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Fasting serum concentration of apolipoprotein B48 represents residual risks in patients with new-onset and chronic coronary artery disease
Clinica Chimica Acta 421 (2013) 51–56
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Fasting serum concentration of apolipoprotein B48 represents residual risks in patients with new-onset and chronic coronary artery disease Kenta Mori a, Tatsuro Ishida a,⁎, Tomoyuki Yasuda a, Tomoko Monguchi a, Maki Sasaki a, Kensuke Kondo a, Minoru Hasokawa a, Hideto Nakajima a, Yoko Haraguchi a, Li Sun a, Masakazu Shinohara a, Ryuji Toh b, Kunihiro Nishimura b, Ken-ichi Hirata a a b
Division of Cardiovascular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Division of Evidence-based Laboratory Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
a r t i c l e
i n f o
Article history: Received 28 November 2012 Received in revised form 22 January 2013 Accepted 6 February 2013 Available online 18 February 2013 Keywords: Apolipoprotein B48 Remnant lipoprotein Coronary artery disease Hyperlipidemia Restenosis LDL cholesterol
a b s t r a c t Background: To identify new therapeutic targets for coronary artery disease (CAD), we investigated whether fasting serum concentration of apolipoprotein (apo) B48 could be a marker for CAD. Methods: Patients with CAD were divided into those with new-onset CAD [i.e., those receiving percutaneous coronary intervention (PCI) for the first time] and those with chronic CAD (i.e., those receiving follow-up coronary angiography). Fasting serum biochemical analyses were performed on admission and 6 months after the PCI. Results: On admission, serum LDL-C concentrations in patients with chronic CAD (n = 138), presumably receiving statin treatment, were lower than in patients with new-onset CAD (n = 50, p b 0.02) or without CAD (n = 71, p b 0.001). Nevertheless, apoB48 was higher in CAD patients than in those without CAD (p b 0.001). After adjusting for classic cardiovascular risk factors, multivariate logistic regression analyses showed apoB48 to be an independent predictor of coronary risk in new-onset or chronic CAD, irrespective of the LDL-C levels. Moreover, apoB48 was markedly increased during the follow-up period in CAD patients having new lesion progression after the prior PCI. Conclusion: Fasting serum apoB48 concentration could be a marker of new onset as well as chronic CAD, and predict new lesion progression in secondary prevention. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Statins, which are the main treatment for hypercholesterolemia, decrease serum concentration of low-density lipoprotein cholesterol (LDL-C) and thus reduce the risk of coronary artery disease (CAD) by 30-40% [1]. However, a residual risk remains, and research aims to identify new therapeutic targets beyond LDL-C. Metabolic syndrome is a major cause of cardiovascular disease and premature death. Patients with metabolic syndrome typically have low high-density lipoprotein cholesterol (HDL-C), qualitative changes in LDL-C, and hypertriglyceridemia. Although triglycerides (TGs) are not directly atherogenic, they are an important marker and modulator of CAD because TGs are associated with atherogenic TG-rich remnant lipoprotein particles. Furthermore,
⁎ Corresponding author. Tel.: +81 78 382 5846; fax: +81 78 382 5859. E-mail addresses: [email protected] (K. Mori), [email protected] (T. Ishida), [email protected] (T. Yasuda), [email protected] (T. Monguchi), [email protected] (M. Sasaki), [email protected] (K. Kondo), [email protected] (M. Hasokawa), [email protected] (H. Nakajima), [email protected] (Y. Haraguchi), [email protected] (L. Sun), [email protected] (M. Shinohara), [email protected] (R. Toh), [email protected] (K. Nishimura), [email protected] (K. Hirata). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.02.005
the TG-rich remnant lipoproteins are increased in postprandial hyperlipidemia [2], which is a risk factor for the development of CAD [3]. Remnant particles are commonly estimated by measuring the concentration of remnant-like particle cholesterol (RLP-C), which is widely heterogeneous in size and composition. However, it is now possible to measure remnant lipoprotein directly using a new homologous assay for apolipoprotein (apo) B48. ApoB48 is present only in intestinally derived lipoproteins such as chylomicron and chylomicron remnants [4]. A high fasting concentration of serum apoB48 reflects the delayed metabolism of TG-rich lipoproteins and the presence of postprandial hyperlipidemia [5]. In addition, fasting serum apoB48 level might be expected to be a marker for CAD [6], because chylomicron remnants can penetrate vessels to form foam cells and thus directly promote the initiation and progression of atherosclerosis [7]. Furthermore, serum apoB48 concentration was reported to correlate with carotid intima-media thickness [8]. 2. Patients and methods 2.1. Patients Patients with and without CAD admitted to Kobe University Hospital, Kobe, Japan, from April 2008 to March 2010, were eligible. Coronary
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lesions were defined as ≥75% narrowing of the coronary luminal diameter, measured by coronary angiography. Patients were registered only once during the study period. Exclusion criteria were emergency admission, heart failure (New York Heart Association functional class 4), cancer in the past 5 y, pulmonary hypertension, kidney failure (serum creatinine concentration >2.0 mg/dl or hemodialysis), and active inflammation (serum C-reactive protein concentration >1 mg/dl). Patients were categorized as those with or without CAD. The CAD patients were further divided into 2 subgroups: patients with new-onset CAD [i.e., those receiving percutaneous coronary intervention (PCI) for the first time] and patients with chronic CAD (i.e., those receiving follow-up coronary angiography 6 months after their first PCI). Moreover, these patients underwent follow-up coronary revascularization over 6 months after the initial PCI, and were divided into three groups; 1) patients without coronary restenosis, 2) patients with new stenotic lesion progression in non-stented sites, and 3) patients with in-stent coronary restenosis. Hypertension was diagnosed in patients with systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg, and was also recorded for patients treated with antihypertensive drugs. Diabetes mellitus was diagnosed in patients with fasting serum glucose >126 mg/dl or hemoglobin A1c value >6.5% (NGSP), according to the clinical guidelines of the Japan Diabetes Society. Diabetes was also recorded for patients treated with antidiabetic drugs. Dyslipidemia was diagnosed in patients with high serum LDL-C concentration, according to the Japan Atherosclerosis Society Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases [9]. Dyslipidemia was also recorded for patients treated with antihyperlipidemic drugs. Metabolic syndrome was defined according to the Japan Atherosclerosis Society guidelines [10]. Alcohol consumption was categorized as moderate (b 60 ml/day) or excessive (≥60 ml/day) [11]. Smoking status was categorized as never smoked, former smoker, or current smoker. Former smokers had not smoked for ≥1 y. 2.2. Ethical considerations The study was done in accordance with the ethical principles of the Declaration of Helsinki and the Ethical Guidelines for Clinical Research, enforced by the Ministry of Health, Labour and Welfare of Japan from 31 July 2008. The protocol was approved by the institutional review board of the Graduate School of Medicine, Kobe University, Japan, and all patients gave their informed written consent before enrolment. 2.3. Biochemical analyses Serum samples were collected after overnight fast at the point of initial admission (baseline), and in some patients, at 6 months after the initial PCI. Serum samples were stored at −80 °C until use, and biochemical analyses were done using standard techniques [12]. ApoAI, apoB and apoE were measured using the immunoturbidity method (Sekisui Medical, Tokyo, Japan). RLP-C was measured by immunoadsorption using the Jimro-II assay kit (Otsuka Pharmaceutical, Tokyo, Japan). ApoB48 was measured using the chemiluminescent enzyme immunoassay using anti-human apoB48 monoclonal antibodies (Fujirebio, Tokyo, Japan) [12]. ApoB100 was calculated by subtracting apoB48 from apoB. Estimated glomerular filtration rate (eGFR) was calculated thus: eGFR (ml/min/1.73 m2)=194×serum creatinine (mg/dl)−1.094 × age (y)−0.287 (×0.739 for women). 2.4. Statistical analysis Values are expressed as mean ± SD or frequencies (%). Variables with skewed distribution were normalized by natural logarithmic transformation, and expressed as median and interquartile range (25th to 75th centiles). One-way ANOVA was used to compare
continuous variables between groups. Chi-square test was used to compare categorical values between groups. Relations between apoB48 and serum lipids, lipoproteins, and other variables were examined by Pearson correlation coefficient and multivariate logistic regression analysis. Stepwise multivariate logistic regression analysis was used to determine the best independent predictor of coronary risk in patients without CAD, with new-onset CAD, and with chronic CAD, with the P value-to-enter set at 0.10. All statistical analyses were done using Stata 11.2 software (Stata, College Station TX). A P b 0.05 was considered statistically significant. We adjusted significance levels by Bonferroni correction in multiple comparison tests.
