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Serum carbonic anhydrase autoantibodies in metabolic syndrome
Diabetes & Metabolic Syndrome: Clinical Research & Reviews 3 (2009) 211–213
Contents lists available at ScienceDirect
Diabetes & Metabolic Syndrome: Clinical Research & Reviews journal homepage: www.elsevier.com/locate/dsx
Original paper
Serum carbonic anhydrase autoantibodies in metabolic syndrome Ahmet Alver a,*, Ahmet Mentes¸e a, Cihangir Erem b, Orhan Deg˘er a, Mustafa Koc¸ak b, E. Edip Keha a a b
Department of Biochemistry, Faculty of Medicine, Karadeniz Technical University (KTU), 61080, Trabzon, Turkey Division of Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, Karadeniz Technical University, 61080, Trabzon, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Metabolic syndrome Carbonic anhydrase Anti-CA II antibody Anti-CA I antibody
Aims: Carbonic anhydrase (CA) autoantibodies have been observed in various autoimmune and idiopathic diseases right now. The aim of this study was to investigate the presence of anti-carbonic anhydrase I and II antibodies in subjects with metabolic syndrome (MetS). Methods: 43 subjects with MetS and 30 non-MetS subjects (controls) were included in the study. Carbonic anhydrase autoantibodies were screened by ELISA. Results: Anti-CA I antibody was detected in 7 of 43 subjects with MetS. The antibody-positive prevalence of MetS group (16%) was found to be significantly higher compared with that of control group (p < 0.05). There were no significant changes in positive rate of anti-CA II antibody. Positive results were obtained in 2 of 43 subjects with MetS. Conclusions: Anti-CA I antibodies were present in sera obtained from MetS subject and these antibodies may be involved in pathogenesis of MetS. ß 2009 Diabetes India. Published by Elsevier Ltd. All rights reserved.
1. Introduction
In the present study, we investigated the presence of anti-CA I and CA II antibodies in MetS and evaluated a possible role of antiCA antibodies in pathogenesis of the syndrome.
The metabolic syndrome (MetS) is a cluster of clinical features characterized by some metabolic abnormality including insulin resistance, diabetes, hypertension, dyslipidemia and obesity. The prevalence of MetS was increased for more than two decades [1]. In our region, MetS prevalence has been reported to be 29.9% [2]. The main proposed reason of MetS is insulin resistance or hyperinsulinemia, although a central role for insulin resistance in the MetS is still controversial. Multiple factors such as sex, race, genetic and environmental conditions play role in development of MetS. Carbonic anhydrases (CA; EC 4.2.1.1) are involved in many important physiological processes in various tissues such as, transport of CO2, pH regulation, renal and male reproductive tract acidification, electrolyte secretion, and ion transport. 15 CA isoenzymes (CA I–XV) have been described up to now and shown that their subcellular localization and tissue distribution are very different [3,4]. It was shown that an autoimmune response to CA II in animal models [5–7] and in following studies, numerous researchers have found CA I and/or CA II autoantibodies in sera from patients with various autoimmune diseases including endometriosis [8], type 1 diabetes [9], primary biliary cirrhosis [10], idiopathic chronic pancreatitis [11], and Graves’ disease [12].
* Corresponding author. Tel.: +90 462 377 54 90; fax: +90 462 325 05 18. E-mail address: [email protected] (A. Alver).
2. Materials and methods 2.1. Study populations The study was carried out in the Departments of Biochemistry and Internal Medicine of the Faculty of Medicine, Karadeniz Technical University. After receiving approval from the Local Ethics Committee of the School of Medicine, informed consents were obtained from all patients. As the criteria of MetS National Cholesterol Education Program’s Adult Treatment Panel III (NCEP: ATP III) was taken and 43 subjects with any of 3 or more of the following, were included to this study: Abdominal obesity: waist girth >102 cm in men and >88 cm in women, Hypertriglyceridemia: serum triglycerides (TG) level 150 mg/ dl (1.69 mmol/l), Low HDL-cholesterol: <40 mg/dl (1.04 mmol/l) in men and <50 mg/dl (1.29 mmol/l) in women. High blood pressure: systolic blood pressure (SBP) 130 mmHg and/or diastolic blood pressure (DBP) 85 mmHg or on treatment for hypertension. High fasting glucose: serum glucose level 110 mg/dl (6.1 mmol/l) or on treatment for diabetes.
