ORIGINAL ARTICLE
Testosterone and high-sensitive C-reactive protein in coronary artery disease patients awaiting coronary artery bypass graft C. M. Wickramatilake1, M. R. Mohideen2, B. P. S. Withanawasam3 & C. Pathirana1 1 Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka; 2 Department of Medicine, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka; 3 Teaching Hospital Kegalle, Kegalle, Sri Lanka
Keywords Coronary artery disease—high-sensitive C-reactive protein—total testosterone Correspondence Dr C. M. Wickramatilake, Department of Biochemistry, Faculty of Medicine, University of Ruhuna, P.O. Box: 70, Galle, Sri Lanka. Tel.: +94 0912234801/803 Extension: 212; Fax: +94 0912222314; E-mail: [email protected] Accepted: March 15, 2014 doi: 10.1111/and.12290
Summary Natural androgens inhibit atherosclerosis in men. This study aimed to examine whether testosterone and high-sensitive C-reactive protein differ between patients with coronary artery disease and those without coronary artery disease and to determine the association with the severity of coronary artery disease. Two hundred and six male subjects were recruited. Serum total testosterone and high-sensitive C-reactive protein were estimated. Severity of coronary artery disease was assessed by angiographic scores. Total testosterone level in patients was significantly different from controls (11.4 2.7 vs. 18.1 7.2 nM P = 0.001) and high-sensitive protein level in cases was significantly higher compared to controls (3.37 1.62 mg l 1 vs. 1.71 0.60 mg l 1, P = 0.001). Testosterone levels were not significantly different with vessel (P = 0.592), Leaman (P = 0.694) and Gensini (P = 0.329) score groups, but high-sensitive C-reactive protein showed significant positive correlation among the respective groups (P = 0.005, P = 0.028, P = 0.015). Testosterone was lower, while high-sensitive C-reactive protein was higher in patients compared to controls. Testosterone showed no correlation with the severity of atherosclerosis, but high-sensitive C-reactive protein showed significant positive correlation.
Introduction The relationship between testosterone and coronary artery disease is a dynamic hot spot in the medical research history. The results of some studies suggest favourable effects of androgens on the cardiovascular system and studies involving humans have shown that the androgen levels of men with CAD are lower than those without (English et al., 2000; Dunajska et al., 2004; Rosano et al., 2007; Turhan et al., 2007; Hu et al., 2011). Further, low endogenous testosterone in men is associated with cardiovascular disease risk factors (Phillips et al., 1993; Laaksonen et al., 2004). Atherosclerosis is a progressive disease and is increasingly recognised as a complex phenomenon involving the interaction between several mechanisms: dyslipidaemia, inflammation, thrombosis and other dysfunctional metabolic syndromes. Inflammation has obtained much attention as an important risk factor in coronary artery disease (Ridker et al., 2000; Ballantyne et al., 2004). © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
The association of testosterone with CAD has not been studied in the Sri Lankan population who has different genetic origin compared to Western population. Hence, this study aimed to examine whether testosterone and high sensitivity C-reactive protein differ between men with coronary artery disease and those without coronary artery disease and to determine the association with the severity of coronary artery disease. Materials and methods Study population and data collection Group 1 comprised 103 consecutive male patients (age range of 30–70 years) with angiographically diagnosed CAD, awaiting coronary artery bypass graft (CABG) at the Cardiothoracic Unit, Teaching Hospital, Karapitiya, Sri Lanka. Group 2 comprised 103 controls (age range of 30–70 years) selected from patients awaiting minor surgery in the surgical units of the same hospital without 1
Testosterone and hs-CRP in atherosclerosis
clinically manifested coronary artery disease. Those with abnormal electrocardiograms were not considered for recruitment as controls. The research project was approved by the Ethical Review Committee of Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka. Informed written consent was obtained from all the participants. An interviewer-administered questionnaire was used to collect the socio-demographic data and the relevant information from the study subjects. Anthropometric and blood pressure measurements were obtained according to the standardised operating procedures. Sample collection and biochemical investigations A sample of venous blood was collected in the morning following an overnight fast (10–12 h). Plasma