still not fully understood. It has been reported that cholesteryl ester transfer protein (CETP) may contribute to the formation of small dense LDL particles via an indirect mechanism involving an enhanced cholesteryl ester transfer from HDL to large VLDL1, which are regarded as precursors of small dense LDL.20 Moreover, it has been shown that both plasma CETP mass and cholesteryl ester transfer out of HDL decrease in response to statin treatment.6 Inhibition of this process would favour an increase in LDL diameter and a density shift towards more buoyant LDL in response to statin therapy, together with a decrease in triglyceride-rich lipoproteins, an important substrate for CETP.20 There are several limitations to the present study. First, the study had a relatively small sample size from a single centre.
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(a)
(b)
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(d)
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Fig. 2 Effects of atorvastatin on low-density lipoprotein (LDL) subfractions analysed by ANOVA or paired samples t-test in (a,d) the control group, (b,d) patients receiving 10 mg/day atorvastatin and (c,f) patients receiving 20 mg/day atorvastatin. (□), large low-density lipoprotein–cholesterol (LDL-C); ( ), intermediate LDL-C; (■), small LDL-C. (a,d) There were no significant differences in LDL subfractions between baseline and at the 8 week follow up in the control group (P > 0.05 for all). (b,e) Afer 8 weeks treatment, 10 mg/day atorvastatin significantly decreased the cholesterol concentration of all LDL subfractions (P = 0.007, P < 0.001 and P = 0.012 for large, intermediate and small LDL-C, respectively), as well as the proportion of each LDL subfraction (P = 0.004, P < 0.001 and P = 0.013, respectively). (c,f) After 8 weeks treatment with 20 mg/day atorvastatin, there was a significant decrease in the cholesterol concentration of all LDL subfractions (P < 0.001, P = 0.001 and P = 0.006 for large, intermediate and small LDL-C, respectively), as well as the proportion of each LDL subfraction (P = 0.010, P = 0.002 and P = 0.003, respectively). Data are the mean SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with baseline (at the same dose).
The study protocol was reviewed and approved by the Ethics Committee of Fu Wai Hospital and Cardiovascular Institute, Beijing, China. All patients and control subjects provided informed consent.
receive either 10 mg/day (n = 17) or 20 mg/day (n = 20) atorvastatin for 8 weeks to evaluate the short-term and dose-dependent effects of low–moderate doses atorvastatin on HDL and LDL subfractions. Healthy subjects who had not received any drug treatment previously were enrolled as the control group (n = 15). Data regarding serum lipid profile and HDL and LDL subfractions were collected at baseline and at Week 8. The inclusion criteria for the present study were as follows: (i) definite diagnostic imaging evidence of atherosclerotic lesions detected by arterial ultrasound, coronary chest tomography or coronary angiography; (ii) no treatment history with statins or other drugs known to affect blood lipids within the previous 4 weeks; and (iii) age 18–70 years. Patients with triglycerides ≥ 500 mg/dL (5.6 mmol/L), previous acute coronary syndrome within 1 month, serious heart failure or arrhythmia, infectious disease within 1 month, serious liver or renal dysfunction, autoimmune disease, malignant disease, those who were pregnant or lactating or those with a psychiatric disorder were excluded from the study. In addition, patients with laboratory values more than threefold the upper limit of normal (ULN) for aspartate aminotransferase or alanine aminotransferase, or more than fivefold the ULN for creatine phosphokinase were also excluded from the study.
Study design and population
Laboratory examinations
In the present randomized controlled open-label prospective study, 37 patients with atherosclerosis were randomly assigned to
Blood samples were obtained from the cubital vein at baseline and after 8 weeks treatment in all subjects after an overnight fast.
Second, the study duration was short, which may have enabled us to fully uncover the effects of atorvastatin on lipoprotein subfractions. Moreover, we did not use age-matched healthy volunteers as the control group. Furthermore, the impact of high-does atorvastatin on lipoprotein was not evaluated in the present study. Finally, the impact of atrovastatin-induced changes in lipoprotein subfractions on clinical outcome was not investigated because of the small sample size. In conclusion, the findings of the present randomized controlled open-label prospective study indicate that 8 weeks therapy with a clinically conventional dose of 20 mg/day atorvastatin results in a favourable modification of the HDL subfraction phenotype in addition to effects on the cholesterol concentration of all LDL subfractions and mean LDL particle size.
METHODS Ethics approval
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All samples were subsequently stored at –80°C and analysed immediately after thawing. Serum concentrations of TC, triglycerides, HDL-C, LDL-C, ApoA-I, ApoB, Lp(a) and free fatty acids (FFA) were measured using an automatic biochemistry analyser (Model 7150; Hitachi, Tokyo, Japan). Total cholesterol, triglycerides, HDL-C, LDL-C and FFA levels were measured by enzymatic assay, whereas ApoA-I, ApoB and Lp(a) were determined by a turbidimetric immunoassay. Analysis of HDL subfractions Blood samples were also used for subfraction analysis. The HDL subclass analysis was performed electrophoretically using highresolution 3% polyacrylamide gel tubes and the Lipoprint HDL System (Quantimetrix, Redondo Beach, CA, USA) according to the manufacturer’s instructions and as described previously.24,25 By this analysis, HDL was divided into 10 subfractions: Subfractions 1–3 represent large HDL particles, Subfractions 4–7 indicate intermediate HDL particles and Subfractions 8–10 indicate small HDL particles. The cholesterol concentration (mg/dL) of each HDL subfraction and the proportion of the cholesterol concentration of each HDL subfraction as a percentage of the HDLC concentration were subsequently determined. Analysis of LDL subfractions The cholesterol content of LDL subfractions was also determined electrophoretically using high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL System, as previously described.25,26 Using this assay, LDL was divided into seven subfractions: Subfraction 1 represents large LDL particles, Subfraction 2 indicates intermediate LDL particles and Subfractions 3–7 refer to small dense LDL particles. The intermediate density lipoprotein can also be detected and the cholesterol mass (mg/dL) of each lipoprotein subfraction, the mean LDL particle size ( A) and the proportion of the cholesterol mass of each lipoprotein subfraction as a percentage of the total cholesterol mass were determined by this assay. Statistical analysis Data are expressed as the mean SD. To compare the dose effect at the same time point, Student’s t-test and one-way ANOVA were used. The significance of differences between baseline and after treatment were evaluated by paired samples t-test. Statistical significance was defined as P < 0.05. All statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).
ACKNOWLEDGEMENTS This work was supported, in part, by grants from the National Natural Scientific Foundation (81070171, 81241121, 81100118), Specialized Research Fund for the Doctoral Program of Higher Education of China (20111106110013, 20101106120007), Capital Special Foundation of Clinical Application Research (Z121107001012015), Capital Health Development Fund (2011400302), Beijing Natural Science Foundation (7131014) and Specialized Research Personnel Fund of Fu Wai Hospital (2012-FWXX02).
DISCLOSURE The authors declare no conflict of interests.
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