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Evaluation of apolipoprotein M as a biomarker of coronary artery disease
Available online at www.sciencedirect.com
Clinical Biochemistry 42 (2009) 365 – 370
Evaluation of apolipoprotein M as a biomarker of coronary artery disease Weiqiang Su b , Guoqing Jiao a,b , Chengjian Yang b , Yizhou Ye a,⁎ a
b
Department of Cardiovascular Surgery, Affiliated Shanghai 1st People's Hospital, Shanghai Jiaotong University, Hongkou, 85 Wujin Road, Shanghai 200080, People's Republic of China Department of Cardiovascular Surgery, Affiliated Wuxi 2nd People's Hospital, Nanjing Medical University, Jiangsu, People's Republic of China Received 1 August 2008; received in revised form 14 November 2008; accepted 15 November 2008 Available online 3 December 2008
Abstract Objective: To investigate the possible role of apolipoprotein M (ApoM) in the development of coronary artery disease (CAD). Design and methods: Case-controlled study, which consisted of 118 CAD patients and 255 unrelated subjects used as control group. Plasma concentration of ApoM was determined by dot blot, severity of CAD was expressed with Gensini score or the numbers of lesioned coronary arteries, and serum lipid levels were also measured. Results and discussion: Our study shows the mean level of plasma ApoM is 1.3757 ± 0.1493 ODu mm− 2 in CAD patients, while it is 1.3502 ± 0.1288 ODu mm− 2 in control group, and there are significant differences in plasma level of ApoM between two groups (t = 0.032, P b 0.05). Concentration of plasma ApoM is positively associated with plasma total cholesterol (r = 0.38, P = 0.025), high density lipoprotein cholesterol (r = 0.29, P = 0.03), low density lipoprotein cholesterol (r = 0.16, P = 0.03) and apolipoproein A–I (r = 0.24, P = 0.03). Multiple logistic and linear regression analysis showed that plasma concentration of ApoM did not correlate either with the number of lesioned coronaries or the Gensini score after adjusted for conventional cardiovascular risk factors (P N 0.05, respectively). Conclusion: The findings suggest that ApoM could not be an independent risk factor but a biomarker of CAD. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: ApoM; Coronary artery disease; HDL; LDL; TC
Introduction Coronary artery disease (CAD) and stroke are the major cause of death and disability in developing countries. A number of traditional risk factors for CAD include aging, sex, obesity, smoking, diabetes, hypertension, physical inactivity, elevated total plasma cholesterol, low-density lipoprotein (LDL), decreased high density lipoprotein (HDL), and lifestyle [1–3]. Adverse levels of serum lipoprotein are traditionally used as predictive of CAD [4]. ApoM was first described in 1999 [5]. The protein is composed of 188 amino acids, with an apparent size of 25 kDa. It belongs to the lipocalin protein superfamily [6,7]. Hepatic nuclear factor (HNF)-1a in mice is required for ApoM expression in vivo [8]. Mutations in HNF-1a, in heterozygous form, are responsible for the development of maturity-onset diabetes of the young (MODY) type 3 in humans, and reduced plasma ⁎ Corresponding author. Fax: +86 021 63253883. E-mail address: [email protected] (Y. Ye).
ApoM levels were found in MODY 3 patients [8]. A sandwich ELISA for the measurement of ApoM in human plasma shows that ApoM content in the healthy human plasma pool is 0.94 mM, or ∼ 23 mg/l, ApoM concentration is strongly correlated to total cholesterol in healthy individuals [9], which roughly corresponds to 1/50th of the mean molar concentration of apolipoproteinA-I(ApoA-I) in plasma [10]. There is a well-established relationship between plasma total cholesterol and the risk of development of myocardial infarction [11–13]. The strong correlation of ApoM with plasma cholesterol, together with the finding that overexpression of ApoM in LDL receptor deficient mice can protect against atherosclerosis [14], make it interesting to investigate whether ApoM is a predictor of cardiovascular disease. Our previous study of T-778C polymorphism of ApoM gene in Han Chinese CAD patients demonstrated that the C allele at nucleotide − 778 in the ApoM gene is associated with increased risk of CAD and CAD patients with the CC and the CT genotype of SNP-778 have higher plasma concentrations of total cholesterol than normal healthy control group [15]. In order to determine the
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W. Su et al. / Clinical Biochemistry 42 (2009) 365–370
relationships between plasma concentrations of ApoM and CAD, we conducted a case-controlled study, which further revealed the pathophysiological function of ApoM in the development of CAD.
distal segment of the CAD or mid-distal of LCX or right coronary artery, and 0.5 for others [16].
Methods
Plasma ApoM concentration was estimated by a dot-blot analysis with monoclonal rabbit anti-human ApoM antibody (ABNOVA). In brief, 10 μL of plasma was diluted with Tris–HCl buffer (1:20) and 5 μL diluted samples applied to the membranes in triplicate (Hybond-C, Amersham Life Science). The membrane was quenched in Tris–HCl buffer with 4% Tween and 3% bovine serum albumin for 2 h, followed by sequential incubations with monoclonal rabbit anti-human ApoM antibody (dilution, 1:1000 in Tris–HCl buffer) ovenight at 4 °C, and then alkaline phosphatase (AP) conjugated secondary antibody for 2 h at room temperature. The development of AP activity was performed with a commercial visualization system following the manufacturer's instructions (Dako). The recombinant ApoM protein (ABNOVA) and ddH2O were used as standard. The relative amount of ApoM was quantified by a scanner using the software of Quantity One (Version 4.2.1, Bio-Rad Laboratories) and presented as adjusted volume (ODu mm− 2). The same experiment was repeated at least three times.
