FEBS Open Bio 5 (2015) 712–716
journal homepage: www.elsevier.com/locate/febsopenbio
Detection of circulating IgG antibodies to apolipoprotein B100 in acute myocardial infarction Xueying Zhang a,b, Xiaohong Zhang b, Mingming Lei b, Yingzi Lin b, Ian L. Megson c, Jun Wei c, Bo Yu a,⇑, Yuanzhe Jin b,⇑ a b c
Department of Cardiovascular Medicine, The First Affiliated Hospital of China Medical University, Shenyang 110001, China Department of Cardiovascular Medicine, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China Department of Diabetes & Cardiovascular Science, University of the Highlands & Islands, Centre for Health Science, Inverness IV2 3JH, UK
a r t i c l e
i n f o
Article history: Received 22 June 2015 Revised 29 July 2015 Accepted 6 August 2015
Keywords: Acute myocardial infarction ApoB100 oxLDL Antibody Atherosclerosis
a b s t r a c t A number of studies have reported an association between increased levels of antibodies against oxidized low-density lipoprotein (oxLDL) and cardiovascular disease, but the anti-oxLDL antibody has not been confirmed to serve as an effective biomarker for prediction of acute myocardial infarction (AMI). Apolipoprotein B100 (ApoB100)-derived peptide fragments generated by proteolytic degradation and aldehyde modification are the major antigens in oxLDL, and so the present work was undertaken to detect circulating IgG for Apo-B100-derived peptide antigens. An in-house enzyme-linked immunosorbent assay (ELISA) was developed with eight ApoB100-derived peptide antigens (Ag1–Ag8) to detect circulating anti-ApoB100 IgG levels in 267 patients with AMI and 201 control subjects. Binary logistic regression analysis revealed that circulating IgG for Ag1 was significantly higher in the patient group than the control group (P < 0.001) after adjustment for age, gender, smoking, hypertension, diabetes and circulating levels of cholesterol, HDL, LDL, ApoA and ApoB100. None of the other seven antigens detected an increase in IgG levels in AMI patients compared with control subjects. Spearman correlation analysis showed no correlation between IgG antibody for Ag1 and clinical characteristics. In conclusion, the linear peptide antigens derived from ApoB100 may be suitable for the development of an ELISA antibody test for prediction of AMI, although further confirmation is still needed in large-scale clinical studies. Ó 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Acute myocardial infarction (AMI) is defined as myocardial cell death due to prolonged ischemia [14], characterized mainly by rupture or erosion of coronary atherosclerotic plaque and subsequent complete or incomplete occlusive thrombosis. Patients can be diagnosed as having AMI when they have increased blood concentrations of sensitive and specific biomarkers, such as circulating troponin or the membrane-bound fraction of creatinine kinase Abbreviations: AMI, acute myocardial infarction; ApoB100, apolipoprotein B100; cAg, control antigen; ELISA, enzyme-linked immunosorbent assay; hAgs, human antigens; HLA, human leukocyte antigen; IgG, immunoglobulin G; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein ⇑ Corresponding authors at: Department of Cardiovascular Medicine, The First Affiliated Hospital of China Medical University, No. 155 Nanjing North Street, Shenyang 110001, China. Tel.: +86 (0) 13940495166; fax: +86 (0) 83282691 (B. Yu). Department of Cardiovascular Medicine, the Fourth Affiliated Hospital of China Medical University, No. 4 Chongshan East Rd, Shenyang 110032, China, Tel.: +86 (0) 18900916111; fax: +86 (0)2462042117 (Y. Jin). E-mail addresses: [email protected] (B. Yu), [email protected] (Y. Jin).