glucose, serum total cholesterol (TCh), triglycerides (TGs) and high-density lipoprotein cholesterol (HDL-Ch) were estimated by a colorimetric method using commercial kit (ProDia International, UAE) on the spectrophotometer. LDL-Ch level was calculated using the Friedewald formula except when TG exceeded 4.5 mM (Friedewald et al., 1972). Serum testosterone concentration was estimated using a commercial enzyme immunoassay kit (PATHOZYME TESTOSTERONE OD 497; OMEGA DIAGNOSTICS LTD, Omega House, UK; Star Fax 1000) according to the manufacturer’s instructions with the lowest detection limit of 0.06 ng ml 1 and coefficient variation of ≤10%. Serum hs-CRP was measured by a test kit based on turbidimetry (DIAgAM, Rue du Parc Industriel, 7822, Ghislenghien, Belgium). The analytical range of the test kit was 0.005–16.00 mg l 1, and the intra-assay and interassay coefficient of variations were 57 to ≤76, >76 to ≤101, >101 to ≤166 which included 26 (25.2%), 26 (25.2%), 28 (27.2%) and 23 (22.3%) patients respectively. The testosterone levels among the four groups did not show significant difference (P = 0.329), but hs-CRP levels showed a significant difference (P = 0.015). Leaman score ranged from 2.5 to 35 and the quartile categories were 2.5≤ to ≤10.5, >10.5 to ≤15.5, >15.5 to ≤20.5, >20.5 to ≤49.5, which included 26 (25.2%), 27 3
Serum hs-CRP concentration (mg l–1)
Testosterone and hs-CRP in atherosclerosis
C. M. Wickramatilake et al.
3.5
r = –0.608 P = 0.001
3.0 2.5 2.0 1.5 1.0 10
15
20
25
30
35
Serum total testosterone concentration (nM)
Fig. 2 Negative correlation between serum concentrations of total testosterone and hs-CRP in controls.
(26.2%), 27 (26.2%) and 23 (22.3%) patients respectively. The severity categories of Leaman score did not show significant difference with testosterone levels (P = 0.694), while hs-CRP levels showed a significant (P = 0.028) difference among the four severity groups. Discussion In the present study, we found that basal serum total testosterone levels in established angiographically documented coronary artery disease patients were significantly lower than that in the controls. These findings are consistent with several reported case–control studies (Dunajska et al., 2004; Rosano et al., 2007; Turhan et al., 2007; Hu et al., 2011) which reveal lower total testosterone levels in patients with proven CAD. There are other studies that show no significant differences in the total testosterone levels, but significant differences have been shown in relation to free testosterone (FT) and bioavailable testosterone (BT) (English et al., 2000; Yang et al., 2005) between cases and controls. Some studies have reported lower TT and FT levels in the CAD group compared to control group (Rosano et al., 2007; Turhan et al., 2007). Furthermore, a study has linked the presence of low testosterone level to the development of premature CAD (Turhan et al., 2007). Among the studies carried out on Western population (English et al., 2000; Dunajska et al., 2004; Rosano et al., 2007), two studies showed significant difference of TT levels between cases and controls (Dunajska et al., 2004; Rosano et al., 2007). Analysis between mean TT level of the case groups of these studies and our case group showed that there was a significant difference (English et al., 2000; Dunajska et al., 2004; Rosano et al., 2007), but only one study showed a significant difference of TT levels between control groups (Rosano et al., 2007). So,
4
this reflects the difference between Western population and Asians. Few studies carried out on Chinese men showed that there was a significant difference of TT levels between cases and controls (Cao et al., 2010; Hu et al., 2011), while others do not (Yang et al., 2005; Zhang et al., 2011). The mean TT testosterone levels of our case group and control group were significantly different compared to the TT level of respective groups of one of their study (Yang et al., 2005), whereas one showed no difference (Hu et al., 2011). Another study carried out on Iranians revealed that there was no significant difference of TT concentration between cases and controls (Davoodig et al., 2007); however, when TT levels of our study groups were compared with their TT levels of the respective groups, there were significant differences. Therefore, it implies that even among the non-Western population, there may be variations in the TT levels in coronary artery disease patients as well as in control subjects according to the ethnicity. It further demonstrates the importance of this study conducted on Sri Lankans. Our study showed a high odds ratio for low testosterone as a risk factor for CAD, reflecting an increased risk. Results also support the hypothesis