Study population Subjects for the present study consisted of 118 CAD patients (89 males and 29 females with a mean age of 61.8 ± 11.4 years) and 225 unrelated individuals (152 males and 73 females with a mean age of 60.4 ± 12.7 years) were enrolled as control group. Criterion for enrolment of CAD patients is set as at least 50% stenosis determined angiographically existed in at least one of the major segments of coronary arteries, i.e. the right coronary artery, left circumflex, or left anterior descending arteries. Controls were acquired from those who have performed a negative coronary artery angiography so that CAD patients are excluded. History of conventional risk factors for CAD, including habitual cigarette smoking, hypertension (systolic blood pressure ≥160 mm Hg and/or diastolic blood pressure ≥95 mm Hg), diabetes mellitus, or hypercholesterolemia (TC ≥ 5.7 mmol/L) were obtained from the patients or their medical records. Exclusion criteria for both CAD patients and control group include familial hypercholesterolemia, diabetes mellitus, cancer, renal disease, and any other chronic illnesses. All individuals in both CAD and control group are Han Chinese. Ethical approval for this investigation was obtained from the Research Ethics Committee, Shanghai Jiaotong University. Written consent was obtained from each subject before blood samples were collected. The investigation complies with the principles outlined in the Declaration of Helsinki. Assessment of coronary atherosclerosis severity Selective coronary angiography was conducted using the Judkins' method. For every artery, at least two views were adopted. The degree of coronary stenosis was assessed by quantitative coronary angiography, and diametrical stenosis more than 50% was deemed as a positive result of CAD. The following two methods were used to assess coronary atherosclerosis severity: (1) The number of lesioned arteries(≥50% stenosis of luminal diameter): lesion in the left anterior descending, left circumflex, right coronary artery or their main branches was recorded as one lesioned artery, and lesion in the left main coronary artery was recorded as two lesioned arteries. The same coronary artery was not counted more than twice. According to the number of lesioned arteries, there were three ranks, namely, 1 lesioned artery, 2 lesioned arteries, and 3 lesioned arteries; (2) Gensini score: 1–25%, 26–50%, 51–75%, 76–90%, 91–99%, and 100% of coronary luminal narrowing were given scores of 1, 2, 4, 8, 16, and 32 respectively. This score was then multiplied by a factor based on the importance of lesion's position in the coronary arterial tree: 5 for the left main coronary artery, 2.5 for proximal segment of the left anterior descending coronary artery (LAD) or the circumflex artery (LCX), 1.5 for mid-segment of LAD, 1 for
Measurement of plasma ApoM concentration
Biochemical analysis Plasma total cholesterol (TC) was measured by the cholesterol oxidase method and triglycerides (TG) by the phosphoglycerol oxidase method with commercial kits (Shanghai Mingdian Bio-Tech Co., China). Plasma low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were determined using the chemical modification enzymatic method (Trinity Biotech, Ireland). Plasma concentration of ApoA-I, apolipoprotein B(ApoB) and Lpα were determined by immunoturbidimetry assay (Shanghai Mingdian Bio-Tech Co., China). Plasma concentration of apolipoprotein E (ApoE) was determined by ELISA using a commercial kit (Shanghai Mingdian Bio-Tech Co., China). Lipid parameters were measured using an automated RA-1000 (Technician, USA). Samples for quality control were provided by RANDOX (England). Statistical analysis Data are presented as mean ± S.E. Student's t test, chi-square test, Pearson or Spearman rank-order correlation were used for the analysis of the data using GraphPad Prism 4.03statistics software. The SPSS statistical software package version 11.5 was used for multiple logistic and linear regres-sion analysis. P ≤ 0.05 levels was considered as statistical significance. Results Clinical characteristics of CAD patients and control group The clinical characteristics of CAD patients and control group are shown in Table 1. Age and gender matched in CAD
W. Su et al. / Clinical Biochemistry 42 (2009) 365–370
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Table 1 Characteristics of the total study population CAD patients (n = 118) Control subjects (n = 225) Age (years) 61.8 ± 11.4 Sex (M/F) 89/29 Habitual 41.5 smoking (%) BMI (kg/m2) 24.7 ± 3.1 Systolic 133.4 ± 31.2 Bp (mmHg) Diastolic 84.6 ± 13.4 Bp (mmHg) TC (mmol/L) 5.4 ± 1.4 TG (mmol/L) 1.7 ± 1.1 LDL-C (mmol/L) 2.5 ± 0.8 HDL-C (mmol/L) 1.1 ± 0.3
P value
60.4 ± 12.7 152/73 40.1
0.587 0.138 0.352
24.3 ± 3.3 124.3 ± 28.7
0.715 b0.001
82.9 ± 15.5
0.06
3.7 ± 1.0 1.5 ± 1.0 1.9 ± 0.7 1.1 ± 0.4
b0.001 0.03 b0.001 0.561
Data are presented in mean ± SD format. BMI, body mass index; TG, triglycerides; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol.
and control group. Several conventional risk factors for CAD, including habitual smoking are identical between two groups. However, systolic blood pressure (P b 0.001), TC (P b 0.001), TG (P = 0.03) and LDL-C (P b 0.001) concentrations in the CAD patients are significantly higher than in control group (Table 1). Plasma concentration of ApoM in CAD patients and control group As shown in Fig. 1, the concentration of plasma ApoM is 1.3757 ± 0.1493 ODu mm− 2 in CAD patients and 1.3502 ± 0.1288 ODu mm− 2 in control group. The concentration of plasma ApoM is higher in CAD patients than that in control group (t = 0.032, P b 0.05). Our previous study of T-778C polymorphism of ApoM gene in Han Chinese CAD patients demonstrated that the C allele at nucleotide − 778 in the ApoM gene is associated with increased risk of CAD [15]. To investigate the effect of T-778C polymorphism of ApoM gene on CAD, the concentration of
Fig. 1. Plasma concentration of ApoM in CAD patients and control group. ApoM concentration was determined by dot blot analysis as described in section Methods. Data which was analyzed by software Quantity One are presented as ODu mm− 2. Other data are presented as means ± S.D. The concentration of plasma ApoM was 1.3757 ± 0.1493 ODu mm− 2 in CAD patients and 1.3502 ± 0.1288 ODu mm− 2 in control group. Plasma concentration of ApoM was higher in CAD patients than that in control group. There was significant differences between CAD patients and controls with student t test (t = 0.032, P b 0.05).