1871-4021/$ – see front matter ß 2009 Diabetes India. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dsx.2009.07.011
A. Alver et al. / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 3 (2009) 211–213
212
30 non-MetS subjects were used as a control group after their clinical and laboratory tests were determined. Blood samples were collected in the morning after 10–12 h fasting for the measurements of the lipid profile and plasma glucose, and the sera were separated after clotting by centrifugation. Aliquots were then taken and stored at 80 8C until the tests were performed. Serum triglycerides were measured using a glycerol oxidase enzymatic method; HDL-cholesterol by a cholesterol oxidase enzymatic method in supernatant after precipitation with phosphotungistic acid–MgCl2; fasting serum glucose was measured using an enzymatic (glucose oxidase) colorimetric method. All determinations were performed with an autoanalyzer (Roche, Modular, Switzerland). Reagents used were supplied by the same manufacturer. 2.2. Enzyme-linked immunosorbent assay (ELISA) for serum antibody to CA I and CA II Serum anti-CA I and anti-CA II antibodies were detected by ELISA according to a previously described method [11], with minor modifications. Briefly, microtiter plates (high binding, flat-bottomed plates; BioscienCE) were coated with 50 ml of 10 mg/ml human CA I or CA II (electrophoretically purified from erythrocytes, were purchased from Sigma Chemical Co.) in carbonate buffer (0.05 mM, pH 9.6) and incubated overnight at 4 8C. The wells were washed with phosphate buffer (pH 7) and blocked with 2% skim milk in phosphate buffer for 2 h at room temperature (RT). After being washed with phosphate buffer containing 0.05% Tween-20, wells were incubated with 100 ml of serum diluted with blocking buffer (1:200) for 2 h at RT. After washing, each well was incubated for 2 h at RT with 100 ml of 1:2000 dilution of peroxidaseconjugated anti-human IgG anti-serum (Sigma Chemical Co.) in blocking buffer. Following washes with phosphate buffer, wells were incubated with 100 ml substrate solution (10 nmol orthophenylenediamine) for 20 min at RT. The reaction was stopped by the addition of 100 ml of 2 M H2SO4 to each well and the absorbances of wells were read at 480 nm. Control wells that were not coated with CA I or CA II were also used for ELISA of each serum studied. All assays were performed in duplicate, and the specific binding of serum antibody to CA I or CA II was calculated by subtracting the average absorbance of control wells from the average absorbance of the antigen coated wells (specific binding = Acoated Acontol). The samples from a positive MetS subject and a control subject were used for determination of intra-assay coefficient of variation. For anti-CA II and anti-CA I antibody assay, the intra-assay coefficient of variation was 7.3% (n = 10) and 5.5% (n = 10) in MetS subject, and 5% (n = 10) and 6% (n = 10) in control subject, respectively.
Fig. 1. Anti-CA I antibodies in sera from subjects with MetS and controls. The dotted line indicates the mean value plus 3SD of control sera (A480 = 0.785). The prevalence of MetS group was significantly higher than that of controls group (p < 0.05).
with MetS. The antibody-positive prevalence of MetS group (16%) was found to be significantly higher compared with that of control group (p < 0.05). No significant difference between the mean absorbance value of MetS group and that of controls group was found (p > 0.05). In addition, there was a positive correlation between anti-CA I titers and systolic blood pressure in MetS group (r = 0.346, p < 0.05). The anti-CA II antibody levels in subjects with MetS and control subjects were shown in Fig. 2. The mean absorbance value for the control subjects was 0.363 0.174 (n = 30) and the absorbance values higher than 0.885, the mean absorbance + 3SD of control subjects, were defined as positive. Positive results were obtained in 2 of 43 subjects with MetS. The mean absorbance value of MetS group (0.546 0.252) was found to be significantly higher compared with that of the control group (p < 0.001). In our study, there was none of the subjects having both positive anti-CA I antibody and anti-CA II antibody together. A good correlation between anti-CA I and anti-CA II titers was found in control groups (r = 0.82, p < 0.0001) (Fig. 3); however, no correlation between anti-CA II titers and other clinical parameters studied was observed (Table 1). 4. Discussion As mentioned previously, CA autoantibodies have been observed in various autoimmune and idiopathic diseases, but, the pathogenic roles of these antibodies have not been clear [8– 12]. In the present study, we found anti-CA I antibody prevalence (16%) in subjects with MetS to be higher than control subjects (Fig. 1). In contrast with CA II, CA I have more restricted tissue distribution. It is expressed mainly in erythrocytes and is the most
2.3. Statistical analysis All results were expressed as means SD. The x2-test and Mann–Whitney’s U-test were used for assessment of prevalence rates and evaluation of the differences in antibody titers, respectively. pValues of less than 0.05 were considered significantly different. 3. Results Anti-CA I antibody levels were quantified in the MetS and control groups by ELISA at a serum dilution of 1:200 (Fig. 1). The mean absorbance value for control group was 0.356 0.143 and that for MetS group was 0.483 0.398. In the present study, any absorbance higher than 0.785 was taken as positive (mean + 3SD of control subjects). Anti-CA I antibody was detected in 7 of 43 subjects
Fig. 2. Anti-CA II antibodies in sera from subjects with MetS and controls. The dotted line indicates the mean value plus 3SD of control sera (A480 = 0.885).