[30]. AMI is the leading cause of death in coronary heart disease, but there are still no effective biomarkers available for prediction of AMI or identification of unstable coronary plaques. Atherosclerosis of coronary arteries is the main cause of AMI. Sub-endothelial retention of low-density lipoprotein (LDL) on account of accumulation of the oxidized form of LDL (oxLDL) in macrophages and formation of foam cells as a critical early event in atherogenesis [10,17]. Many immunocytochemical studies have identified both oxLDL epitopes and anti-oxLDL immunoglobulins within atherosclerotic lesions [36]. Normal levels of oxLDL are as low as 0.1 ng/lg LDL protein in healthy people [13], making measurement difficult. In addition, spot oxLDL measurements only provide a snap-shot of concentration at a particular moment and are likely to be susceptible to extraneous factors, including diet, smoking and exercise. However, the oxidative modification of LDL leads to the formation of immunogenic epitopes that can induce the secretion of specific antibodies against oxLDL [29,1]. Circulating antibodies against oxLDL represent a durable measure of lipid peroxidation [21].
http://dx.doi.org/10.1016/j.fob.2015.08.006 2211-5463/Ó 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
713
X. Zhang et al. / FEBS Open Bio 5 (2015) 712–716
Circulating anti-oxLDL antibodies are natural antibodies that can be detected almost in every individual, and the concentrations of anti-oxLDL antibodies often peak just before the onset of clinical disease [32]. There are numerous studies demonstrating the association between circulating anti-oxLDL antibodies and cardiovascular disease, but the results reported to date have been inconsistent. Some studies have determined a positive correlation between the levels of circulating anti-oxLDL antibodies and atherosclerotic diseases, whereas others have shown an opposite result [3,12,11,33,16,2,34]. The reasons for inconsistent findings may be due to the difficulties in standardizing the LDL antigens used for testing of antibodies to oxLDL, which can also be altered on account of oxidation ex vivo [11,20]. An approach to overcome the problem of standardization is to detect antibodies against specific native or MDA-modified ApoB100 peptide sequences [5,4,19]. ApoB100 is the only component permanently associated with formation of LDL in the body. Its peptide fragments generated by the proteolytic degradation and aldehyde-modification are the major antigens in oxLDL [25]. These antigens have the advantage of being specific to oxLDL for the unique amino acid sequence. The mature ApoB100 protein is composed of 4563 amino acids in a single polypeptide chain that may carry many human leukocyte antigen (HLA)-restricted epitopes. The present work was thus undertaken to develop an in-house enzyme-linked immunosorbent assay (ELISA) with ApoB100-derived linear peptide antigens to detect circulating IgG antibodies against these linear peptides in AMI. 2. Methods 2.1. Subjects A total of 267 patients who were diagnosed with AMI between January 2012 and January 2014 were recruited by the Department of Cardiovascular Medicine, Fourth Affiliated Hospital of China Medical University, Shenyang, China. Of these 267 patients aged 60.89 ± 11.01 years, 200 were male and 67 were female. They were all diagnosed with spontaneous AMI (namely type 1) based on the Third Universal Definition of Myocardial Infarction published by the European Society of Cardiology [31], including both ST elevation myocardial infarction and non-ST-elevation myocardial infarction, one of whom had atrial septal defect without any symptom Table 1 Clinical characteristics of patients with AMI and control subjects. Characteristics
AMI (n = 267)
Control (n = 201)
P value
Sex (Male) Age (years) Smoker (%) Drinker (%) Hypertension (%) Diabetes (%) Obesity (%) SBP (mmHg) DBP (mmHg) Heart rate (ppm) BMI Cholesterol (mM) Triglyceride (mM) HDL (mM) LDL (mM) ApoA (mM) ApoB (mM) Gensini score