Fig. 2. Relationship between plasma concentration of ApoM and total cholesterol in both CAD patients and control group (r = 0.38, P = 0.021). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are presented as ODu mm− 2. Total cholesterol was measured by the cholesterol oxidase method using an automated RA-100. Pearson correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
plasma ApoM was determined to see if it changes as a function of the genotypes. For both groups, plasma concentration of ApoM with TT genotype (1.3697 ± 0.1141 ODu mm− 2) was slightly higher than that in those with CT (1.3618 ± 0.1401 ODu mm− 2) or CC (1.3628 ± 0.1302 ODu mm− 2) genotype, but there were no significant differences among the genotypes (t = 0.4112, P = 0.073) (not shown). Correlation of ApoM and other lipid parameters In our study several common clinical lipid parameters were measured for each subject. These measurements are used to study covariation of ApoM concentration. Among these parameters, Lpα and TG, data distribution are significantly skewed, thus the nonparametric Spearman rank-order correlation coefficient is calculated. The Pearson correlation coefficient is calculated for other parameters. Our analysis shows ApoM level is correlated with plasma TC (r = 0.38, P = 0.021) (Fig. 2), LDL-C(r = 0.16, P = 0.03) (Fig. 3), HDL-C(r = 0.29, P = 0.01) (Fig. 4) and ApoA-I(r = 0.2357, P = 0.0327) (Table 2). However, there is no statistically significant (P N 0.05) correlation between ApoM concentration and TG, ApoB, apolipoprotein E (ApoE) and Lpα (Table 2).
Fig. 3. Relationship between plasma concentration of ApoM and LDL cholesterol in both CAD patients and control group (r = 0.16, P = 0.03). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are expressed as ODu mm− 2. Plasma concentration of LDL cholesterol was determined by the chemical modification enzymatic method using an automated RA-100. Pearson correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
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Fig. 4. Relationship between plasma concentration of ApoM and HDL cholesterol in both CAD patients and control group (r = 0.29, P = 0.03). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software of Quantity One are expressed as ODu mm− 2. Plasma concentration of HDL cholesterol levels was determined by the chemical modification enzymatic method using an automated RA-100. Pearson correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
Fig. 5. Concentration of plasma ApoM in different ranks of CAD patients according to the numbers of lesioned arteries. ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are expressed as ODu mm− 2. Other data are presented as means ± S.D. There were no significant differences among different ranks of CAD patients with student t test (t = 0.382, P N 0.05).
after adjusted for conventional cardiovascular risk factors (P N 0.05, respectively).
Plasma concentration of ApoM and coronary atherosclerosis severity
Discussion Quantitative coronary angiography demonstrated that 22 subjects had lesions in 1 artery, 46 had lesions in 2 arteries, and 50 had lesions in 3 arteries. The concentration of plasma ApoM is 1.4065 ± 0.1794 ODu mm− 2, 1.3523 ± 0.1504 ODu mm− 2 and 1.3657 ± 0.1438 ODu mm− 2 in patients with 1 lesioned artery, 2 lesioned arteries and 3 lesioned arteries respectively. No significant difference is detected among these three ranks (t = 0.382, P N 0.05) as illustrated in Fig. 5. Nonparametric Spearman rank-order correlation coefficient is calculated to assess the relationship between plasma level of ApoM and Gensini score for CAD patients, and result shows no relationship between plasma concentrations of ApoM and Gensini score of coronary atherosclerotic heart disease. (r = 0.1226, P = 0.1716) (Fig. 6). Multiple logistic and linear regression analysis was also applied to adjustment for age, sex, BMI, and history of smoking, hypertension and hypercholesterolemia. It showed that plasma concentration of ApoM did not correlate either with the number of lesioned coronaries or the Gensini score
ApoM was originally found in chylomicrons and is found in all major lipoprotein classes [5], it is highly hydrophobic and must co-circulate with lipoprotein particles in the blood stream. As a member of lipocalin protein superfamily, the signal peptide of ApoM remains uncleaved in the circulating protein [6], which is necessary for ApoM's ability to associate with lipoprotein [17]. ApoM plays an important role in lipid metabolism and atherosclerosis. It has been recently described in preβ-HDL formation [18]. In the same study, overexpression of ApoM in LDL receptor knockout mice protected against atherosclerosis when the mice were challenged with a cholesterol-enriched diet [18]. In the setting of low density lipoprotein receptor deficiency, ApoM-Tg mice with approximately 2-fold increased plasma ApoM concentrations developed smaller atherosclerotic lesions than control group [19]. Compared with ApoM-free HDL, human ApoM-containing HDL
Table 2 Results from correlation studies of ApoM plasma concentration and TG, ApoA-I, ApoB, ApoE, Lpα Lipid parameter
r
P value
95% CI
TG ⁎ ApoA-I ApoB ApoE Lpα ⁎
− 0.06856 0.2357 − 0.08189 0.03549 0.06549
0.4260 0.0327 0.3433 0.6817 0.4487
− 0.2336 to − 0.0552 to − 0.2469 to − 0.1337 to − 0.1040 to
0.1003 0.2815 0.08770 0.2026 0.2313
ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are presented as ODu mm− 2. ⁎ TG and Lpα display a skewed distribution, and Spearman rank-order correlation coefficient was calculated. Pearson correlation coefficient was calculated for ApoA-I, ApoB and ApoE, using GraphPad Prism 4.03statistics software.