that hypotestosteronaemia is likely to increase the risk of CAD, for consideration as an independent risk factor. The latter is supported by two studies, which have shown low testosterone as an independent predictor of CAD (Turhan et al., 2007; Hu et al., 2011). In our study, the prevalence of risk factors among cases was higher than in the controls. Recent studies have shown that low endogenous testosterone was not only related to cardiovascular disease, but also to its risk factors, including hypertension, obesity, dyslipidaemia, diabetes mellitus and metabolic syndrome (Phillips et al., 1993; Laaksonen et al., 2004). These findings strongly suggest that low testosterone level may be involved in the pathogenesis of CAD through the mediation of the traditional risk factors. The serum lipid and plasma glucose levels are also significantly higher in the CAD patient group than in the control group. Therefore, it is possible to state that the lower levels of testosterone among our cases are associated with a higher prevalence of CAD risk factors. In this case–control study, significant differences in serum testosterone and the lipid concentrations were observed consistently, between case and control groups, which remained unchanged following adjustments for covariates. It was shown that there was a significant positive correlation between the serum levels of HDL-Ch and testosterone, while a significant negative correlation was elicited between concentrations of LDL-Ch and testosterone in patients with established coronary artery disease.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
C. M. Wickramatilake et al.
There are several ways by which testosterone may affect lipid metabolism and atherosclerosis. One proposed mechanism is that testosterone acts through modulation of lipid factors, supported by findings of a favourable relationship with HDL-Ch. Our finding of a potentially antiatherogenic effect of natural endogenous testosterone within the physiological range by modulating the HDL cholesterol level is in agreement with earlier reports of studies carried out on healthy men (Van Pottelbergh et al., 2003; Bataille et al., 2005). Hence, it appears that testosterone levels within the physiological range have a beneficial effect on the lipid profile and on the prevention of atherosclerosis. Serum hs-CRP level was significantly higher in patients compared to controls, supporting hs-CRP as a marker of vascular inflammation and a risk factor of CAD (Ridker et al., 2000; : Ballantyne et al., 2004). The inverse relationship between serum total testosterone and hs-CRP may indicate that low levels of endogenous testosterone may encourage low-grade chronic inflammation creating an atherogenic environment. Reports on the association of testosterone and the hs-CRP are restricted to few, all showing negative correlation but with variable strength of relationships (Yang et al., 2005; Davoodig et al., 2007; Hu et al., 2011). Therefore, the negative correlation existing between these two variables supports the notion that lower testosterone status may induce an inflammation contributing to atherosclerosis. In our study, the severity of CAD assessed by the vessel score, Gensini score and the Leaman score did not show significant association with the serum total testosterone concentrations. These observations are consistent with several previous reports where the degree of CAD assessed by coronary angiogram was not correlated with serum testosterone levels (English et al., 2000; Dunajska et al., 2004). Yet, there are studies that have found significant negative correlation between angiographically assessed CAD severity and the serum testosterone level (Rosano et al., 2007; Hu et al., 2011). Moreover, there is limited evidence on the association of hs-CRP with the atherosclerotic burden. These studies show variable associations between C-reactive protein concentration and the extent of atherosclerosis. Some studies show that serum hs-CRP concentrations are correlated with the severity of peripheral arterial disease assessed by ankle brachial pressure index (Vainas et al., 2005; Owens et al., 2007), while others show a correlation with the extent and severity of CAD (Zebrack et al., 2002, 2003). In contrast, there are studies suggesting that serum concentration of hs-CRP is not associated with the severity of CAD assessed angiographically (Sukhija et al., 2007; Niccoli et al., 2008). Our study showed a significant positive correlation between hs-CRP and © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
Testosterone and hs-CRP in atherosclerosis