3. Results 3.1. Patient baseline characteristics A total of 259 patients were enrolled in this study. Of these, 188 patients had CAD: 39 men and 11 women with new-onset CAD, and 114 men and 24 women with chronic CAD. The 71 patients (60 men) without CAD had arrhythmia, valvular heart disease, or cardiomyopathy. Table 1 shows patient characteristics at baseline. Patients without CAD were significantly younger than those with new-onset or chronic CAD. We found no significant difference in gender, body mass index, waist circumference, family history of CAD, or history of stroke between the three groups. Metabolic syndrome, hypertension, diabetes mellitus, and dyslipidemia were significantly more prevalent in patients with CAD than in those without CAD. Significantly more patients with new-onset or chronic CAD than those without CAD were being treated with statins toward the achievement of the lower target LDL-C concentrations for CAD patients. Consequently, patients with new-onset or chronic CAD had significantly lower total cholesterol than patients without CAD (Table 1). Furthermore, LDL-C in patients with chronic CAD, who were presumably receiving aggressive statin treatment for secondary prevention, was significantly lower than in patients with new-onset CAD or without CAD. The concentration of apoAI, apoB and apoB100 was significantly lower in patients with new-onset or chronic CAD than in those without CAD. That is, most atherogenic lipoproteins and associated apolipoproteins were higher in patients without CAD than in patients with CAD. In contrast, HDL-C was significantly lower in patients with new-onset or chronic CAD than in patients without CAD. The distribution of serum apoB48 concentration was skewed to the left (Sup. Fig. 1), so in further statistical analyses we normalized apoB48 values by logarithmic transformation. Analysis confirmed that apoB48 was significantly higher in patients with new-onset or chronic CAD than in those without CAD (Fig. 1a). Furthermore, in patients with low LDL-C (b100 mg/dl) as a result of statin treatment, apoB48 was approximately twice as higher in patients with new-onset or chronic CAD than in those without CAD [4.0 (2.4–6.7) or 4.0 (2.4–5.7) vs. 2.2 (0.7–2.9) μg/ml, respectively] (Fig. 1b). Moreover, apoB48 in patients taking a statin was similar to that in patients not taking a statin (data not shown).
3.2. Relations between apolipoprotein B48 concentration and other risk factors To analyze relations between apoB48 concentration and other established cardiovascular risk factors, we normalized data for TGs, lipoprotein(a), RLP-C, apoB, apoB100, apoE, fasting serum glucose, and high-sensitivity C-reactive protein by logarithmic transformation to control for skewed distribution (data not shown). We found that ln(apoB48) was positively correlated with ln(TGs), ln(RLP-C), and body mass index (Sup. Fig. 2a–c). In contrast, ln(apoB48) was negatively correlated with HDL-C and eGFR (Sup. Fig. 2d and e).
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Table 1 Patient baseline characteristics.a Variable
Patients without CAD (n = 71)
Patients with new-onset CAD (n = 50)
Patients with chronic CAD (n = 138)
Male, n (%) Age (y) Body mass index (kg/m2) Metabolic syndrome, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Dyslipidemia, n (%) Alcohol consumption None (n = 142, 54.8%) Moderate (n = 102, 39.0%) Excessive (n = 15, 5.8%)b Smoking status Never smoked (n = 88, 34.0%) Former smoker (n = 104, 40.1%)c Current smoker (n = 67, 25.9%) Family history of CAD, n (%) History of stroke, n (%) Lipid-lowering therapy, n (%) Statin Fibrate Eicosapentaenoic acid Ezetimibe Serum concentration Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Triglycerides (mg/dl)d Lipoprotein(a)d RLP-C (mg/dl)d ApoAI (mg/dl) ApoB (mg/dl)d ApoB48 (μg/ml)d ApoB100 (mg/dl)d ApoE (mg/dl)d LDL-C:HDL-C ratio Non–HDL-C (mg/dl) Fasting serum glucose (mg/dl)d eGFR (ml/min/1.73 m2) hs-CRP (mg/dl)d
60 (84.5) 59.3 ± 13.7 24.0 ± 4.0 12 (16.9) 32 (45.1) 9 (12.7) 31 (44.3)
39 (78.0) 67.7 ± 10.1⁎ 25.0 ± 3.2 23 (46.0)⁎ 37 (74.0)⁎ 20 (40.0)⁎ 39 (78.0)⁎
114 (82.6) 67.5 ± 10.0⁎ 24.4 ± 2.9 67 (48.6)⁎ 122 (88.4)⁎ 69 (50.0)⁎ 117 (85.4)⁎
32 (22.5) 32 (31.4) 7 (46.7)
26 (18.3) 22 (21.6) 2 (13.3)
84 (32.4) 48 (47.1) 6 (40.0)
33 (37.5) 20 (19.2) 18 (26.7) 10 (18.5) 4 (5.7)
17 (19.3) 20 (19.2) 13 (19.4) 11 (20.4) 5 (10.0)
38 (43.2)⁎ 64 (61.5)⁎ 36 (53.7) 33 (61.1) 8 (6.0)
12 (16.9) 0 0 1 (1.4)
30 (60.0)⁎ 2 (4.0) 3 (6.0) 0
99 (71.7)⁎ 2 (1.5) 6 (4.4) 2 (1.5)
193.4 ± 37.3 55.1 ± 14.6 115.2 ± 33.0 110 (85–155) 13.9 (7.6–23.7) 6.5 (4.4–9.9) 142.0 ± 29.1 80 (71–91) 2.8 (1.5–4.3) 79.8 (70.9–90.9) 4.0 (3.4–4.8) 2.25 ± 0.97 138.2 ± 37.4 95 (86–103) 72.3 ± 17.3 0.05 (0.03–0.10)
177.1 ± 38.0⁎ 46.2 ± 11.3⁎ 107.3 ± 32.2 120 (103–187) 21.45 (10.2–32.6) 7.3 (4.5–11) 130.4 ± 24.7⁎
164.2 ± 28.6⁎ 48.3 ± 13.8⁎ 93.7 ± 22.8⁎,† 115.5 (87–150) 16.2 (9.5–28.5) 5.6 (3.9–8.7) 132.1 ± 24.3⁎ 70 (60–84)⁎,† 4.0 (2.4–5.9)⁎
81 (67–94) 3.8 (2.1–6.6)⁎ 80.2 (66.6–93.8)⁎ 4.1 (3.6–4.6) 2.43 ± 0.86 131.0 ± 36.1 96 (89–102) 63.8 ± 16.3⁎ 0.05 (0.03–0.11)
69.1 (59.8–83.1)† 3.8 (3.2–4.4) 2.07 ± 0.68† 115.7 ± 26.0⁎,† 97 (88–116)⁎ 63.9 ± 17.4⁎ 0.06 (0.03–0.14)
Apo, apolipoprotein; CAD, coronary artery disease; eGFR, estimated glomerular filtration rate; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; RLP-C, remnant-like particle cholesterol. a Values expressed as mean ± SD or median (25th to 75th centiles). b Alcohol consumption ≥60 ml/day. c Former smokers had not smoked for ≥1 y. d Based on one-way ANOVA (with Bonferroni correction) normalized by logarithmic transformation, median (5th to 95th centiles). ⁎ p b 0.05 vs. without CAD. † p b 0.05 vs. new-onset CAD. P value was based on χ2 test, categorical values or one-way ANOVA with Bonferroni correction; continuous variables, mean ± SD, unless otherwise specified.