A. Alver et al. / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 3 (2009) 211–213
Fig. 3. Correlation between anti-CA I antibody titer and anti-CA II antibody titer in study populations. The lines indicate the mean value + 3SD of control sera absorbance (A480) in both antibodies as described in results and in legends to Figs. 1 and 2.
213
similar result was reported by Taniguchi et al. in patients with type 2 diabetes [9]. In their study, a high prevalence of autoantibodies against CA II was observed in type 1 diabetes, while no positive subject with type 2 diabetes was found. Anti-CA I antibodies were not determined at the same study. To explain the significance of anti-CA I antibody in the pathogenesis of MetS is difficult. However, CAs play an important role in biochemical processes such as pH homeostasis and electrolyte secretion and blocking of the active site of the enzyme by its antibody prevents to play these roles [16]. Furthermore, autoantibodies can be taken by the cells via receptor-mediated transcytosis [17]. Thus, anti-CA I antibody may enter to target cell and inhibit CA activity. In this way, intracellular pH may be changed and as a result of these changes ion balance and metabolism may be altered. In summary, anti-CA I antibody is detected in MetS subjects, but the pathogenic role of that antibody remains uncertain. Further investigations are required to determine the significance of CA autoantibody production in MetS subjects. Acknowledgment This work was supported by Karadeniz Technical University research fund (project number: 2007.114.001.1).
abundant non-hemoglobin protein [3]. In some diseases, including Systemic Lupus Erythmatosus, Sjo¨gren’s Syndrome and primary biliary cirrhosis, positive anti-CA I antibody values in patients’ sera have been reported together with positive anti-CA II antibody [10,11] and this situation was explained with a cross-reactivity because of homology between CA I and CA II. However, we observed that CA I autoantibodies were independent of anti-CA II antibodies in subject with MetS (Fig. 3). In addition, there was a weak positive correlation between anti-CA I antibody titers and systolic blood pressure in MetS group (r = 0.346, p < 0.05). The high prevalence of this antibody in this group may indicate that anti-CA I antibodies is an epiphenomenon secondary to the vascular structural changes. Because, CA I is present in vessel and may have an important role in regulation of vessel tone [13,14]. Another reason of immune response to CA I may arise from high levels of glucose in subjects with MetS. Glycosylation of CAs was found to be increased in erythrocytes from diabetics [15], therefore, antigenic properties of CA I may be altered. CA II is present in the cytosol of every tissue, and autoimmune sialoadenitis was successfully induced in mice by immunization with CA II [3,6]. Thus, it could be a target antigen in the autoimmune diseases related to some tissues or organs. Although we found high levels antibody titers against to CA II, the positive antibody titers was observed for only two MetS subjects (Fig. 2). A
Table 1 Demographic and clinical parameters of subjects with MetS and controls. n
Age (year) Sex (m/f) Triglyceride (mmol/l) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Fasting Glucose (mmol/l) SBP (mmHg) DBP (mmHg) Waist girth (cm) Prevalence of anti-CA I antibody (%) (p/n)a Prevalence of anti-CA II antibody (%) (p/n) a
Controls
MetS
30
43
47.03 8.21 (11/17) 1.07 0.13 4.58 0.39 1.51 0.27 2.91 0.40 4.58 0.50 120.66 8.97 76.50 8.62 90.20 13.59 0 (0/30) 0 (0/30)
50.65 9.97 (19/24) 3.00 1.06 5.42 1.08 1.01 0.15 3.72 0.97 8.65 3.31 149.10 21.12 91.20 12.69 109.74 9.32 16 (7/43) 5 (2/43)
(p/n): (anti-CA antibody-positive subjects number/total subjects number).