Fig. 6. Relationships between plasma concentration of ApoM and Gensini score of CAD patients(r = 0.1226, P = 0.1716). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software of Quantity One are expressed as ODu mm− 2. Gensini score was used to assess coronary atherosclerosis severity according to selective coronary angiography with the Judkins' method. It indicated that plasma concentration of ApoM had no relationship with Gensini score of coronary artery disease, and the nonparametric Spearman rank-order correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
W. Su et al. / Clinical Biochemistry 42 (2009) 365–370
particles had an enhanced ability to reduce LDL oxidation and promote cholesterol efflux [20]. However, as ApoM-containing HDL represents only a small fraction of total HDL in plasma the actual biological implications of these findings are uncertain. It is the first time that the plasma concentration of ApoM in CAD patients and control group are compared in the study of ApoM functionality. Our analysis shows that concentration of plasma ApoM in CAD patients was higher than that in control group. Correlation analysis between the concentration of plasma ApoM and lipid parameters shows that plasma level of ApoM is positively associated with TC, LDL-C and HDL-C. This is in line with the earlier results from experiment based on sandwich ELISA for the quantification of ApoM in human plasma using two monoclonal antibodies [9]. Plasma level of ApoA-I was also positively correlated with plasma concentration of ApoM in our study. Previous experiment indicated that significantly reduced plasma ApoM levels, only 33% of normal concentration, have been discovered in mice lacking ApoA-I. The majority of ApoM is associated with HDL and two or three ApoA-I molecules are associated with each HDL particle [21]. Both our current study and previous experiments suggest that ApoM is involved in the metabolism of ApoA-I. The isolated human plasma ApoM is located mainly in HDL and to a minor extent in LDL [20]. ApoM is presented in ∼5% of HDL particles and in b 2% of LDL particles of human plasma. ApoM-containing lipoproteins contained Apolipoprotein J, ApoA-I, ApoA-II, ApoC-I, ApoC-II, ApoC-III, paraoxonase 1, and ApoB. The apolipoprotein composition varies between ApoM-containing particles and ApoM-free particles such as ApoJ and ApoM-containing HDL contains significantly more free cholesterol than HDL lacking ApoM [20]. Plasma LDL-C is positively associated with plasma concentration of ApoM, while there has no relationship between plasma level of ApoM and LDL-related ApoB in the present study. Whether the amount of cholesterol and ApoB has difference between ApoMcontaining LDL particles and ApoM-free LDL particles is to be further investigated. SNP site is located in promoter region, which may affect gene transcription as well as the protein level in the blood and, therefore, may contribute to its association with some diseases. Our previous study shows that T-778C polymorphism of ApoM gene is associated with CAD [15], we evaluated the impact of the T-778C polymorphism on plasma concentration of ApoM in the present study. We did not find any association between the T-778C polymorphism and plasma levels of ApoM, which suggests that the T-778C polymorphism had no effect on the expression of ApoM. The mechanism of T-778C polymorphism of ApoM gene on CAD is still to be elucided. To assess the role of plasma ApoM in the development of CAD, relationship between concentration of plasma ApoM and severity of CAD was analyzed in our study. There were no significant differences in concentration of plasma ApoM among three ranks of CAD. Gensini score of CAD has no correlation with level of plasma ApoM. Logistic and multiple linear regression analysis show that in all subjects plasma concentration of ApoM did not correlate with the number of lesioned
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coronaries and Gensini score adjusted for conventional cardiovascular risk factors. These findings suggested that ApoM is not an independent risk factor for the development of CAD but could be a biomarker of CAD. The strong correlation of plasma level of ApoM with plasma cholesterol, together with the finding that concentration of plasma ApoM in CAD patients is higher than that in control group indicates that ApoM could be a biomarker of CAD. An in-depth structural analysis may unravel important role in physiological and pathological processes of lipid metabolism. Many isoforms of ApoM have been detected associated with different lipoproteins [22–24]. To assess the role of ApoM in lipid metabolism and development of CAD different isoforms of ApoM should be further elucidated. In conclusion, our study shows that plasma level of ApoM in CAD patients is higher than that in normal healthy control group, plasma concentration of ApoM is not associated with severity of CAD. Plasma level of ApoM is associated with total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and ApoA-I. Plasma level of ApoM could not be an independent risk factor but a biomarker of CAD. Acknowledgments We thank Dr. JunBo Ge of Affiliated Shanghai Zhongshan Hospital, Shanghai Fudan University, Shanghai, China for the collection of studied samples. We also thank Prof. Qing-Bo He of Department of Biomedical Statistics, Shanghai Jiaotong University, Shanghai, China for the statistical instruction in the present study. References [1] Lahoz C, Schaefer EJ, Cupples LA, Wilson PW, Levy D, Osgood D, et al. Apolipoprotein E genotyping and cardiovascular disease in the Framingham Heart study. Atherosclerosis 2001;154:529–37. [2] Frishman WH. Biologic markers as predictors of cardiovascular disease. Am J Med 1998;104:185–275. [3] Centers for Disease Control. Effectiveness in disease and injury prevention: public health focus: physical activity and the prevention of coronary heart disease. Morb Mort Wkly Rep 1993;42:669–72. [4] Srinivasan SR, Ehnholm C, Elkasabany A, et al. Influence of apolipoprotein E polymorphism on serum lipids and lipoprotein changes from childhood to adulthood the Bogalusa Heart study. Atherosclerosis 1999; 143:435–43. [5] Xu N, Dahlback B. A novel human apolipoprotein (ApoM). J Biol Chem 1999;274:31286–90. [6] Duan J, Dahlback B, Villoutreix BO. Proposed lipocalin fold for apolipoprotein M based on bioinformatics and site-directed mutagenesis. FEBS Lett 2001;499:127–32. [7] Ahnström J, Faber K, Axler O, Dahlbäck B. Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M. J Lipid Res 2007;48: 1754–62. [8] Richter S, Shih DQ, Pearson ER, Wolfrum C, Fajans SS, Hattersley AT, et al. Regulation of apolipoprotein M gene expression by MODY3 gene hepatocyte nuclear factor-1alpha: haploinsufficiency is associated with reduced serum apolipoprotein M levels. Diabetes 2003;52:2989–95. [9] Axler O, Ahnström J, Dahlbäck B. An ELISA for apolipoprotein M reveals a strong correlation to total cholesterol in human plasma. J Lipid Res 2007; 48:1772–80.
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[10] Jungner I, Marcovina SM, Walldius G, Holme I, Kolar W, Steiner E. Apolipoprotein B and A-I values in 147576 Swedish males and females, standardized according to the World Health Organization-International Federation of Clinical Chemistry First International Reference Materials. Clin Chem 1998;44:1641–9. [11] Kannel WB, Castelli WP, Gordon T, McNamara PM. Serum cholesterol, lipoproteins, and the risk of coronary heart disease. The Framingham Study. Ann Intern Med 1971;74:1–12. [12] Kannel WB, Neaton JD, Wentworth D, Thomas HE, Stamler J, Hulley SB, et al. Overall and coronary heart disease mortality rates in relation to major risk factors in 325,348 men screened for the MRFIT. Multiple Risk Factor Intervention Trial. Am Heart J 1986;112:825–36. [13] Mensah GA, Brown DW, Croft JB, Greenlund KJ. Major coronary risk factors and death from coronary heart disease: baseline and follow-up mortality data from the Second National Health and Nutrition Examination Survey (NHANES II). Am J Prev Med 2005;29:68–74. [14] Wolfrum C, Poy MN, Stoffel M. Apolipoprotein M is required for prebetaHDL formation and cholesterol efflux to HDL and protects against atherosclerosis. Nat Med 2005;11:418–22. [15] Jiao GQ, Yuan ZX, Xue YS, Yang CJ, Lu CB, Lu ZQ, et al. A prospective evaluation of apolipoprotein M gene T-778C polymorphism in relation to coronary artery disease in Han Chinese. Clin Biochem 2007;40:1108–12. [16] Genisi GG. A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol 1983;51:606.