3.3. Independence of apolipoprotein B48 as a predictor of coronary risk After adjusting for cardiovascular risk factors, univariate, multivariate and stepwise logistic regression analyses were done between patients without CAD and those with new-onset CAD, and between patients without CAD and those with chronic CAD. Univariate logistic regression analysis showed high apoB48 to be a significant coronary risk factor for both groups of CAD patients, and multivariate and stepwise logistic regression models confirmed the independence of classic and known risk factors (Table 2). Model 1 used apoB48 as an independent predictor of coronary risk for patients with new-onset or chronic CAD after adjusting for classic risk factors. Model 2 also adjusted for classical risks and alcohol consumption, family history of CAD, history of stroke, body mass index, metabolic syndrome, LDL-C, ln(TGs), eGFR, and ln(apoB48). After stepwise selection, apoB48 remained a strong predictor for CAD in patients with new-onset or chronic CAD (Table 2). Next, same analyses were done in the patients with statin-treated cases in CAD (excluding the cases with fibrate and EPA) (CAD/with statin group, n =120), and those without CAD cases without statins (without CAD/no statin, n =59). ApoB48 levels in CAD/with statin were significantly higher than those in without CAD/no statin [3.9 (2.4–5.7) vs. 3.0
(1.9–4.7) μg/ml, p=0.003]. LDL-C levels in CAD/with statin were significantly lower than those in without CAD/no statin [91.2±2 4.8 vs. 118.8±31.7 mg/ml, pb 0.001], suggesting that statins used in this study did not efficiently reduce the high level of apoB48 in the CAD patients. Moreover, high apoB48 was an independent risk factor for CAD during statin treatment (Table 3). Finally, when applied to data for patients with low LDL-C (b 100 mg/dl), the same analyses showed high apoB48 to be an independent risk factor for CAD after adjusting for those risk factors (Table 4). 3.4. Association of apolipoprotein B48 and triglyceride concentration Next, patients were divided into 4 groups according to apoB48 and TG concentration, with cutoff values of 3.6 μg/ml (median of its distribution) and 150 mg/dl (high end of normal range), respectively (Fig. 1). The high concentration of both apoB48 and TGs, which is a typical characteristic of postprandial hyperlipidemia, appeared more common in patients with new-onset or chronic CAD than in patients without CAD. Furthermore, significantly more patients with CAD patients (28.0% with new-onset CAD and 37.0% with chronic CAD) had high apoB48 and low TG levels than those without CAD (16.9%). We also found that
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p = 0.021
a
Table 3 Logistic regression analysis for CAD and statin treatment in relation to cardiovascular risk factors and ln(apoB48).a
p < 0.001
3
Analysis and variable(s)
Without CAD/no statin vs. with CAD/with statin
ln(apoB48)
2 1 0 12Without CAD
b
New-onset CAD
Chronic CAD
p = 0.002 p < 0.001
Odds ratio (95% CI)
p value
1.88 (1.18–2.99)
0.01⁎
3.40 (1.01–11.69)
b0.05⁎
1.09 (1.02–1.17) 0.07 (0.01–0.45) 1.26 (1.03–1.55) 11.37 (1.53–13.11) 4.41 (1.19–16.30) 0.95 (0.92–0.97) 3.11 (1.49–6.46)
0.01⁎ 0.01⁎ 0.03⁎ b0.01† 0.03⁎ b0.01† 0.04⁎
ApoB48, apolipoprotein B48; CAD, coronary artery disease; LDL-C, low-density lipoprotein cholesterol. a Analyzed between the patients without CAD/no statin (n = 59) vs. the patients with CAD/with statin (n = 120). b Adjusted for classic cardiovascular risk factors and also alcohol consumption, family history of CAD, history of stroke, body mass index, metabolic syndrome, LDL-C, triglycerides, and estimated glomerular filtration rate. ⁎ p b 0.05. † p b 0.01.
3 2
ln(apoB48)
Univariate analysis ln(apoB48) Multivariate analyses (classic and known risk factors)b ln(apoB48) Stepwise multivariate analysisb Age Female Body mass index Metabolic syndrome Hypertension Total cholesterol ln(apoB48)
1 0
3.5. Increase in serum apoB48 was accompanied with coronary lesion progression
-1 -2
Without CAD
New-onset CAD
Chronic CAD
Fig. 1. Fasting serum apoB48 level was elevated in patients with CAD. The apoB48 concentration was normalized by logarithmic transformation, and compared in patients with and without CAD: a, in all patients; and b, in patients with low low-density lipoprotein cholesterol (b100 mg/dl). New-onset CAD represents patients receiving percutaneous coronary intervention (PCI) for the first time, and chronic CAD patients receiving follow-up coronary angiography 6 months after their first PCI. Horizontal line, median; box, 25th to 75th centiles; ends of whiskers, 5th and 95th centiles. ApoB48, apolipoprotein B48; CAD, coronary artery disease.
the apoB48:TGs ratio, a marker for small chylomicron remnants, was higher in CAD patients than in those without CAD [0.034 (0.022–0.048) vs. 0.024 (0.014–0.043), pb 0.01].
Finally, we investigated whether the change of apoB48 concentration may affect in-stent restenosis or progression of new atherosclerotic lesions after PCI. When apoB48 concentration was evaluated during the follow-up period, apoB48 was 81% increased from baseline at 6 months after the PCI in patients having new stenotic lesion progression. In contrast, it was unchanged in the no restenosis group or in the in-stent restenosis group during the follow-up period (Fig. 3). 4. Discussion Although apoB48 is a marker of intestinally derived lipids including chylomicron remnants, the significance of apoB48 in CAD was not fully understood. It was previously reported that the fasting apoB48 concentration was similar between patients with and without CAD
Table 2 Logistic regression analysis for new onset- or chronic CAD in relation to cardiovascular risk factors and ln(apoB48).a Analysis and variable(s)
Without CAD vs. new-onset CAD Odds ratio (95% CI)
Univariate analysis ln(apoB48) Multivariate analyses Model 1 (classic risk factors)b ln(apoB48) Model 2 (classic and known risk factors)c ln(apoB48) Stepwise multivariate analysisc Age Hypertension Metabolic syndrome Total cholesterol ln(apoB48)
Without CAD vs. chronic CAD p value
Odds ratio (95% CI)
p value
1.77 (1.10–2.84)
0.02⁎
2.13 (1.43–3.17)
b0.01†
1.95 (1.05–3.60)
0.03⁎
2.55 (1.45–4.48)
b0.01†
5.56 (1.51–20.47)
0.01⁎
3.58 (1.25–10.25)
0.02⁎
0.01⁎ 0.02⁎ b0.01†
1.04 (1.00–1.09) 4.41 (1.74–11.19) None remained 0.97 (0.96–0.99) 2.58 (1.44–4.63)
0.04⁎ b0.01†
None remained None remained 4.48 (1.53–13.11) 0.95 (0.90–0.99) 3.11 (1.49–6.46)
b0.01† b0.01†
ApoB48, apolipoprotein B48; CAD, coronary artery disease; LDL-C, low-density lipoprotein cholesterol. a Analyzed between the patients between the patients without CAD (n = 71) vs. with new-onset CAD (n = 50), and without CAD vs. with chronic CAD (n = 138). b Adjusted for age, gender, hypertension, diabetes mellitus, total cholesterol, high-density lipoprotein cholesterol, and smoking status (non-current or current). c Adjusted for classic cardiovascular risk factors and also alcohol consumption, family history of CAD, history of stroke, body mass index, metabolic syndrome, LDL-C, triglycerides, and estimated glomerular filtration rate. ⁎ p b 0.05. † p b 0.01.
K. Mori et al. / Clinica Chimica Acta 421 (2013) 51–56 Table 4 Logistic regression analysis for patients with low serum LDL-C concentration (b100 mg/dl) in relation to cardiovascular risk factors and ln(apoB48).a Variable
Without CAD vs. new-onset CAD
0.03⁎ 0.02⁎
2.13 (1.43–3.17)
7.28 (2.08–25.40) None remained 0.97 (0.94–0.99) 3.67 (1.71–7.80)
b0.01†
b0.01†
Patients (%)
None remained 7.31 (1.28–41.73) None remained 3.88 (1.25–12.02)
0.02⁎
High apoB48, high TGs
Low apoB48, low TGs
Low apoB48, high TGs
80
60
40
0.04⁎ b0.01†
Apo, apolipoprotein; CAD, coronary artery disease; HDL-C, high-density lipoprotein cholesterol; LDL-C, high-density lipoprotein cholesterol. a Analyzed between the patients without CAD (n = 23) vs. with new-onset CAD (n = 20), and without CAD vs. with chronic CAD (n = 81). b Adjusted for age, gender, hypertension, diabetes mellitus, total cholesterol, high-density lipoprotein cholesterol, and smoking status (non-current or current). ⁎ pb 0.05. † pb 0.01.