References [1] Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet 2005;365: 1415–28. [2] Erem C, Hacıhasanog˘lu A, Deg˘er O, Topbas¸ M, Hos¸ver I, Erso¨z HO, et al. Prevalence of metabolic syndrome and associated risk factors in among Turkish adults: Trabzon MetS study. Endocrine 2008;33:9–20. [3] Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995;64:375–401. [4] Supuran CT, Briganti F, Tilli S, Chegwidden WR, Scozzafava A. Carbonic anhydrase inhibitors: sulfonamides as tumor agent? Bioorg Med Chem 2001;9:703–14. [5] Inagaki Y, Jinno-Yoshida Y, Hamasaki Y, Ueki H. A novel autoantibody reactive with carbonic anhydrase in sera from patients with systemic lupus erythematosus and Sjo¨gren’s syndrome. J Dermatol Sci 1991;2:147–54. [6] Nishimori I, Bratonova T, Toshkov I, Caffrey T, Mogaki M, Shibata Y, et al. Induction of Experimental autoimmune sialodenitis by immunization of PL/J mice with carbonic anhydrase II. J immunol 1995;154:4865–73. [7] Uchida K, Okazaki K, Nishi T, Uose S, Nakase H, Ohana M, et al. Experimental immune-mediated pancreatitis in neonatally thymectomized mice immunized with carbonic anhydrase II and lactoferrin. Lab Invest 2002;82:411–24. [8] Kiechle FL, Quattrocıocchı-Longe TM, Brinton DA. Carbonic anhydrase antibody in sera from patients with endometriosis. Am J Clin Pathol 1994;101: 611–5. [9] Taniguchi T, Okazaki K, Okamoto M, Seko S, Tanaka J, Uchida K, et al. High prevalence of autoantibodies against carbonic anhydrase II and lactoferrin in type 1 diabetes: concept of autoimmune exocrinopathy and endocrinopathy of pancreas. Pancreas 2003;27:26–30. [10] Invernizzi P, Battezzati PM, Crosignani A, Zermiani P, Bignotto M, Del Papa N, et al. Antibody to carbonic anhydrase II is present in primary biliary cirrhosis irrespective of antimitochondrial antibody status. Clin Exp Immunol 1998; 144:448–54. [11] Kino-Ohsaki J, Nishimori I, Morita M, Okazaki K, Yamamoto Y, Onish S, et al. Serum antibodies to carbonic anhydrase I and II in patients with idiopathic chronic pancreatitis and Sjo¨gren’s syndrome. Gastroenterology 1996;110: 1579–86. [12] Alver A, Mentes¸e A, Karahan SC, Erem C, Keha EE, Arıkan MK, et al. Increased serum anti-carbonic anhydrase II antibodies in patients with Graves’ disease. Exp Clin Endocrinol 2007;115:287–91. [13] Pickkers P, Garcha RS, Schachter M, Smits P, Hughes AD. Inhibition of carbonic anhydrase accounts for the direct vascular effects of hydrochlorothiazidine. Hypertension 1999;33:1043–8. [14] Puscas I, Gilau L, Coltau M, Pasca R, Domuta G, Baican M, et al. Calcium channel blockers reduce blood pressure in part by inhibiting vascular smooth muscle carbonic anhydrase I. Cardivasc Drugs Ther 2000;14:523–8. [15] Kondo T, Murakami K, Ohtsuka Y. Estimation of characterization of glycosylated carbonic anhydrase I in erythrocytes from patients with diabetes mellitus. Clin Chim Acta 1987;166:227–36. [16] Botre´ F, Botre´ C, Podesta E, Podda M, Invernizzi P. Effect of anti-carbonic anhydrase antibodies on carbonic anhydrase I and II. Clin Chem 2003;49: 1221–3. [17] Rojas R, Apodaca G. Immunoglobulin transport across polarized epithelial cells. Nat Rev Mol Cell Biol 2002;3:944–55.