the CAD severity assessed by the three types of scoring systems with a significant difference among the severity categories. In conclusion, total testosterone levels were low, while hs-CRP levels were high in patients with coronary artery disease compared to controls. The hs-CRP level was negatively correlated with that of total testosterone level. The low levels of total testosterone, high levels of hs-CRP and abnormal lipid profile appeared to be risk factors in atherogenesis. The positive correlation between testosterone and the HDL-Ch levels reflected a protective role for testosterone in CAD. Testosterone level did not show a correlation with the severity of CAD, while hs-CRP level did show a significant positive correlation with the severity. Due to financial constraints and limited facilities, additional assays such as sex hormone binding globulin and free and bio-available testosterone could not be performed. Acknowledgements We wish to acknowledge the University Grants Commission, Sri Lanka, for the financial assistance provided for the project, Mrs. DABN Amerasekara (Statistician, Applied Statistic Association, Sri Lanka), Senior Lecturer, Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Sri Lanka, for the statistical advice provided, and Mrs. Kawmadhi Abeywaradana, Technical Officer, Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Sri Lanka, for the support offered in the biochemical assays. Conflict of interest The authors declare that they have no conflict of interest concerning this article. References Ballantyne CM, Hoogeveen RC, Bang H, Coresh J, Folsom AR, Heiss G, Sharrett AR (2004) Lipoprotein-associated phospholipase A2, high- sensitivity C-reactive protein, and risk for incident coronary heart disease in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study. Circulation 109:837–842. Bataille V, Perret B, Evans A, Amouyel P, Arveiler D, Ducimetiere P, Bard JM, Ferrieres J (2005) Sex hormonebinding globulin is a major determinant of the lipid profile: the PRIME study. Atherosclerosis 179:369–373. Cao J, Zou H, Zhu BP, Wang H, Li J, Ding Y, Li XY (2010) Sex hormones and androgen receptor: risk factors of coronary heart disease in elderly men. Chin Med Sci J 25:44–49.
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Testosterone and hs-CRP in atherosclerosis
Davoodig G, Amirezadegan A, Borumand MA, Dehkordimr MR, Kazemisaeid A, Yaminisharif A (2007) The relationship between level of androgenic hormones and coronary artery disease in men. Cardiovasc J Afr 18:362–366. Dunajska K, Milewicz A, Szymczak J, Jedrzejuk D, Kuliczkowski W, Salomon P, Nowicki P (2004) Evaluation of sex hormone levels and some metabolic factors in men with coronary atherosclerosis. Aging Male 7:197–204. English KM, Mandour O, Steeds RP, Diver MJ, Jones TH, Channer KS (2000) Men with coronary artery disease have lower levels of androgens than men with normal coronary angiograms. Eur Heart J 21:890–894. Friedewald WT, Levy RI, Fredrickson DS (1972) Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 18:499–502. Gensini GG (1983) A more meaningful scoring system for determinating the severity of coronary heart disease. Am J Cardiol 51:606. Hu X, Rui L, Zhu T, Xia H, Yang X, Wang X, Liu H, Lu Z, Jiang H (2011) Low testosterone level in middle-aged male patients with coronary artery disease. Eur J Intern Med 22: e133–e136. Laaksonen DE, Niskanen L, Punnonen K, Nyyss€ onen K, Tuomainen TP, Valkonen VP, Salonen R, Salonen JT (2004) Testosterone and sex hormone-binding globulin predict the metabolic syndrome and diabetes in middle-aged men. Diabetes Care 27:1036–1041. Leaman DM, Brower RW, Meester GT, Serruys P, van den Brand M (1981) Coronary artery atherosclerosis: severity of the disease, severity of angina pectoris and compromised left ventricular function. Circulation 63:285–299. Niccoli G, Biasucci LM, Biscione C, Fusco B, Porto I, Leone AM, Bona RD, Dato I, Liuzzo G, Crea F (2008) Independent prognostic value of C-reactive protein and coronary artery disease extent in patients affected by unstable angina. Atherosclerosis 196:779–785. Owens CD, Ridker PM, Belkin M, Hamdan AD, Pomposelli F, Logerfo F, Creager MA, Conte MS (2007) Elevated Creactive protein levels are associated with postoperative events in patients undergoing lower extremity vein bypass surgery. J Vasc Surg 45:2–9. Petak SM, Nankin HR, Spark RF, Swerdloff RS, RodriguezRigau LJ; American Association of Clinical Endocrinologists (2002) American Association of Clinical Endocrinologists Medical Guidelines for clinical practice for the evaluation and treatment of hypogonadism in adult male patients–2002 update. Endocr Pract 8:440–456. Phillips GB, Jing TY, Resnick LM, Barbagallo M, Laragh JH, Sealey JE (1993) Sex hormones and haemostatic risk factors for coronary heart disease in men with hypertension. J Hypertens 11:699–702.