[13,14], while those studies excluded patients with multiple risk factors or treated with anti-hyperlipidemic drugs. In contrast, the present study included CAD patients with multiple risk factors or treated with statins, and has demonstrated that fasting serum concentration of apoB48 was significantly increased in patients with new-onset or chronic CAD, which is consistent with a recent study [15]. Thus, we have provided direct evidence that apoB48 is a marker both for new-onset or chronic CAD. In the present study, apoB48 levels in CAD patients were twice as high as those without CAD, even when target LDL-C level (b100 mg/dl) was achieved. It has been reported that apoB48 is high under conditions including impaired glucose tolerance, chronic kidney disease, and obesity [16]. Conversely, apoB48 is decreased by medications such as statins, ezetimibe, fibrates, eicosapentaenoic acid [17], glucagon-like peptide 1, and dipeptidyl peptidase-4 inhibitors [18,19]. Given the regulated production by various factors, serum apolipoprotein B48 concentration probably increases in proportion to the severity of metabolic abnormalities and may therefore reflect the coronary risk remaining during LDL-Clowering therapy. It is of note that the present study did not document a difference in serum apoB48 levels in patients with and without statin treatment, in contrast to the previous findings in which atorvastatin (20–80 mg/day) and rosuvastatin (40 mg/day) markedly decreased apoB48 as well as LDL-C [20,21]. All patients enrolled in the present study were Japanese taking low doses of statins, for example, atorvastatin 5–10 mg/day, pitavastatin 2–4 mg/day, pravastatin 5–10 mg/day, or rosuvastatin 2.5–5 mg/day. We speculate that these low statin doses may account for the lack of apoB48-decreasing effect in this study. Inappropriate control of serum lipid profile is known to evoke vascular inflammation [22]. However, in the present study the concentration of high-sensitive C-reactive protein did not correlate with apoB48 or the presence of CAD. This is probably because most of the CAD patients were already being treated with statins and their LDL-C was maintained below individual target levels. In this context, fasting serum apoB48 concentration may reflect residual coronary risk independent of statin treatment or classic risk factors. Moreover, our findings imply the importance of intestinally derived exogenous lipoproteins in addition to serum LDL-C levels in the secondary prevention of CAD. In fact we have directly shown that the apoB48 concentration was increased during follow up periods in patients with new progression of coronary stenotic lesions in non-stented sites, while it was not changed in patients without major restenosis or with in-stent restenosis. This finding suggests that insufficient control of intestinally derived lipoproteins/lipids may lead to new lesion progression even if the patients are treated with statins. Thus, the change
20
*
*
0
Without CAD
New-onset CAD
Chronic CAD
Fig. 2. Serum levels of apoB48 and TG in patients with CAD. Percentage of patients according to fasting serum concentration of apoB48 (cutoff value, 3.6 μg/ml) and triglycerides (TG, cutoff value, 150 mg/dl) were shown. ApoB48 was increased in new-onset and chronic CAD patients compared to non-CAD patients even if the TGs levels were low. *pb 0.01 vs. patients without CAD. ApoB48, apolipoprotein B48; CAD, coronary artery disease, TGs, triglycerides.
of serum apoB48 levels may reflect the residual risks and predict lesion progression in secondary prevention of CAD. In particular, increase of apoB48 after PCI may predict lesion progression of CAD during statin treatment. Apolipoprotein B48 has been considered to be a sensitive marker for postprandial hyperlipidemia, and our findings support the notion that apoB48 reflects the concentration of TG-rich remnant lipoprotein particles. High apoB48 suggests delayed metabolism of TG-rich lipoproteins [23], which are commonly associated with insulin resistance and abdominal obesity. In this study, apoB48 in patients with type 2 diabetes mellitus was higher than that in patients without diabetes (data not shown). Also, HOMA-IR was positively correlated with apoB48 in patients without diabetes, while it was not correlated with apoB48 in diabetic patients with treated with anti-diabetic drugs. These findings suggest that the apoB48 would be useful to evaluate the prevalence of diabetic dyslipidemia or metabolic syndrome.
ApoB48 (µg/dL) 160
Change of apoB48 and LDL-C (%)
1.77 (1.10–2.84)
High apoB48, low TGs
100
Without CAD vs. chronic CAD
Odds ratio (95% CI) p value Odds ratio (95% CI) p value Univariate analysis ln(apoB48) Stepwise multivariate analysisb Hypertension Diabetes mellitus Total cholesterol ln(apoB48)
55
P < 0.05
LDL-C (mg/dL) P < 0.05
120
80
40
0
-40
-80
No restenosis
New lesion progression
In-stent restenosis
Fig. 3. Increase of apoB48 was associated with progression of coronary lesion. ApoB48 was measured at baseline and 6 months after coronary intervention. ApoB48 was increased during the follow-up period in patients having new lesion progression (n=12), while it was not changed in patients without restenosis (n=54) or with in-stent restenosis (n=17). The change of LDL-C was not statistically different among the three groups. ApoB48, apolipoprotein B48; LDL-C, low-density lipoprotein cholesterol.
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It has been reported that the apoB48:TGs ratio may reflect the concentration of atherogenic small chylomicron remnants [8]. In the present study, the high ratio of apoB48 to TGs in CAD patients suggests increased small chylomicron remnants in this group. Furthermore, both new-onset and chronic CAD patients were higher in fasting serum apoB48 levels than non-CAD patients irrespective of fasting TG levels (Fig. 2). It is considered that fasting serum apoB levels may represent delayed catabolism of TG-rich lipoproteins, which is not known simply by serum TG levels. 4.1. Conclusion Fasting serum apoB48 is higher in patients with new-onset or chronic CAD, independent of serum LDL-C levels, and the increase in apoB48 concentration is associated with coronary lesion progression in post-PCI patients undergoing LDL-lowering therapy. Therefore, apoB48 could be useful as a new marker for CAD in the primary and secondary prevention of this disease, as well as a possible therapeutic target. Acknowledgments We thank Fujirebio Inc. (Tokyo, Japan) for measuring serum apoB48 concentration. This work was supported by Grants-In-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the grants from the Senshin Medical Research Foundation, and the VRI Research Grant from AstraZeneca. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.cca.2013.02.005. References [1] Cholesterol Treatment Realists Collaborators, Mihaylova B, Emerson J, et al. The effects of lowering LDL cholesterol with statin therapy in people at low risk of vascular disease: meta-analysis of individual data from 27 randomised trials. Lancet 2012;380:581–90. [2] Miller M, Stone NJ, Ballantyne C, et al. Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association. Circulation 2011;123: 2292–333. [3] Nakamura T, Kugiyama K. Triglycerides and remnant particles as risk factors for coronary artery disease. Curr Atheroscler Rep 2006;8:107–10. [4] Sakai N, Uchida Y, Ohashi K, et al. Measurement of fasting serum apoB-48 levels in normolipidemic and hyperlipidemic subjects by ELISA. J Lipid Res 2003;44: 1256–62. [5] Masuda D, Sakai N, Sugimoto T, et al. Fasting serum apolipoprotein B-48 can be a marker of postprandial hyperlipidemia. J Atheroscler Thromb 2011;18:1062–70.