Contents lists available at ScienceDirect
Diabetes & Metabolic Syndrome: Clinical Research & Reviews journal homepage: www.elsevier.com/locate/dsx
Original paper
Serum carbonic anhydrase autoantibodies in metabolic syndrome Ahmet Alver a,*, Ahmet Mentes¸e a, Cihangir Erem b, Orhan Deg˘er a, Mustafa Koc¸ak b, E. Edip Keha a a b
Department of Biochemistry, Faculty of Medicine, Karadeniz Technical University (KTU), 61080, Trabzon, Turkey Division of Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, Karadeniz Technical University, 61080, Trabzon, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Metabolic syndrome Carbonic anhydrase Anti-CA II antibody Anti-CA I antibody
Aims: Carbonic anhydrase (CA) autoantibodies have been observed in various autoimmune and idiopathic diseases right now. The aim of this study was to investigate the presence of anti-carbonic anhydrase I and II antibodies in subjects with metabolic syndrome (MetS). Methods: 43 subjects with MetS and 30 non-MetS subjects (controls) were included in the study. Carbonic anhydrase autoantibodies were screened by ELISA. Results: Anti-CA I antibody was detected in 7 of 43 subjects with MetS. The antibody-positive prevalence of MetS group (16%) was found to be significantly higher compared with that of control group (p < 0.05). There were no significant changes in positive rate of anti-CA II antibody. Positive results were obtained in 2 of 43 subjects with MetS. Conclusions: Anti-CA I antibodies were present in sera obtained from MetS subject and these antibodies may be involved in pathogenesis of MetS. ß 2009 Diabetes India. Published by Elsevier Ltd. All rights reserved.
1. Introduction
In the present study, we investigated the presence of anti-CA I and CA II antibodies in MetS and evaluated a possible role of antiCA antibodies in pathogenesis of the syndrome.
The metabolic syndrome (MetS) is a cluster of clinical features characterized by some metabolic abnormality including insulin resistance, diabetes, hypertension, dyslipidemia and obesity. The prevalence of MetS was increased for more than two decades [1]. In our region, MetS prevalence has been reported to be 29.9% [2]. The main proposed reason of MetS is insulin resistance or hyperinsulinemia, although a central role for insulin resistance in the MetS is still controversial. Multiple factors such as sex, race, genetic and environmental conditions play role in development of MetS. Carbonic anhydrases (CA; EC 4.2.1.1) are involved in many important physiological processes in various tissues such as, transport of CO2, pH regulation, renal and male reproductive tract acidification, electrolyte secretion, and ion transport. 15 CA isoenzymes (CA I–XV) have been described up to now and shown that their subcellular localization and tissue distribution are very different [3,4]. It was shown that an autoimmune response to CA II in animal models [5–7] and in following studies, numerous researchers have found CA I and/or CA II autoantibodies in sera from patients with various autoimmune diseases including endometriosis [8], type 1 diabetes [9], primary biliary cirrhosis [10], idiopathic chronic pancreatitis [11], and Graves’ disease [12].
* Corresponding author. Tel.: +90 462 377 54 90; fax: +90 462 325 05 18. E-mail address: [email protected] (A. Alver).
2. Materials and methods 2.1. Study populations The study was carried out in the Departments of Biochemistry and Internal Medicine of the Faculty of Medicine, Karadeniz Technical University. After receiving approval from the Local Ethics Committee of the School of Medicine, informed consents were obtained from all patients. As the criteria of MetS National Cholesterol Education Program’s Adult Treatment Panel III (NCEP: ATP III) was taken and 43 subjects with any of 3 or more of the following, were included to this study: Abdominal obesity: waist girth >102 cm in men and >88 cm in women, Hypertriglyceridemia: serum triglycerides (TG) level 150 mg/ dl (1.69 mmol/l), Low HDL-cholesterol: <40 mg/dl (1.04 mmol/l) in men and <50 mg/dl (1.29 mmol/l) in women. High blood pressure: systolic blood pressure (SBP) 130 mmHg and/or diastolic blood pressure (DBP) 85 mmHg or on treatment for hypertension. High fasting glucose: serum glucose level 110 mg/dl (6.1 mmol/l) or on treatment for diabetes.