[17] Axler O, Ahnström J, Dahlbäck B. Apolipoprotein M associates to lipoproteins through its retained signal peptide. FEBS Lett 2008;582:826–8. [18] Wolfrum C, Poy MN, Stoffel M. Apolipoprotein M is required for prebetaHDL formation and cholesterol efflux to HDL and protects against atherosclerosis. Nat Med 2005;11:418–22. [19] Christoffersen C, Jauhiainen M, Moser M, Porse B, Ehnholm C, Boesl M, et al. Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knock-out mice. J Biol Chem 2008;25:1839–47. [20] Christoffersen C, Nielsen LB, Axler O, Andersson A, Johnsen AH, Dahlbäck B. Isolation and characterization of human apolipoprotein M-containing lipoproteins. J Lipid Res 2006;47:1833–43. [21] Faber K, Hvidberg V, Moestrup SK, Dahlbäck B, Nielsen LB. Megalin is a receptor for apolipoprotein M, and kidney-specific megalin-deficiency confers urinary excretion of apolipoprotein M. Mol Endocrinol 2006;20:212–8. [22] Karlsson H, Leanderson P, Tagesson C, Lindahl M. Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry. Proteomics 2005;5:551–65. [23] Karlsson H, Lindqvist H, Tagesson C, Lindahl M. Characterization of apolipoprotein M isoforms in low-density lipoprotein. J Proteome Res 2006;5: 2685–90. [24] Mancone C, Amicone L, Fimia GM, Bravo E, Piacentini M, Tripodi M. Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF. Proteomics 2007;7:143–54.
Clinical Biochemistry 42 (2009) 365 – 370
Evaluation of apolipoprotein M as a biomarker of coronary artery disease Weiqiang Su b , Guoqing Jiao a,b , Chengjian Yang b , Yizhou Ye a,⁎ a
b
Department of Cardiovascular Surgery, Affiliated Shanghai 1st People's Hospital, Shanghai Jiaotong University, Hongkou, 85 Wujin Road, Shanghai 200080, People's Republic of China Department of Cardiovascular Surgery, Affiliated Wuxi 2nd People's Hospital, Nanjing Medical University, Jiangsu, People's Republic of China Received 1 August 2008; received in revised form 14 November 2008; accepted 15 November 2008 Available online 3 December 2008
Abstract Objective: To investigate the possible role of apolipoprotein M (ApoM) in the development of coronary artery disease (CAD). Design and methods: Case-controlled study, which consisted of 118 CAD patients and 255 unrelated subjects used as control group. Plasma concentration of ApoM was determined by dot blot, severity of CAD was expressed with Gensini score or the numbers of lesioned coronary arteries, and serum lipid levels were also measured. Results and discussion: Our study shows the mean level of plasma ApoM is 1.3757 ± 0.1493 ODu mm− 2 in CAD patients, while it is 1.3502 ± 0.1288 ODu mm− 2 in control group, and there are significant differences in plasma level of ApoM between two groups (t = 0.032, P b 0.05). Concentration of plasma ApoM is positively associated with plasma total cholesterol (r = 0.38, P = 0.025), high density lipoprotein cholesterol (r = 0.29, P = 0.03), low density lipoprotein cholesterol (r = 0.16, P = 0.03) and apolipoproein A–I (r = 0.24, P = 0.03). Multiple logistic and linear regression analysis showed that plasma concentration of ApoM did not correlate either with the number of lesioned coronaries or the Gensini score after adjusted for conventional cardiovascular risk factors (P N 0.05, respectively). Conclusion: The findings suggest that ApoM could not be an independent risk factor but a biomarker of CAD. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: ApoM; Coronary artery disease; HDL; LDL; TC
Introduction Coronary artery disease (CAD) and stroke are the major cause of death and disability in developing countries. A number of traditional risk factors for CAD include aging, sex, obesity, smoking, diabetes, hypertension, physical inactivity, elevated total plasma cholesterol, low-density lipoprotein (LDL), decreased high density lipoprotein (HDL), and lifestyle [1–3]. Adverse levels of serum lipoprotein are traditionally used as predictive of CAD [4]. ApoM was first described in 1999 [5]. The protein is composed of 188 amino acids, with an apparent size of 25 kDa. It belongs to the lipocalin protein superfamily [6,7]. Hepatic nuclear factor (HNF)-1a in mice is required for ApoM expression in vivo [8]. Mutations in HNF-1a, in heterozygous form, are responsible for the development of maturity-onset diabetes of the young (MODY) type 3 in humans, and reduced plasma ⁎ Corresponding author. Fax: +86 021 63253883. E-mail address: [email protected] (Y. Ye).
ApoM levels were found in MODY 3 patients [8]. A sandwich ELISA for the measurement of ApoM in human plasma shows that ApoM content in the healthy human plasma pool is 0.94 mM, or ∼ 23 mg/l, ApoM concentration is strongly correlated to total cholesterol in healthy individuals [9], which roughly corresponds to 1/50th of the mean molar concentration of apolipoproteinA-I(ApoA-I) in plasma [10]. There is a well-established relationship between plasma total cholesterol and the risk of development of myocardial infarction [11–13]. The strong correlation of ApoM with plasma cholesterol, together with the finding that overexpression of ApoM in LDL receptor deficient mice can protect against atherosclerosis [14], make it interesting to investigate whether ApoM is a predictor of cardiovascular disease. Our previous study of T-778C polymorphism of ApoM gene in Han Chinese CAD patients demonstrated that the C allele at nucleotide − 778 in the ApoM gene is associated with increased risk of CAD and CAD patients with the CC and the CT genotype of SNP-778 have higher plasma concentrations of total cholesterol than normal healthy control group [15]. In order to determine the
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relationships between plasma concentrations of ApoM and CAD, we conducted a case-controlled study, which further revealed the pathophysiological function of ApoM in the development of CAD.
distal segment of the CAD or mid-distal of LCX or right coronary artery, and 0.5 for others [16].