[6] Otokozawa S, Ai M, Diffenderfer MR, et al. Fasting and postprandial apolipoprotein B-48 levels in healthy, obese, and hyperlipidemic subjects. Metabolism 2009;58: 1536–42. [7] Nakano T, Nakajima K, Niimi M, et al. Detection of apolipoproteins B-48 and B-100 carrying particles in lipoprotein fractions extracted from human aortic atherosclerotic plaques in sudden cardiac death cases. Clin Chim Acta 2008;390:38–43. [8] Nakatani K, Sugimoto T, Masuda D, et al. Serum apolipoprotein B-48 levels are correlated with carotid intima-media thickness in subjects with normal serum triglyceride levels. Atherosclerosis 2011;218:226–32. [9] Teramoto T, Sasaki J, Ueshima H, et al. Diagnostic criteria for dyslipidemia — executive summary of Japan Atherosclerosis Society (JAS) guideline for diagnosis and prevention of atherosclerotic cardiovascular diseases for Japanese. J Atheroscler Thromb 2007;14: 155–8. [10] Matsuzawa Y. Metabolic syndrome — definition and diagnostic criteria in Japan. J Atheroscler Thromb 2005;12:301. [11] Yamada Y, Noborisaka Y, Suzuki H, Ishizaki M, Yamada S. Alcohol consumption, serum gamma-glutamyltransferase levels, and coronary risk factors in a middle-aged occupational population. J Occup Health 2003;45:293–9. [12] Hanada H, Mugii S, Okubo M, et al. Establishment of chemiluminescence enzyme immunoassay for apolipoprotein B-48 and its clinical applications for evaluation of impaired chylomicron remnant metabolism. Clin Chim Acta 2012;413:160–5. [13] Valero R, Lorec AM, Paganelli F, et al. Fasting apoprotein B-48 level and coronary artery disease in a population without frank fasting hypertriglyceridemia. Metabolism 2005;54:1442–7. [14] Reyes-Soffer G, Holleran S, Karmally W, et al. Measures of postprandial lipoproteins are not associated with coronary artery disease in patients with type 2 diabetes mellitus. J Lipid Res 2009;50:1901–9. [15] Masuda D, Sugimoto T, Tsujii KI, et al. Correlation of fasting serum apolipoprotein B-48 with coronary artery disease prevalence. Eur J Clin Invest 2012;42:992–9. [16] Kinoshita M, Ohnishi H, Maeda T, et al. Increased serum apolipoprotein B48 concentration in patients with metabolic syndrome. J Atheroscler Thromb 2009;16:517–22. [17] Valdivielso P, Rioja J, Garcia-Arias C, Sanchez-Chaparro MA, Gonzalez-Santos P. Omega 3 fatty acids induce a marked reduction of apolipoprotein B48 when added to fluvastatin in patients with type 2 diabetes and mixed hyperlipidemia: a preliminary report. Cardiovasc Diabetol 2009;8:1. [18] Hsieh J, Longuet C, Baker CL, et al. The glucagon-like peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice. Diabetologia 2010;53:552–61. [19] Xiao C, Bandsma RH, Dash S, Szeto L, Lewis GF. Exenatide, a glucagon-like peptide-1 receptor agonist, acutely inhibits intestinal lipoprotein production in healthy humans. Arterioscler Thromb Vasc Biol 2012;32:1513–9. [20] Otokozawa S, Ai M, Van Himbergen T, et al. Effects of intensive atorvastatin and rosuvastatin treatment on apolipoprotein B-48 and remnant lipoprotein cholesterol levels. Atherosclerosis 2009;205:197–201. [21] Lamon-Fava S, Diffenderfer MR, Barrett PH, et al. Effects of different doses of atorvastatin on human apolipoprotein B-100, B-48, and A-I metabolism. J Lipid Res 2007;48:1746–53. [22] Ridker PM, Danielson E, Fonseca FA, et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med 2008;359: 2195–207. [23] Sato I, Ishikawa Y, Ishimoto A, et al. Significance of measuring serum concentrations of remnant lipoproteins and apolipoprotein B-48 in fasting period. J Atheroscler Thromb 2009;16:12–20.
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Fasting serum concentration of apolipoprotein B48 represents residual risks in patients with new-onset and chronic coronary artery disease Kenta Mori a, Tatsuro Ishida a,⁎, Tomoyuki Yasuda a, Tomoko Monguchi a, Maki Sasaki a, Kensuke Kondo a, Minoru Hasokawa a, Hideto Nakajima a, Yoko Haraguchi a, Li Sun a, Masakazu Shinohara a, Ryuji Toh b, Kunihiro Nishimura b, Ken-ichi Hirata a a b
Division of Cardiovascular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Division of Evidence-based Laboratory Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
a r t i c l e
i n f o
Article history: Received 28 November 2012 Received in revised form 22 January 2013 Accepted 6 February 2013 Available online 18 February 2013 Keywords: Apolipoprotein B48 Remnant lipoprotein Coronary artery disease Hyperlipidemia Restenosis LDL cholesterol
a b s t r a c t Background: To identify new therapeutic targets for coronary artery disease (CAD), we investigated whether fasting serum concentration of apolipoprotein (apo) B48 could be a marker for CAD. Methods: Patients with CAD were divided into those with new-onset CAD [i.e., those receiving percutaneous coronary intervention (PCI) for the first time] and those with chronic CAD (i.e., those receiving follow-up coronary angiography). Fasting serum biochemical analyses were performed on admission and 6 months after the PCI. Results: On admission, serum LDL-C concentrations in patients with chronic CAD (n = 138), presumably receiving statin treatment, were lower than in patients with new-onset CAD (n = 50, p b 0.02) or without CAD (n = 71, p b 0.001). Nevertheless, apoB48 was higher in CAD patients than in those without CAD (p b 0.001). After adjusting for classic cardiovascular risk factors, multivariate logistic regression analyses showed apoB48 to be an independent predictor of coronary risk in new-onset or chronic CAD, irrespective of the LDL-C levels. Moreover, apoB48 was markedly increased during the follow-up period in CAD patients having new lesion progression after the prior PCI. Conclusion: Fasting serum apoB48 concentration could be a marker of new onset as well as chronic CAD, and predict new lesion progression in secondary prevention. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Statins, which are the main treatment for hypercholesterolemia, decrease serum concentration of low-density lipoprotein cholesterol (LDL-C) and thus reduce the risk of coronary artery disease (CAD) by 30-40% [1]. However, a residual risk remains, and research aims to identify new therapeutic targets beyond LDL-C. Metabolic syndrome is a major cause of cardiovascular disease and premature death. Patients with metabolic syndrome typically have low high-density lipoprotein cholesterol (HDL-C), qualitative changes in LDL-C, and hypertriglyceridemia. Although triglycerides (TGs) are not directly atherogenic, they are an important marker and modulator of CAD because TGs are associated with atherogenic TG-rich remnant lipoprotein particles. Furthermore,
⁎ Corresponding author. Tel.: +81 78 382 5846; fax: +81 78 382 5859. E-mail addresses: [email protected] (K. Mori), [email protected] (T. Ishida), [email protected] (T. Yasuda), [email protected] (T. Monguchi), [email protected] (M. Sasaki), [email protected] (K. Kondo), [email protected] (M. Hasokawa), [email protected] (H. Nakajima), [email protected] (Y. Haraguchi), [email protected] (L. Sun), [email protected] (M. Shinohara), [email protected] (R. Toh), [email protected] (K. Nishimura), [email protected] (K. Hirata). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.02.005
the TG-rich remnant lipoproteins are increased in postprandial hyperlipidemia [2], which is a risk factor for the development of CAD [3]. Remnant particles are commonly estimated by measuring the concentration of remnant-like particle cholesterol (RLP-C), which is widely heterogeneous in size and composition. However, it is now possible to measure remnant lipoprotein directly using a new homologous assay for apolipoprotein (apo) B48. ApoB48 is present only in intestinally derived lipoproteins such as chylomicron and chylomicron remnants [4]. A high fasting concentration of serum apoB48 reflects the delayed metabolism of TG-rich lipoproteins and the presence of postprandial hyperlipidemia [5]. In addition, fasting serum apoB48 level might be expected to be a marker for CAD [6], because chylomicron remnants can penetrate vessels to form foam cells and thus directly promote the initiation and progression of atherosclerosis [7]. Furthermore, serum apoB48 concentration was reported to correlate with carotid intima-media thickness [8]. 2. Patients and methods 2.1. Patients Patients with and without CAD admitted to Kobe University Hospital, Kobe, Japan, from April 2008 to March 2010, were eligible. Coronary
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lesions were defined as ≥75% narrowing of the coronary luminal diameter, measured by coronary angiography. Patients were registered only once during the study period. Exclusion criteria were emergency admission, heart failure (New York Heart Association functional class 4), cancer in the past 5 y, pulmonary hypertension, kidney failure (serum creatinine concentration >2.0 mg/dl or hemodialysis), and active inflammation (serum C-reactive protein concentration >1 mg/dl). Patients were categorized as those with or without CAD. The CAD patients were further divided into 2 subgroups: patients with new-onset CAD [i.e., those receiving percutaneous coronary intervention (PCI) for the first time] and patients with chronic CAD (i.e., those receiving follow-up coronary angiography 6 months after their first PCI). Moreover, these patients underwent follow-up coronary revascularization over 6 months after the initial PCI, and were divided into three groups; 1) patients without coronary restenosis, 2) patients with new stenotic lesion progression in non-stented sites, and 3) patients with in-stent coronary restenosis. Hypertension was diagnosed in patients with systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg, and was also recorded for patients treated with antihypertensive drugs. Diabetes mellitus was diagnosed in patients with fasting serum glucose >126 mg/dl or hemoglobin A1c value >6.5% (NGSP), according to the clinical guidelines of the Japan Diabetes Society. Diabetes was also recorded for patients treated with antidiabetic drugs. Dyslipidemia was diagnosed in patients with high serum LDL-C concentration, according to the Japan Atherosclerosis Society Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases [9]. Dyslipidemia was also recorded for patients treated with antihyperlipidemic drugs. Metabolic syndrome was defined according to the Japan Atherosclerosis Society guidelines [10]. Alcohol consumption was categorized as moderate (b 60 ml/day) or excessive (≥60 ml/day) [11]. Smoking status was categorized as never smoked, former smoker, or current smoker. Former smokers had not smoked for ≥1 y. 2.2. Ethical considerations The study was done in accordance with the ethical principles of the Declaration of Helsinki and the Ethical Guidelines for Clinical Research, enforced by the Ministry of Health, Labour and Welfare of Japan from 31 July 2008. The protocol was approved by the institutional review board of the Graduate School of Medicine, Kobe University, Japan, and all patients gave their informed written consent before enrolment. 2.3. Biochemical analyses Serum samples were collected after overnight fast at the point of initial admission (baseline), and in some patients, at 6 months after the initial PCI. Serum samples were stored at −80 °C until use, and biochemical analyses were done using standard techniques [12]. ApoAI, apoB and apoE were measured using the immunoturbidity method (Sekisui Medical, Tokyo, Japan). RLP-C was measured by immunoadsorption using the Jimro-II assay kit (Otsuka Pharmaceutical, Tokyo, Japan). ApoB48 was measured using the chemiluminescent enzyme immunoassay using anti-human apoB48 monoclonal antibodies (Fujirebio, Tokyo, Japan) [12]. ApoB100 was calculated by subtracting apoB48 from apoB. Estimated glomerular filtration rate (eGFR) was calculated thus: eGFR (ml/min/1.73 m2)=194×serum creatinine (mg/dl)−1.094 × age (y)−0.287 (×0.739 for women). 2.4. Statistical analysis Values are expressed as mean ± SD or frequencies (%). Variables with skewed distribution were normalized by natural logarithmic transformation, and expressed as median and interquartile range (25th to 75th centiles). One-way ANOVA was used to compare
continuous variables between groups. Chi-square test was used to compare categorical values between groups. Relations between apoB48 and serum lipids, lipoproteins, and other variables were examined by Pearson correlation coefficient and multivariate logistic regression analysis. Stepwise multivariate logistic regression analysis was used to determine the best independent predictor of coronary risk in patients without CAD, with new-onset CAD, and with chronic CAD, with the P value-to-enter set at 0.10. All statistical analyses were done using Stata 11.2 software (Stata, College Station TX). A P b 0.05 was considered statistically significant. We adjusted significance levels by Bonferroni correction in multiple comparison tests.