200 (74.9%) 60.9 ± 11.0 160 (59.9%) 77 (28.8%) 138 (51.7) 71 (26.6%) 39 (17.3%) 129.2 ± 22.9 77.5 ± 14.5 73.2 ± 15.8 24.7 ± 3.7 4.50 ± 1.09 1.69 ± 1.28 1.01 ± 0.25 3.02 ± 0.96 0.92 ± 0.43 0.73 ± 0.38 55.2 ± 26.7
136 (67.7%) 59.4 ± 5.4 67 (37.4%) 42 (21.1%) 46 (22.9%) 30 (15.1%) 38 (19.0%) 130.9 ± 18.1 78.4 ± 10.8 76.2 ± 11.6 25.3 ± 3.1 5.27 ± 0.97 1.91 ± 1.92 1.29 ± 0.33 3.13 ± 0.81 1.34 ± 0.22 0.91 ± 0.18 –
0.085 0.086
journal homepage: www.elsevier.com/locate/febsopenbio
Detection of circulating IgG antibodies to apolipoprotein B100 in acute myocardial infarction Xueying Zhang a,b, Xiaohong Zhang b, Mingming Lei b, Yingzi Lin b, Ian L. Megson c, Jun Wei c, Bo Yu a,⇑, Yuanzhe Jin b,⇑ a b c
Department of Cardiovascular Medicine, The First Affiliated Hospital of China Medical University, Shenyang 110001, China Department of Cardiovascular Medicine, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China Department of Diabetes & Cardiovascular Science, University of the Highlands & Islands, Centre for Health Science, Inverness IV2 3JH, UK
a r t i c l e
i n f o
Article history: Received 22 June 2015 Revised 29 July 2015 Accepted 6 August 2015
Keywords: Acute myocardial infarction ApoB100 oxLDL Antibody Atherosclerosis
a b s t r a c t A number of studies have reported an association between increased levels of antibodies against oxidized low-density lipoprotein (oxLDL) and cardiovascular disease, but the anti-oxLDL antibody has not been confirmed to serve as an effective biomarker for prediction of acute myocardial infarction (AMI). Apolipoprotein B100 (ApoB100)-derived peptide fragments generated by proteolytic degradation and aldehyde modification are the major antigens in oxLDL, and so the present work was undertaken to detect circulating IgG for Apo-B100-derived peptide antigens. An in-house enzyme-linked immunosorbent assay (ELISA) was developed with eight ApoB100-derived peptide antigens (Ag1–Ag8) to detect circulating anti-ApoB100 IgG levels in 267 patients with AMI and 201 control subjects. Binary logistic regression analysis revealed that circulating IgG for Ag1 was significantly higher in the patient group than the control group (P < 0.001) after adjustment for age, gender, smoking, hypertension, diabetes and circulating levels of cholesterol, HDL, LDL, ApoA and ApoB100. None of the other seven antigens detected an increase in IgG levels in AMI patients compared with control subjects. Spearman correlation analysis showed no correlation between IgG antibody for Ag1 and clinical characteristics. In conclusion, the linear peptide antigens derived from ApoB100 may be suitable for the development of an ELISA antibody test for prediction of AMI, although further confirmation is still needed in large-scale clinical studies. Ó 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Acute myocardial infarction (AMI) is defined as myocardial cell death due to prolonged ischemia [14], characterized mainly by rupture or erosion of coronary atherosclerotic plaque and subsequent complete or incomplete occlusive thrombosis. Patients can be diagnosed as having AMI when they have increased blood concentrations of sensitive and specific biomarkers, such as circulating troponin or the membrane-bound fraction of creatinine kinase Abbreviations: AMI, acute myocardial infarction; ApoB100, apolipoprotein B100; cAg, control antigen; ELISA, enzyme-linked immunosorbent assay; hAgs, human antigens; HLA, human leukocyte antigen; IgG, immunoglobulin G; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein ⇑ Corresponding authors at: Department of Cardiovascular Medicine, The First Affiliated Hospital of China Medical University, No. 155 Nanjing North Street, Shenyang 110001, China. Tel.: +86 (0) 13940495166; fax: +86 (0) 83282691 (B. Yu). Department of Cardiovascular Medicine, the Fourth Affiliated Hospital of China Medical University, No. 4 Chongshan East Rd, Shenyang 110032, China, Tel.: +86 (0) 18900916111; fax: +86 (0)2462042117 (Y. Jin). E-mail addresses: [email protected] (B. Yu), [email protected] (Y. Jin).