Methods
Plasma ApoM concentration was estimated by a dot-blot analysis with monoclonal rabbit anti-human ApoM antibody (ABNOVA). In brief, 10 μL of plasma was diluted with Tris–HCl buffer (1:20) and 5 μL diluted samples applied to the membranes in triplicate (Hybond-C, Amersham Life Science). The membrane was quenched in Tris–HCl buffer with 4% Tween and 3% bovine serum albumin for 2 h, followed by sequential incubations with monoclonal rabbit anti-human ApoM antibody (dilution, 1:1000 in Tris–HCl buffer) ovenight at 4 °C, and then alkaline phosphatase (AP) conjugated secondary antibody for 2 h at room temperature. The development of AP activity was performed with a commercial visualization system following the manufacturer's instructions (Dako). The recombinant ApoM protein (ABNOVA) and ddH2O were used as standard. The relative amount of ApoM was quantified by a scanner using the software of Quantity One (Version 4.2.1, Bio-Rad Laboratories) and presented as adjusted volume (ODu mm− 2). The same experiment was repeated at least three times.
Study population Subjects for the present study consisted of 118 CAD patients (89 males and 29 females with a mean age of 61.8 ± 11.4 years) and 225 unrelated individuals (152 males and 73 females with a mean age of 60.4 ± 12.7 years) were enrolled as control group. Criterion for enrolment of CAD patients is set as at least 50% stenosis determined angiographically existed in at least one of the major segments of coronary arteries, i.e. the right coronary artery, left circumflex, or left anterior descending arteries. Controls were acquired from those who have performed a negative coronary artery angiography so that CAD patients are excluded. History of conventional risk factors for CAD, including habitual cigarette smoking, hypertension (systolic blood pressure ≥160 mm Hg and/or diastolic blood pressure ≥95 mm Hg), diabetes mellitus, or hypercholesterolemia (TC ≥ 5.7 mmol/L) were obtained from the patients or their medical records. Exclusion criteria for both CAD patients and control group include familial hypercholesterolemia, diabetes mellitus, cancer, renal disease, and any other chronic illnesses. All individuals in both CAD and control group are Han Chinese. Ethical approval for this investigation was obtained from the Research Ethics Committee, Shanghai Jiaotong University. Written consent was obtained from each subject before blood samples were collected. The investigation complies with the principles outlined in the Declaration of Helsinki. Assessment of coronary atherosclerosis severity Selective coronary angiography was conducted using the Judkins' method. For every artery, at least two views were adopted. The degree of coronary stenosis was assessed by quantitative coronary angiography, and diametrical stenosis more than 50% was deemed as a positive result of CAD. The following two methods were used to assess coronary atherosclerosis severity: (1) The number of lesioned arteries(≥50% stenosis of luminal diameter): lesion in the left anterior descending, left circumflex, right coronary artery or their main branches was recorded as one lesioned artery, and lesion in the left main coronary artery was recorded as two lesioned arteries. The same coronary artery was not counted more than twice. According to the number of lesioned arteries, there were three ranks, namely, 1 lesioned artery, 2 lesioned arteries, and 3 lesioned arteries; (2) Gensini score: 1–25%, 26–50%, 51–75%, 76–90%, 91–99%, and 100% of coronary luminal narrowing were given scores of 1, 2, 4, 8, 16, and 32 respectively. This score was then multiplied by a factor based on the importance of lesion's position in the coronary arterial tree: 5 for the left main coronary artery, 2.5 for proximal segment of the left anterior descending coronary artery (LAD) or the circumflex artery (LCX), 1.5 for mid-segment of LAD, 1 for
Measurement of plasma ApoM concentration
Biochemical analysis Plasma total cholesterol (TC) was measured by the cholesterol oxidase method and triglycerides (TG) by the phosphoglycerol oxidase method with commercial kits (Shanghai Mingdian Bio-Tech Co., China). Plasma low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were determined using the chemical modification enzymatic method (Trinity Biotech, Ireland). Plasma concentration of ApoA-I, apolipoprotein B(ApoB) and Lpα were determined by immunoturbidimetry assay (Shanghai Mingdian Bio-Tech Co., China). Plasma concentration of apolipoprotein E (ApoE) was determined by ELISA using a commercial kit (Shanghai Mingdian Bio-Tech Co., China). Lipid parameters were measured using an automated RA-1000 (Technician, USA). Samples for quality control were provided by RANDOX (England). Statistical analysis Data are presented as mean ± S.E. Student's t test, chi-square test, Pearson or Spearman rank-order correlation were used for the analysis of the data using GraphPad Prism 4.03statistics software. The SPSS statistical software package version 11.5 was used for multiple logistic and linear regres-sion analysis. P ≤ 0.05 levels was considered as statistical significance. Results Clinical characteristics of CAD patients and control group The clinical characteristics of CAD patients and control group are shown in Table 1. Age and gender matched in CAD
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Table 1 Characteristics of the total study population CAD patients (n = 118) Control subjects (n = 225) Age (years) 61.8 ± 11.4 Sex (M/F) 89/29 Habitual 41.5 smoking (%) BMI (kg/m2) 24.7 ± 3.1 Systolic 133.4 ± 31.2 Bp (mmHg) Diastolic 84.6 ± 13.4 Bp (mmHg) TC (mmol/L) 5.4 ± 1.4 TG (mmol/L) 1.7 ± 1.1 LDL-C (mmol/L) 2.5 ± 0.8 HDL-C (mmol/L) 1.1 ± 0.3
P value
60.4 ± 12.7 152/73 40.1
0.587 0.138 0.352
24.3 ± 3.3 124.3 ± 28.7
0.715 b0.001
82.9 ± 15.5
0.06
3.7 ± 1.0 1.5 ± 1.0 1.9 ± 0.7 1.1 ± 0.4
b0.001 0.03 b0.001 0.561
Data are presented in mean ± SD format. BMI, body mass index; TG, triglycerides; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol.
and control group. Several conventional risk factors for CAD, including habitual smoking are identical between two groups. However, systolic blood pressure (P b 0.001), TC (P b 0.001), TG (P = 0.03) and LDL-C (P b 0.001) concentrations in the CAD patients are significantly higher than in control group (Table 1). Plasma concentration of ApoM in CAD patients and control group As shown in Fig. 1, the concentration of plasma ApoM is 1.3757 ± 0.1493 ODu mm− 2 in CAD patients and 1.3502 ± 0.1288 ODu mm− 2 in control group. The concentration of plasma ApoM is higher in CAD patients than that in control group (t = 0.032, P b 0.05). Our previous study of T-778C polymorphism of ApoM gene in Han Chinese CAD patients demonstrated that the C allele at nucleotide − 778 in the ApoM gene is associated with increased risk of CAD [15]. To investigate the effect of T-778C polymorphism of ApoM gene on CAD, the concentration of
Fig. 1. Plasma concentration of ApoM in CAD patients and control group. ApoM concentration was determined by dot blot analysis as described in section Methods. Data which was analyzed by software Quantity One are presented as ODu mm− 2. Other data are presented as means ± S.D. The concentration of plasma ApoM was 1.3757 ± 0.1493 ODu mm− 2 in CAD patients and 1.3502 ± 0.1288 ODu mm− 2 in control group. Plasma concentration of ApoM was higher in CAD patients than that in control group. There was significant differences between CAD patients and controls with student t test (t = 0.032, P b 0.05).