1871-4021/$ – see front matter ß 2009 Diabetes India. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dsx.2009.07.011
A. Alver et al. / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 3 (2009) 211–213
212
30 non-MetS subjects were used as a control group after their clinical and laboratory tests were determined. Blood samples were collected in the morning after 10–12 h fasting for the measurements of the lipid profile and plasma glucose, and the sera were separated after clotting by centrifugation. Aliquots were then taken and stored at 80 8C until the tests were performed. Serum triglycerides were measured using a glycerol oxidase enzymatic method; HDL-cholesterol by a cholesterol oxidase enzymatic method in supernatant after precipitation with phosphotungistic acid–MgCl2; fasting serum glucose was measured using an enzymatic (glucose oxidase) colorimetric method. All determinations were performed with an autoanalyzer (Roche, Modular, Switzerland). Reagents used were supplied by the same manufacturer. 2.2. Enzyme-linked immunosorbent assay (ELISA) for serum antibody to CA I and CA II Serum anti-CA I and anti-CA II antibodies were detected by ELISA according to a previously described method [11], with minor modifications. Briefly, microtiter plates (high binding, flat-bottomed plates; BioscienCE) were coated with 50 ml of 10 mg/ml human CA I or CA II (electrophoretically purified from erythrocytes, were purchased from Sigma Chemical Co.) in carbonate buffer (0.05 mM, pH 9.6) and incubated overnight at 4 8C. The wells were washed with phosphate buffer (pH 7) and blocked with 2% skim milk in phosphate buffer for 2 h at room temperature (RT). After being washed with phosphate buffer containing 0.05% Tween-20, wells were incubated with 100 ml of serum diluted with blocking buffer (1:200) for 2 h at RT. After washing, each well was incubated for 2 h at RT with 100 ml of 1:2000 dilution of peroxidaseconjugated anti-human IgG anti-serum (Sigma Chemical Co.) in blocking buffer. Following washes with phosphate buffer, wells were incubated with 100 ml substrate solution (10 nmol orthophenylenediamine) for 20 min at RT. The reaction was stopped by the addition of 100 ml of 2 M H2SO4 to each well and the absorbances of wells were read at 480 nm. Control wells that were not coated with CA I or CA II were also used for ELISA of each serum studied. All assays were performed in duplicate, and the specific binding of serum antibody to CA I or CA II was calculated by subtracting the average absorbance of control wells from the average absorbance of the antigen coated wells (specific binding = Acoated Acontol). The samples from a positive MetS subject and a control subject were used for determination of intra-assay coefficient of variation. For anti-CA II and anti-CA I antibody assay, the intra-assay coefficient of variation was 7.3% (n = 10) and 5.5% (n = 10) in MetS subject, and 5% (n = 10) and 6% (n = 10) in control subject, respectively.
Fig. 1. Anti-CA I antibodies in sera from subjects with MetS and controls. The dotted line indicates the mean value plus 3SD of control sera (A480 = 0.785). The prevalence of MetS group was significantly higher than that of controls group (p < 0.05).
with MetS. The antibody-positive prevalence of MetS group (16%) was found to be significantly higher compared with that of control group (p < 0.05). No significant difference between the mean absorbance value of MetS group and that of controls group was found (p > 0.05). In addition, there was a positive correlation between anti-CA I titers and systolic blood pressure in MetS group (r = 0.346, p < 0.05). The anti-CA II antibody levels in subjects with MetS and control subjects were shown in Fig. 2. The mean absorbance value for the control subjects was 0.363 0.174 (n = 30) and the absorbance values higher than 0.885, the mean absorbance + 3SD of control subjects, were defined as positive. Positive results were obtained in 2 of 43 subjects with MetS. The mean absorbance value of MetS group (0.546 0.252) was found to be significantly higher compared with that of the control group (p < 0.001). In our study, there was none of the subjects having both positive anti-CA I antibody and anti-CA II antibody together. A good correlation between anti-CA I and anti-CA II titers was found in control groups (r = 0.82, p < 0.0001) (Fig. 3); however, no correlation between anti-CA II titers and other clinical parameters studied was observed (Table 1). 4. Discussion As mentioned previously, CA autoantibodies have been observed in various autoimmune and idiopathic diseases, but, the pathogenic roles of these antibodies have not been clear [8– 12]. In the present study, we found anti-CA I antibody prevalence (16%) in subjects with MetS to be higher than control subjects (Fig. 1). In contrast with CA II, CA I have more restricted tissue distribution. It is expressed mainly in erythrocytes and is the most
2.3. Statistical analysis All results were expressed as means SD. The x2-test and Mann–Whitney’s U-test were used for assessment of prevalence rates and evaluation of the differences in antibody titers, respectively. pValues of less than 0.05 were considered significantly different. 3. Results Anti-CA I antibody levels were quantified in the MetS and control groups by ELISA at a serum dilution of 1:200 (Fig. 1). The mean absorbance value for control group was 0.356 0.143 and that for MetS group was 0.483 0.398. In the present study, any absorbance higher than 0.785 was taken as positive (mean + 3SD of control subjects). Anti-CA I antibody was detected in 7 of 43 subjects
Fig. 2. Anti-CA II antibodies in sera from subjects with MetS and controls. The dotted line indicates the mean value plus 3SD of control sera (A480 = 0.885).