3. Results 3.1. Patient baseline characteristics A total of 259 patients were enrolled in this study. Of these, 188 patients had CAD: 39 men and 11 women with new-onset CAD, and 114 men and 24 women with chronic CAD. The 71 patients (60 men) without CAD had arrhythmia, valvular heart disease, or cardiomyopathy. Table 1 shows patient characteristics at baseline. Patients without CAD were significantly younger than those with new-onset or chronic CAD. We found no significant difference in gender, body mass index, waist circumference, family history of CAD, or history of stroke between the three groups. Metabolic syndrome, hypertension, diabetes mellitus, and dyslipidemia were significantly more prevalent in patients with CAD than in those without CAD. Significantly more patients with new-onset or chronic CAD than those without CAD were being treated with statins toward the achievement of the lower target LDL-C concentrations for CAD patients. Consequently, patients with new-onset or chronic CAD had significantly lower total cholesterol than patients without CAD (Table 1). Furthermore, LDL-C in patients with chronic CAD, who were presumably receiving aggressive statin treatment for secondary prevention, was significantly lower than in patients with new-onset CAD or without CAD. The concentration of apoAI, apoB and apoB100 was significantly lower in patients with new-onset or chronic CAD than in those without CAD. That is, most atherogenic lipoproteins and associated apolipoproteins were higher in patients without CAD than in patients with CAD. In contrast, HDL-C was significantly lower in patients with new-onset or chronic CAD than in patients without CAD. The distribution of serum apoB48 concentration was skewed to the left (Sup. Fig. 1), so in further statistical analyses we normalized apoB48 values by logarithmic transformation. Analysis confirmed that apoB48 was significantly higher in patients with new-onset or chronic CAD than in those without CAD (Fig. 1a). Furthermore, in patients with low LDL-C (b100 mg/dl) as a result of statin treatment, apoB48 was approximately twice as higher in patients with new-onset or chronic CAD than in those without CAD [4.0 (2.4–6.7) or 4.0 (2.4–5.7) vs. 2.2 (0.7–2.9) μg/ml, respectively] (Fig. 1b). Moreover, apoB48 in patients taking a statin was similar to that in patients not taking a statin (data not shown).
3.2. Relations between apolipoprotein B48 concentration and other risk factors To analyze relations between apoB48 concentration and other established cardiovascular risk factors, we normalized data for TGs, lipoprotein(a), RLP-C, apoB, apoB100, apoE, fasting serum glucose, and high-sensitivity C-reactive protein by logarithmic transformation to control for skewed distribution (data not shown). We found that ln(apoB48) was positively correlated with ln(TGs), ln(RLP-C), and body mass index (Sup. Fig. 2a–c). In contrast, ln(apoB48) was negatively correlated with HDL-C and eGFR (Sup. Fig. 2d and e).
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Table 1 Patient baseline characteristics.a Variable
Patients without CAD (n = 71)
Patients with new-onset CAD (n = 50)
Patients with chronic CAD (n = 138)
Male, n (%) Age (y) Body mass index (kg/m2) Metabolic syndrome, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Dyslipidemia, n (%) Alcohol consumption None (n = 142, 54.8%) Moderate (n = 102, 39.0%) Excessive (n = 15, 5.8%)b Smoking status Never smoked (n = 88, 34.0%) Former smoker (n = 104, 40.1%)c Current smoker (n = 67, 25.9%) Family history of CAD, n (%) History of stroke, n (%) Lipid-lowering therapy, n (%) Statin Fibrate Eicosapentaenoic acid Ezetimibe Serum concentration Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Triglycerides (mg/dl)d Lipoprotein(a)d RLP-C (mg/dl)d ApoAI (mg/dl) ApoB (mg/dl)d ApoB48 (μg/ml)d ApoB100 (mg/dl)d ApoE (mg/dl)d LDL-C:HDL-C ratio Non–HDL-C (mg/dl) Fasting serum glucose (mg/dl)d eGFR (ml/min/1.73 m2) hs-CRP (mg/dl)d
60 (84.5) 59.3 ± 13.7 24.0 ± 4.0 12 (16.9) 32 (45.1) 9 (12.7) 31 (44.3)
39 (78.0) 67.7 ± 10.1⁎ 25.0 ± 3.2 23 (46.0)⁎ 37 (74.0)⁎ 20 (40.0)⁎ 39 (78.0)⁎
114 (82.6) 67.5 ± 10.0⁎ 24.4 ± 2.9 67 (48.6)⁎ 122 (88.4)⁎ 69 (50.0)⁎ 117 (85.4)⁎
32 (22.5) 32 (31.4) 7 (46.7)
26 (18.3) 22 (21.6) 2 (13.3)
84 (32.4) 48 (47.1) 6 (40.0)
33 (37.5) 20 (19.2) 18 (26.7) 10 (18.5) 4 (5.7)
17 (19.3) 20 (19.2) 13 (19.4) 11 (20.4) 5 (10.0)
38 (43.2)⁎ 64 (61.5)⁎ 36 (53.7) 33 (61.1) 8 (6.0)
12 (16.9) 0 0 1 (1.4)
30 (60.0)⁎ 2 (4.0) 3 (6.0) 0
99 (71.7)⁎ 2 (1.5) 6 (4.4) 2 (1.5)
193.4 ± 37.3 55.1 ± 14.6 115.2 ± 33.0 110 (85–155) 13.9 (7.6–23.7) 6.5 (4.4–9.9) 142.0 ± 29.1 80 (71–91) 2.8 (1.5–4.3) 79.8 (70.9–90.9) 4.0 (3.4–4.8) 2.25 ± 0.97 138.2 ± 37.4 95 (86–103) 72.3 ± 17.3 0.05 (0.03–0.10)
177.1 ± 38.0⁎ 46.2 ± 11.3⁎ 107.3 ± 32.2 120 (103–187) 21.45 (10.2–32.6) 7.3 (4.5–11) 130.4 ± 24.7⁎
164.2 ± 28.6⁎ 48.3 ± 13.8⁎ 93.7 ± 22.8⁎,† 115.5 (87–150) 16.2 (9.5–28.5) 5.6 (3.9–8.7) 132.1 ± 24.3⁎ 70 (60–84)⁎,† 4.0 (2.4–5.9)⁎
81 (67–94) 3.8 (2.1–6.6)⁎ 80.2 (66.6–93.8)⁎ 4.1 (3.6–4.6) 2.43 ± 0.86 131.0 ± 36.1 96 (89–102) 63.8 ± 16.3⁎ 0.05 (0.03–0.11)
69.1 (59.8–83.1)† 3.8 (3.2–4.4) 2.07 ± 0.68† 115.7 ± 26.0⁎,† 97 (88–116)⁎ 63.9 ± 17.4⁎ 0.06 (0.03–0.14)
Apo, apolipoprotein; CAD, coronary artery disease; eGFR, estimated glomerular filtration rate; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; RLP-C, remnant-like particle cholesterol. a Values expressed as mean ± SD or median (25th to 75th centiles). b Alcohol consumption ≥60 ml/day. c Former smokers had not smoked for ≥1 y. d Based on one-way ANOVA (with Bonferroni correction) normalized by logarithmic transformation, median (5th to 95th centiles). ⁎ p b 0.05 vs. without CAD. † p b 0.05 vs. new-onset CAD. P value was based on χ2 test, categorical values or one-way ANOVA with Bonferroni correction; continuous variables, mean ± SD, unless otherwise specified.