Fig. 2. Relationship between plasma concentration of ApoM and total cholesterol in both CAD patients and control group (r = 0.38, P = 0.021). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are presented as ODu mm− 2. Total cholesterol was measured by the cholesterol oxidase method using an automated RA-100. Pearson correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
plasma ApoM was determined to see if it changes as a function of the genotypes. For both groups, plasma concentration of ApoM with TT genotype (1.3697 ± 0.1141 ODu mm− 2) was slightly higher than that in those with CT (1.3618 ± 0.1401 ODu mm− 2) or CC (1.3628 ± 0.1302 ODu mm− 2) genotype, but there were no significant differences among the genotypes (t = 0.4112, P = 0.073) (not shown). Correlation of ApoM and other lipid parameters In our study several common clinical lipid parameters were measured for each subject. These measurements are used to study covariation of ApoM concentration. Among these parameters, Lpα and TG, data distribution are significantly skewed, thus the nonparametric Spearman rank-order correlation coefficient is calculated. The Pearson correlation coefficient is calculated for other parameters. Our analysis shows ApoM level is correlated with plasma TC (r = 0.38, P = 0.021) (Fig. 2), LDL-C(r = 0.16, P = 0.03) (Fig. 3), HDL-C(r = 0.29, P = 0.01) (Fig. 4) and ApoA-I(r = 0.2357, P = 0.0327) (Table 2). However, there is no statistically significant (P N 0.05) correlation between ApoM concentration and TG, ApoB, apolipoprotein E (ApoE) and Lpα (Table 2).
Fig. 3. Relationship between plasma concentration of ApoM and LDL cholesterol in both CAD patients and control group (r = 0.16, P = 0.03). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are expressed as ODu mm− 2. Plasma concentration of LDL cholesterol was determined by the chemical modification enzymatic method using an automated RA-100. Pearson correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
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Fig. 4. Relationship between plasma concentration of ApoM and HDL cholesterol in both CAD patients and control group (r = 0.29, P = 0.03). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software of Quantity One are expressed as ODu mm− 2. Plasma concentration of HDL cholesterol levels was determined by the chemical modification enzymatic method using an automated RA-100. Pearson correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
Fig. 5. Concentration of plasma ApoM in different ranks of CAD patients according to the numbers of lesioned arteries. ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are expressed as ODu mm− 2. Other data are presented as means ± S.D. There were no significant differences among different ranks of CAD patients with student t test (t = 0.382, P N 0.05).
after adjusted for conventional cardiovascular risk factors (P N 0.05, respectively).
Plasma concentration of ApoM and coronary atherosclerosis severity
Discussion Quantitative coronary angiography demonstrated that 22 subjects had lesions in 1 artery, 46 had lesions in 2 arteries, and 50 had lesions in 3 arteries. The concentration of plasma ApoM is 1.4065 ± 0.1794 ODu mm− 2, 1.3523 ± 0.1504 ODu mm− 2 and 1.3657 ± 0.1438 ODu mm− 2 in patients with 1 lesioned artery, 2 lesioned arteries and 3 lesioned arteries respectively. No significant difference is detected among these three ranks (t = 0.382, P N 0.05) as illustrated in Fig. 5. Nonparametric Spearman rank-order correlation coefficient is calculated to assess the relationship between plasma level of ApoM and Gensini score for CAD patients, and result shows no relationship between plasma concentrations of ApoM and Gensini score of coronary atherosclerotic heart disease. (r = 0.1226, P = 0.1716) (Fig. 6). Multiple logistic and linear regression analysis was also applied to adjustment for age, sex, BMI, and history of smoking, hypertension and hypercholesterolemia. It showed that plasma concentration of ApoM did not correlate either with the number of lesioned coronaries or the Gensini score
ApoM was originally found in chylomicrons and is found in all major lipoprotein classes [5], it is highly hydrophobic and must co-circulate with lipoprotein particles in the blood stream. As a member of lipocalin protein superfamily, the signal peptide of ApoM remains uncleaved in the circulating protein [6], which is necessary for ApoM's ability to associate with lipoprotein [17]. ApoM plays an important role in lipid metabolism and atherosclerosis. It has been recently described in preβ-HDL formation [18]. In the same study, overexpression of ApoM in LDL receptor knockout mice protected against atherosclerosis when the mice were challenged with a cholesterol-enriched diet [18]. In the setting of low density lipoprotein receptor deficiency, ApoM-Tg mice with approximately 2-fold increased plasma ApoM concentrations developed smaller atherosclerotic lesions than control group [19]. Compared with ApoM-free HDL, human ApoM-containing HDL
Table 2 Results from correlation studies of ApoM plasma concentration and TG, ApoA-I, ApoB, ApoE, Lpα Lipid parameter
r
P value
95% CI
TG ⁎ ApoA-I ApoB ApoE Lpα ⁎
− 0.06856 0.2357 − 0.08189 0.03549 0.06549
0.4260 0.0327 0.3433 0.6817 0.4487
− 0.2336 to − 0.0552 to − 0.2469 to − 0.1337 to − 0.1040 to
0.1003 0.2815 0.08770 0.2026 0.2313
ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software Quantity One are presented as ODu mm− 2. ⁎ TG and Lpα display a skewed distribution, and Spearman rank-order correlation coefficient was calculated. Pearson correlation coefficient was calculated for ApoA-I, ApoB and ApoE, using GraphPad Prism 4.03statistics software.