A. Alver et al. / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 3 (2009) 211–213
Fig. 3. Correlation between anti-CA I antibody titer and anti-CA II antibody titer in study populations. The lines indicate the mean value + 3SD of control sera absorbance (A480) in both antibodies as described in results and in legends to Figs. 1 and 2.
213
similar result was reported by Taniguchi et al. in patients with type 2 diabetes [9]. In their study, a high prevalence of autoantibodies against CA II was observed in type 1 diabetes, while no positive subject with type 2 diabetes was found. Anti-CA I antibodies were not determined at the same study. To explain the significance of anti-CA I antibody in the pathogenesis of MetS is difficult. However, CAs play an important role in biochemical processes such as pH homeostasis and electrolyte secretion and blocking of the active site of the enzyme by its antibody prevents to play these roles [16]. Furthermore, autoantibodies can be taken by the cells via receptor-mediated transcytosis [17]. Thus, anti-CA I antibody may enter to target cell and inhibit CA activity. In this way, intracellular pH may be changed and as a result of these changes ion balance and metabolism may be altered. In summary, anti-CA I antibody is detected in MetS subjects, but the pathogenic role of that antibody remains uncertain. Further investigations are required to determine the significance of CA autoantibody production in MetS subjects. Acknowledgment This work was supported by Karadeniz Technical University research fund (project number: 2007.114.001.1).
abundant non-hemoglobin protein [3]. In some diseases, including Systemic Lupus Erythmatosus, Sjo¨gren’s Syndrome and primary biliary cirrhosis, positive anti-CA I antibody values in patients’ sera have been reported together with positive anti-CA II antibody [10,11] and this situation was explained with a cross-reactivity because of homology between CA I and CA II. However, we observed that CA I autoantibodies were independent of anti-CA II antibodies in subject with MetS (Fig. 3). In addition, there was a weak positive correlation between anti-CA I antibody titers and systolic blood pressure in MetS group (r = 0.346, p < 0.05). The high prevalence of this antibody in this group may indicate that anti-CA I antibodies is an epiphenomenon secondary to the vascular structural changes. Because, CA I is present in vessel and may have an important role in regulation of vessel tone [13,14]. Another reason of immune response to CA I may arise from high levels of glucose in subjects with MetS. Glycosylation of CAs was found to be increased in erythrocytes from diabetics [15], therefore, antigenic properties of CA I may be altered. CA II is present in the cytosol of every tissue, and autoimmune sialoadenitis was successfully induced in mice by immunization with CA II [3,6]. Thus, it could be a target antigen in the autoimmune diseases related to some tissues or organs. Although we found high levels antibody titers against to CA II, the positive antibody titers was observed for only two MetS subjects (Fig. 2). A
Table 1 Demographic and clinical parameters of subjects with MetS and controls. n
Age (year) Sex (m/f) Triglyceride (mmol/l) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Fasting Glucose (mmol/l) SBP (mmHg) DBP (mmHg) Waist girth (cm) Prevalence of anti-CA I antibody (%) (p/n)a Prevalence of anti-CA II antibody (%) (p/n) a
Controls
MetS
30
43
47.03 8.21 (11/17) 1.07 0.13 4.58 0.39 1.51 0.27 2.91 0.40 4.58 0.50 120.66 8.97 76.50 8.62 90.20 13.59 0 (0/30) 0 (0/30)
50.65 9.97 (19/24) 3.00 1.06 5.42 1.08 1.01 0.15 3.72 0.97 8.65 3.31 149.10 21.12 91.20 12.69 109.74 9.32 16 (7/43) 5 (2/43)
(p/n): (anti-CA antibody-positive subjects number/total subjects number).
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