3.3. Independence of apolipoprotein B48 as a predictor of coronary risk After adjusting for cardiovascular risk factors, univariate, multivariate and stepwise logistic regression analyses were done between patients without CAD and those with new-onset CAD, and between patients without CAD and those with chronic CAD. Univariate logistic regression analysis showed high apoB48 to be a significant coronary risk factor for both groups of CAD patients, and multivariate and stepwise logistic regression models confirmed the independence of classic and known risk factors (Table 2). Model 1 used apoB48 as an independent predictor of coronary risk for patients with new-onset or chronic CAD after adjusting for classic risk factors. Model 2 also adjusted for classical risks and alcohol consumption, family history of CAD, history of stroke, body mass index, metabolic syndrome, LDL-C, ln(TGs), eGFR, and ln(apoB48). After stepwise selection, apoB48 remained a strong predictor for CAD in patients with new-onset or chronic CAD (Table 2). Next, same analyses were done in the patients with statin-treated cases in CAD (excluding the cases with fibrate and EPA) (CAD/with statin group, n =120), and those without CAD cases without statins (without CAD/no statin, n =59). ApoB48 levels in CAD/with statin were significantly higher than those in without CAD/no statin [3.9 (2.4–5.7) vs. 3.0
(1.9–4.7) μg/ml, p=0.003]. LDL-C levels in CAD/with statin were significantly lower than those in without CAD/no statin [91.2±2 4.8 vs. 118.8±31.7 mg/ml, pb 0.001], suggesting that statins used in this study did not efficiently reduce the high level of apoB48 in the CAD patients. Moreover, high apoB48 was an independent risk factor for CAD during statin treatment (Table 3). Finally, when applied to data for patients with low LDL-C (b 100 mg/dl), the same analyses showed high apoB48 to be an independent risk factor for CAD after adjusting for those risk factors (Table 4). 3.4. Association of apolipoprotein B48 and triglyceride concentration Next, patients were divided into 4 groups according to apoB48 and TG concentration, with cutoff values of 3.6 μg/ml (median of its distribution) and 150 mg/dl (high end of normal range), respectively (Fig. 1). The high concentration of both apoB48 and TGs, which is a typical characteristic of postprandial hyperlipidemia, appeared more common in patients with new-onset or chronic CAD than in patients without CAD. Furthermore, significantly more patients with CAD patients (28.0% with new-onset CAD and 37.0% with chronic CAD) had high apoB48 and low TG levels than those without CAD (16.9%). We also found that
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p = 0.021
a
Table 3 Logistic regression analysis for CAD and statin treatment in relation to cardiovascular risk factors and ln(apoB48).a
p < 0.001
3
Analysis and variable(s)
Without CAD/no statin vs. with CAD/with statin
ln(apoB48)
2 1 0 12Without CAD
b
New-onset CAD
Chronic CAD
p = 0.002 p < 0.001
Odds ratio (95% CI)
p value
1.88 (1.18–2.99)
0.01⁎
3.40 (1.01–11.69)
b0.05⁎
1.09 (1.02–1.17) 0.07 (0.01–0.45) 1.26 (1.03–1.55) 11.37 (1.53–13.11) 4.41 (1.19–16.30) 0.95 (0.92–0.97) 3.11 (1.49–6.46)
0.01⁎ 0.01⁎ 0.03⁎ b0.01† 0.03⁎ b0.01† 0.04⁎
ApoB48, apolipoprotein B48; CAD, coronary artery disease; LDL-C, low-density lipoprotein cholesterol. a Analyzed between the patients without CAD/no statin (n = 59) vs. the patients with CAD/with statin (n = 120). b Adjusted for classic cardiovascular risk factors and also alcohol consumption, family history of CAD, history of stroke, body mass index, metabolic syndrome, LDL-C, triglycerides, and estimated glomerular filtration rate. ⁎ p b 0.05. † p b 0.01.
3 2
ln(apoB48)
Univariate analysis ln(apoB48) Multivariate analyses (classic and known risk factors)b ln(apoB48) Stepwise multivariate analysisb Age Female Body mass index Metabolic syndrome Hypertension Total cholesterol ln(apoB48)
1 0
3.5. Increase in serum apoB48 was accompanied with coronary lesion progression
-1 -2
Without CAD
New-onset CAD
Chronic CAD
Fig. 1. Fasting serum apoB48 level was elevated in patients with CAD. The apoB48 concentration was normalized by logarithmic transformation, and compared in patients with and without CAD: a, in all patients; and b, in patients with low low-density lipoprotein cholesterol (b100 mg/dl). New-onset CAD represents patients receiving percutaneous coronary intervention (PCI) for the first time, and chronic CAD patients receiving follow-up coronary angiography 6 months after their first PCI. Horizontal line, median; box, 25th to 75th centiles; ends of whiskers, 5th and 95th centiles. ApoB48, apolipoprotein B48; CAD, coronary artery disease.
the apoB48:TGs ratio, a marker for small chylomicron remnants, was higher in CAD patients than in those without CAD [0.034 (0.022–0.048) vs. 0.024 (0.014–0.043), pb 0.01].
Finally, we investigated whether the change of apoB48 concentration may affect in-stent restenosis or progression of new atherosclerotic lesions after PCI. When apoB48 concentration was evaluated during the follow-up period, apoB48 was 81% increased from baseline at 6 months after the PCI in patients having new stenotic lesion progression. In contrast, it was unchanged in the no restenosis group or in the in-stent restenosis group during the follow-up period (Fig. 3). 4. Discussion Although apoB48 is a marker of intestinally derived lipids including chylomicron remnants, the significance of apoB48 in CAD was not fully understood. It was previously reported that the fasting apoB48 concentration was similar between patients with and without CAD
Table 2 Logistic regression analysis for new onset- or chronic CAD in relation to cardiovascular risk factors and ln(apoB48).a Analysis and variable(s)
Without CAD vs. new-onset CAD Odds ratio (95% CI)
Univariate analysis ln(apoB48) Multivariate analyses Model 1 (classic risk factors)b ln(apoB48) Model 2 (classic and known risk factors)c ln(apoB48) Stepwise multivariate analysisc Age Hypertension Metabolic syndrome Total cholesterol ln(apoB48)
Without CAD vs. chronic CAD p value
Odds ratio (95% CI)
p value
1.77 (1.10–2.84)
0.02⁎
2.13 (1.43–3.17)
b0.01†
1.95 (1.05–3.60)
0.03⁎
2.55 (1.45–4.48)
b0.01†
5.56 (1.51–20.47)
0.01⁎
3.58 (1.25–10.25)
0.02⁎
0.01⁎ 0.02⁎ b0.01†
1.04 (1.00–1.09) 4.41 (1.74–11.19) None remained 0.97 (0.96–0.99) 2.58 (1.44–4.63)
0.04⁎ b0.01†
None remained None remained 4.48 (1.53–13.11) 0.95 (0.90–0.99) 3.11 (1.49–6.46)
b0.01† b0.01†
ApoB48, apolipoprotein B48; CAD, coronary artery disease; LDL-C, low-density lipoprotein cholesterol. a Analyzed between the patients between the patients without CAD (n = 71) vs. with new-onset CAD (n = 50), and without CAD vs. with chronic CAD (n = 138). b Adjusted for age, gender, hypertension, diabetes mellitus, total cholesterol, high-density lipoprotein cholesterol, and smoking status (non-current or current). c Adjusted for classic cardiovascular risk factors and also alcohol consumption, family history of CAD, history of stroke, body mass index, metabolic syndrome, LDL-C, triglycerides, and estimated glomerular filtration rate. ⁎ p b 0.05. † p b 0.01.