[30]. AMI is the leading cause of death in coronary heart disease, but there are still no effective biomarkers available for prediction of AMI or identification of unstable coronary plaques. Atherosclerosis of coronary arteries is the main cause of AMI. Sub-endothelial retention of low-density lipoprotein (LDL) on account of accumulation of the oxidized form of LDL (oxLDL) in macrophages and formation of foam cells as a critical early event in atherogenesis [10,17]. Many immunocytochemical studies have identified both oxLDL epitopes and anti-oxLDL immunoglobulins within atherosclerotic lesions [36]. Normal levels of oxLDL are as low as 0.1 ng/lg LDL protein in healthy people [13], making measurement difficult. In addition, spot oxLDL measurements only provide a snap-shot of concentration at a particular moment and are likely to be susceptible to extraneous factors, including diet, smoking and exercise. However, the oxidative modification of LDL leads to the formation of immunogenic epitopes that can induce the secretion of specific antibodies against oxLDL [29,1]. Circulating antibodies against oxLDL represent a durable measure of lipid peroxidation [21].
http://dx.doi.org/10.1016/j.fob.2015.08.006 2211-5463/Ó 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
713
X. Zhang et al. / FEBS Open Bio 5 (2015) 712–716
Circulating anti-oxLDL antibodies are natural antibodies that can be detected almost in every individual, and the concentrations of anti-oxLDL antibodies often peak just before the onset of clinical disease [32]. There are numerous studies demonstrating the association between circulating anti-oxLDL antibodies and cardiovascular disease, but the results reported to date have been inconsistent. Some studies have determined a positive correlation between the levels of circulating anti-oxLDL antibodies and atherosclerotic diseases, whereas others have shown an opposite result [3,12,11,33,16,2,34]. The reasons for inconsistent findings may be due to the difficulties in standardizing the LDL antigens used for testing of antibodies to oxLDL, which can also be altered on account of oxidation ex vivo [11,20]. An approach to overcome the problem of standardization is to detect antibodies against specific native or MDA-modified ApoB100 peptide sequences [5,4,19]. ApoB100 is the only component permanently associated with formation of LDL in the body. Its peptide fragments generated by the proteolytic degradation and aldehyde-modification are the major antigens in oxLDL [25]. These antigens have the advantage of being specific to oxLDL for the unique amino acid sequence. The mature ApoB100 protein is composed of 4563 amino acids in a single polypeptide chain that may carry many human leukocyte antigen (HLA)-restricted epitopes. The present work was thus undertaken to develop an in-house enzyme-linked immunosorbent assay (ELISA) with ApoB100-derived linear peptide antigens to detect circulating IgG antibodies against these linear peptides in AMI. 2. Methods 2.1. Subjects A total of 267 patients who were diagnosed with AMI between January 2012 and January 2014 were recruited by the Department of Cardiovascular Medicine, Fourth Affiliated Hospital of China Medical University, Shenyang, China. Of these 267 patients aged 60.89 ± 11.01 years, 200 were male and 67 were female. They were all diagnosed with spontaneous AMI (namely type 1) based on the Third Universal Definition of Myocardial Infarction published by the European Society of Cardiology [31], including both ST elevation myocardial infarction and non-ST-elevation myocardial infarction, one of whom had atrial septal defect without any symptom Table 1 Clinical characteristics of patients with AMI and control subjects. Characteristics
AMI (n = 267)
Control (n = 201)
P value
Sex (Male) Age (years) Smoker (%) Drinker (%) Hypertension (%) Diabetes (%) Obesity (%) SBP (mmHg) DBP (mmHg) Heart rate (ppm) BMI Cholesterol (mM) Triglyceride (mM) HDL (mM) LDL (mM) ApoA (mM) ApoB (mM) Gensini score
200 (74.9%) 60.9 ± 11.0 160 (59.9%) 77 (28.8%) 138 (51.7) 71 (26.6%) 39 (17.3%) 129.2 ± 22.9 77.5 ± 14.5 73.2 ± 15.8 24.7 ± 3.7 4.50 ± 1.09 1.69 ± 1.28 1.01 ± 0.25 3.02 ± 0.96 0.92 ± 0.43 0.73 ± 0.38 55.2 ± 26.7
136 (67.7%) 59.4 ± 5.4 67 (37.4%) 42 (21.1%) 46 (22.9%) 30 (15.1%) 38 (19.0%) 130.9 ± 18.1 78.4 ± 10.8 76.2 ± 11.6 25.3 ± 3.1 5.27 ± 0.97 1.91 ± 1.92 1.29 ± 0.33 3.13 ± 0.81 1.34 ± 0.22 0.91 ± 0.18 –
0.085 0.086