Fig. 6. Relationships between plasma concentration of ApoM and Gensini score of CAD patients(r = 0.1226, P = 0.1716). ApoM concentration was determined by dot blot analysis as described in section Methods. Data that was analyzed by software of Quantity One are expressed as ODu mm− 2. Gensini score was used to assess coronary atherosclerosis severity according to selective coronary angiography with the Judkins' method. It indicated that plasma concentration of ApoM had no relationship with Gensini score of coronary artery disease, and the nonparametric Spearman rank-order correlation coefficient was calculated using GraphPad Prism 4.03statistics software.
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particles had an enhanced ability to reduce LDL oxidation and promote cholesterol efflux [20]. However, as ApoM-containing HDL represents only a small fraction of total HDL in plasma the actual biological implications of these findings are uncertain. It is the first time that the plasma concentration of ApoM in CAD patients and control group are compared in the study of ApoM functionality. Our analysis shows that concentration of plasma ApoM in CAD patients was higher than that in control group. Correlation analysis between the concentration of plasma ApoM and lipid parameters shows that plasma level of ApoM is positively associated with TC, LDL-C and HDL-C. This is in line with the earlier results from experiment based on sandwich ELISA for the quantification of ApoM in human plasma using two monoclonal antibodies [9]. Plasma level of ApoA-I was also positively correlated with plasma concentration of ApoM in our study. Previous experiment indicated that significantly reduced plasma ApoM levels, only 33% of normal concentration, have been discovered in mice lacking ApoA-I. The majority of ApoM is associated with HDL and two or three ApoA-I molecules are associated with each HDL particle [21]. Both our current study and previous experiments suggest that ApoM is involved in the metabolism of ApoA-I. The isolated human plasma ApoM is located mainly in HDL and to a minor extent in LDL [20]. ApoM is presented in ∼5% of HDL particles and in b 2% of LDL particles of human plasma. ApoM-containing lipoproteins contained Apolipoprotein J, ApoA-I, ApoA-II, ApoC-I, ApoC-II, ApoC-III, paraoxonase 1, and ApoB. The apolipoprotein composition varies between ApoM-containing particles and ApoM-free particles such as ApoJ and ApoM-containing HDL contains significantly more free cholesterol than HDL lacking ApoM [20]. Plasma LDL-C is positively associated with plasma concentration of ApoM, while there has no relationship between plasma level of ApoM and LDL-related ApoB in the present study. Whether the amount of cholesterol and ApoB has difference between ApoMcontaining LDL particles and ApoM-free LDL particles is to be further investigated. SNP site is located in promoter region, which may affect gene transcription as well as the protein level in the blood and, therefore, may contribute to its association with some diseases. Our previous study shows that T-778C polymorphism of ApoM gene is associated with CAD [15], we evaluated the impact of the T-778C polymorphism on plasma concentration of ApoM in the present study. We did not find any association between the T-778C polymorphism and plasma levels of ApoM, which suggests that the T-778C polymorphism had no effect on the expression of ApoM. The mechanism of T-778C polymorphism of ApoM gene on CAD is still to be elucided. To assess the role of plasma ApoM in the development of CAD, relationship between concentration of plasma ApoM and severity of CAD was analyzed in our study. There were no significant differences in concentration of plasma ApoM among three ranks of CAD. Gensini score of CAD has no correlation with level of plasma ApoM. Logistic and multiple linear regression analysis show that in all subjects plasma concentration of ApoM did not correlate with the number of lesioned
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coronaries and Gensini score adjusted for conventional cardiovascular risk factors. These findings suggested that ApoM is not an independent risk factor for the development of CAD but could be a biomarker of CAD. The strong correlation of plasma level of ApoM with plasma cholesterol, together with the finding that concentration of plasma ApoM in CAD patients is higher than that in control group indicates that ApoM could be a biomarker of CAD. An in-depth structural analysis may unravel important role in physiological and pathological processes of lipid metabolism. Many isoforms of ApoM have been detected associated with different lipoproteins [22–24]. To assess the role of ApoM in lipid metabolism and development of CAD different isoforms of ApoM should be further elucidated. In conclusion, our study shows that plasma level of ApoM in CAD patients is higher than that in normal healthy control group, plasma concentration of ApoM is not associated with severity of CAD. Plasma level of ApoM is associated with total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and ApoA-I. Plasma level of ApoM could not be an independent risk factor but a biomarker of CAD. Acknowledgments We thank Dr. JunBo Ge of Affiliated Shanghai Zhongshan Hospital, Shanghai Fudan University, Shanghai, China for the collection of studied samples. We also thank Prof. Qing-Bo He of Department of Biomedical Statistics, Shanghai Jiaotong University, Shanghai, China for the statistical instruction in the present study. References [1] Lahoz C, Schaefer EJ, Cupples LA, Wilson PW, Levy D, Osgood D, et al. Apolipoprotein E genotyping and cardiovascular disease in the Framingham Heart study. Atherosclerosis 2001;154:529–37. [2] Frishman WH. Biologic markers as predictors of cardiovascular disease. Am J Med 1998;104:185–275. [3] Centers for Disease Control. Effectiveness in disease and injury prevention: public health focus: physical activity and the prevention of coronary heart disease. Morb Mort Wkly Rep 1993;42:669–72. [4] Srinivasan SR, Ehnholm C, Elkasabany A, et al. Influence of apolipoprotein E polymorphism on serum lipids and lipoprotein changes from childhood to adulthood the Bogalusa Heart study. Atherosclerosis 1999; 143:435–43. [5] Xu N, Dahlback B. A novel human apolipoprotein (ApoM). J Biol Chem 1999;274:31286–90. [6] Duan J, Dahlback B, Villoutreix BO. Proposed lipocalin fold for apolipoprotein M based on bioinformatics and site-directed mutagenesis. FEBS Lett 2001;499:127–32. [7] Ahnström J, Faber K, Axler O, Dahlbäck B. Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M. J Lipid Res 2007;48: 1754–62. [8] Richter S, Shih DQ, Pearson ER, Wolfrum C, Fajans SS, Hattersley AT, et al. Regulation of apolipoprotein M gene expression by MODY3 gene hepatocyte nuclear factor-1alpha: haploinsufficiency is associated with reduced serum apolipoprotein M levels. Diabetes 2003;52:2989–95. [9] Axler O, Ahnström J, Dahlbäck B. An ELISA for apolipoprotein M reveals a strong correlation to total cholesterol in human plasma. J Lipid Res 2007; 48:1772–80.
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