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Ridker PM, Hennekens CH, Buring JE, Rifai N (2000) Creactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 342:836–843. Ringqvist I, Fisher LD, Mock M, Davis KB, Wedel H, Chaitman BR, Passamani E, Russell RO Jr, Alderman EL, Kouchoukas NT, Kaiser GC, Ryan TJ, Killip T, Fray D (1983) Prognostic value of angiographic indices of coronary artery disease from the Coronary Artery Surgery Study (CASS). J Clin Invest 71:1854–1866. Rosano GMC, Sheiban I, Massaro R, Pagnotta P, Marazzi G, Vitale C, Mercuro G, Volterrani M, Aversa A, Fini M (2007) Low testosterone levels are associated with coronary artery disease in male patients with angina. Int J Impot Res 19:176–182. Sukhija R, Fahdi I, Garza L, Fink L, Scott M, Aude W, Pacheco R, Bursac Z, Grant A, Mehta JL (2007) Inflammatory markers, angiographic severity of coronary artery disease and patient outcome. Am J Cardiol 99:879– 884. Turhan S, Tulunay C, Gulec S, Ozdol C, Kilickap M, Altin T, Gerede M, Erol C (2007) The association between androgen levels and premature coronary artery disease in men. Coron Artery Dis 18:159–162. Vainas T, Stassen FR, de Graaf R, Twiss EL, Herngreen SB, Welten RJ, van den Akker LH, van Dieijen-Visser MP, Bruggeman CA (2005) C-reactive protein in peripheral arterial disease: relation to severity of the disease and to future cardiovascular events. J Vasc Surg 42:243–251. Van Pottelbergh I, Braeckman L, De Bacquer D, De Backer G, Kaufman JM (2003) Differential contribution of testosterone and estradiol in the determination of cholesterol and lipoprotein profile in healthy middle-aged men. Atherosclerosis 166:95–102. Yang YM, Lv XY, Huang WD, Xu ZR, Wu LJ (2005) Study of androgen and atherosclerosis in old-age male. J Zhejiang Univ Sci B 6:931–935. Zebrack JS, Muhlestein JB, Horne BD, Anderson JL; Intermountain Heart Collaboration Study Group (2002) C-reactive protein and angiographic coronary artery disease: independent and additive predictors of risk in subjects with angina. J Am Coll Cardiol 39: 632–637. Zebrack JS, Anderson JL, Beddhu S, Horne BD, Bair TL, Cheung A, Muhlestein JB; Intermountain Heart Collaborative Study Group (2003) Do associations with C-reactive protein and extent of coronary artery disease account for the increased cardiovascular risk of renal insufficiency? J Am Coll Cardiol 42: 57–63. Zhang XJ, Li XY, Cao TT, Ye L (2011) Correlation of endogenous androgen and androgen receptor level with coronary artery diseases in elderly males. Zhonghua Yi Xue Za Zhi 91:984–986.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
Testosterone and high-sensitive C-reactive protein in coronary artery disease patients awaiting coronary artery bypass graft C. M. Wickramatilake1, M. R. Mohideen2, B. P. S. Withanawasam3 & C. Pathirana1 1 Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka; 2 Department of Medicine, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka; 3 Teaching Hospital Kegalle, Kegalle, Sri Lanka
Keywords Coronary artery disease—high-sensitive C-reactive protein—total testosterone Correspondence Dr C. M. Wickramatilake, Department of Biochemistry, Faculty of Medicine, University of Ruhuna, P.O. Box: 70, Galle, Sri Lanka. Tel.: +94 0912234801/803 Extension: 212; Fax: +94 0912222314; E-mail: [email protected] Accepted: March 15, 2014 doi: 10.1111/and.12290