K. Mori et al. / Clinica Chimica Acta 421 (2013) 51–56 Table 4 Logistic regression analysis for patients with low serum LDL-C concentration (b100 mg/dl) in relation to cardiovascular risk factors and ln(apoB48).a Variable
Without CAD vs. new-onset CAD
0.03⁎ 0.02⁎
2.13 (1.43–3.17)
7.28 (2.08–25.40) None remained 0.97 (0.94–0.99) 3.67 (1.71–7.80)
b0.01†
b0.01†
Patients (%)
None remained 7.31 (1.28–41.73) None remained 3.88 (1.25–12.02)
0.02⁎
High apoB48, high TGs
Low apoB48, low TGs
Low apoB48, high TGs
80
60
40
0.04⁎ b0.01†
Apo, apolipoprotein; CAD, coronary artery disease; HDL-C, high-density lipoprotein cholesterol; LDL-C, high-density lipoprotein cholesterol. a Analyzed between the patients without CAD (n = 23) vs. with new-onset CAD (n = 20), and without CAD vs. with chronic CAD (n = 81). b Adjusted for age, gender, hypertension, diabetes mellitus, total cholesterol, high-density lipoprotein cholesterol, and smoking status (non-current or current). ⁎ pb 0.05. † pb 0.01.
[13,14], while those studies excluded patients with multiple risk factors or treated with anti-hyperlipidemic drugs. In contrast, the present study included CAD patients with multiple risk factors or treated with statins, and has demonstrated that fasting serum concentration of apoB48 was significantly increased in patients with new-onset or chronic CAD, which is consistent with a recent study [15]. Thus, we have provided direct evidence that apoB48 is a marker both for new-onset or chronic CAD. In the present study, apoB48 levels in CAD patients were twice as high as those without CAD, even when target LDL-C level (b100 mg/dl) was achieved. It has been reported that apoB48 is high under conditions including impaired glucose tolerance, chronic kidney disease, and obesity [16]. Conversely, apoB48 is decreased by medications such as statins, ezetimibe, fibrates, eicosapentaenoic acid [17], glucagon-like peptide 1, and dipeptidyl peptidase-4 inhibitors [18,19]. Given the regulated production by various factors, serum apolipoprotein B48 concentration probably increases in proportion to the severity of metabolic abnormalities and may therefore reflect the coronary risk remaining during LDL-Clowering therapy. It is of note that the present study did not document a difference in serum apoB48 levels in patients with and without statin treatment, in contrast to the previous findings in which atorvastatin (20–80 mg/day) and rosuvastatin (40 mg/day) markedly decreased apoB48 as well as LDL-C [20,21]. All patients enrolled in the present study were Japanese taking low doses of statins, for example, atorvastatin 5–10 mg/day, pitavastatin 2–4 mg/day, pravastatin 5–10 mg/day, or rosuvastatin 2.5–5 mg/day. We speculate that these low statin doses may account for the lack of apoB48-decreasing effect in this study. Inappropriate control of serum lipid profile is known to evoke vascular inflammation [22]. However, in the present study the concentration of high-sensitive C-reactive protein did not correlate with apoB48 or the presence of CAD. This is probably because most of the CAD patients were already being treated with statins and their LDL-C was maintained below individual target levels. In this context, fasting serum apoB48 concentration may reflect residual coronary risk independent of statin treatment or classic risk factors. Moreover, our findings imply the importance of intestinally derived exogenous lipoproteins in addition to serum LDL-C levels in the secondary prevention of CAD. In fact we have directly shown that the apoB48 concentration was increased during follow up periods in patients with new progression of coronary stenotic lesions in non-stented sites, while it was not changed in patients without major restenosis or with in-stent restenosis. This finding suggests that insufficient control of intestinally derived lipoproteins/lipids may lead to new lesion progression even if the patients are treated with statins. Thus, the change
20
*
*
0
Without CAD
New-onset CAD
Chronic CAD
Fig. 2. Serum levels of apoB48 and TG in patients with CAD. Percentage of patients according to fasting serum concentration of apoB48 (cutoff value, 3.6 μg/ml) and triglycerides (TG, cutoff value, 150 mg/dl) were shown. ApoB48 was increased in new-onset and chronic CAD patients compared to non-CAD patients even if the TGs levels were low. *pb 0.01 vs. patients without CAD. ApoB48, apolipoprotein B48; CAD, coronary artery disease, TGs, triglycerides.
of serum apoB48 levels may reflect the residual risks and predict lesion progression in secondary prevention of CAD. In particular, increase of apoB48 after PCI may predict lesion progression of CAD during statin treatment. Apolipoprotein B48 has been considered to be a sensitive marker for postprandial hyperlipidemia, and our findings support the notion that apoB48 reflects the concentration of TG-rich remnant lipoprotein particles. High apoB48 suggests delayed metabolism of TG-rich lipoproteins [23], which are commonly associated with insulin resistance and abdominal obesity. In this study, apoB48 in patients with type 2 diabetes mellitus was higher than that in patients without diabetes (data not shown). Also, HOMA-IR was positively correlated with apoB48 in patients without diabetes, while it was not correlated with apoB48 in diabetic patients with treated with anti-diabetic drugs. These findings suggest that the apoB48 would be useful to evaluate the prevalence of diabetic dyslipidemia or metabolic syndrome.
ApoB48 (µg/dL) 160
Change of apoB48 and LDL-C (%)
1.77 (1.10–2.84)
High apoB48, low TGs
100
Without CAD vs. chronic CAD
Odds ratio (95% CI) p value Odds ratio (95% CI) p value Univariate analysis ln(apoB48) Stepwise multivariate analysisb Hypertension Diabetes mellitus Total cholesterol ln(apoB48)
55
P < 0.05
LDL-C (mg/dL) P < 0.05
120
80
40
0
-40
-80
No restenosis
New lesion progression
In-stent restenosis
Fig. 3. Increase of apoB48 was associated with progression of coronary lesion. ApoB48 was measured at baseline and 6 months after coronary intervention. ApoB48 was increased during the follow-up period in patients having new lesion progression (n=12), while it was not changed in patients without restenosis (n=54) or with in-stent restenosis (n=17). The change of LDL-C was not statistically different among the three groups. ApoB48, apolipoprotein B48; LDL-C, low-density lipoprotein cholesterol.
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It has been reported that the apoB48:TGs ratio may reflect the concentration of atherogenic small chylomicron remnants [8]. In the present study, the high ratio of apoB48 to TGs in CAD patients suggests increased small chylomicron remnants in this group. Furthermore, both new-onset and chronic CAD patients were higher in fasting serum apoB48 levels than non-CAD patients irrespective of fasting TG levels (Fig. 2). It is considered that fasting serum apoB levels may represent delayed catabolism of TG-rich lipoproteins, which is not known simply by serum TG levels. 4.1. Conclusion Fasting serum apoB48 is higher in patients with new-onset or chronic CAD, independent of serum LDL-C levels, and the increase in apoB48 concentration is associated with coronary lesion progression in post-PCI patients undergoing LDL-lowering therapy. Therefore, apoB48 could be useful as a new marker for CAD in the primary and secondary prevention of this disease, as well as a possible therapeutic target. Acknowledgments We thank Fujirebio Inc. (Tokyo, Japan) for measuring serum apoB48 concentration. This work was supported by Grants-In-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the grants from the Senshin Medical Research Foundation, and the VRI Research Grant from AstraZeneca. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.cca.2013.02.005. References [1] Cholesterol Treatment Realists Collaborators, Mihaylova B, Emerson J, et al. The effects of lowering LDL cholesterol with statin therapy in people at low risk of vascular disease: meta-analysis of individual data from 27 randomised trials. Lancet 2012;380:581–90. [2] Miller M, Stone NJ, Ballantyne C, et al. Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association. Circulation 2011;123: 2292–333. [3] Nakamura T, Kugiyama K. Triglycerides and remnant particles as risk factors for coronary artery disease. Curr Atheroscler Rep 2006;8:107–10. [4] Sakai N, Uchida Y, Ohashi K, et al. Measurement of fasting serum apoB-48 levels in normolipidemic and hyperlipidemic subjects by ELISA. J Lipid Res 2003;44: 1256–62. [5] Masuda D, Sakai N, Sugimoto T, et al. Fasting serum apolipoprotein B-48 can be a marker of postprandial hyperlipidemia. J Atheroscler Thromb 2011;18:1062–70.
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