Summary Natural androgens inhibit atherosclerosis in men. This study aimed to examine whether testosterone and high-sensitive C-reactive protein differ between patients with coronary artery disease and those without coronary artery disease and to determine the association with the severity of coronary artery disease. Two hundred and six male subjects were recruited. Serum total testosterone and high-sensitive C-reactive protein were estimated. Severity of coronary artery disease was assessed by angiographic scores. Total testosterone level in patients was significantly different from controls (11.4 2.7 vs. 18.1 7.2 nM P = 0.001) and high-sensitive protein level in cases was significantly higher compared to controls (3.37 1.62 mg l 1 vs. 1.71 0.60 mg l 1, P = 0.001). Testosterone levels were not significantly different with vessel (P = 0.592), Leaman (P = 0.694) and Gensini (P = 0.329) score groups, but high-sensitive C-reactive protein showed significant positive correlation among the respective groups (P = 0.005, P = 0.028, P = 0.015). Testosterone was lower, while high-sensitive C-reactive protein was higher in patients compared to controls. Testosterone showed no correlation with the severity of atherosclerosis, but high-sensitive C-reactive protein showed significant positive correlation.
Introduction The relationship between testosterone and coronary artery disease is a dynamic hot spot in the medical research history. The results of some studies suggest favourable effects of androgens on the cardiovascular system and studies involving humans have shown that the androgen levels of men with CAD are lower than those without (English et al., 2000; Dunajska et al., 2004; Rosano et al., 2007; Turhan et al., 2007; Hu et al., 2011). Further, low endogenous testosterone in men is associated with cardiovascular disease risk factors (Phillips et al., 1993; Laaksonen et al., 2004). Atherosclerosis is a progressive disease and is increasingly recognised as a complex phenomenon involving the interaction between several mechanisms: dyslipidaemia, inflammation, thrombosis and other dysfunctional metabolic syndromes. Inflammation has obtained much attention as an important risk factor in coronary artery disease (Ridker et al., 2000; Ballantyne et al., 2004). © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
The association of testosterone with CAD has not been studied in the Sri Lankan population who has different genetic origin compared to Western population. Hence, this study aimed to examine whether testosterone and high sensitivity C-reactive protein differ between men with coronary artery disease and those without coronary artery disease and to determine the association with the severity of coronary artery disease. Materials and methods Study population and data collection Group 1 comprised 103 consecutive male patients (age range of 30–70 years) with angiographically diagnosed CAD, awaiting coronary artery bypass graft (CABG) at the Cardiothoracic Unit, Teaching Hospital, Karapitiya, Sri Lanka. Group 2 comprised 103 controls (age range of 30–70 years) selected from patients awaiting minor surgery in the surgical units of the same hospital without 1
Testosterone and hs-CRP in atherosclerosis
clinically manifested coronary artery disease. Those with abnormal electrocardiograms were not considered for recruitment as controls. The research project was approved by the Ethical Review Committee of Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka. Informed written consent was obtained from all the participants. An interviewer-administered questionnaire was used to collect the socio-demographic data and the relevant information from the study subjects. Anthropometric and blood pressure measurements were obtained according to the standardised operating procedures. Sample collection and biochemical investigations A sample of venous blood was collected in the morning following an overnight fast (10–12 h). Plasma glucose, serum total cholesterol (TCh), triglycerides (TGs) and high-density lipoprotein cholesterol (HDL-Ch) were estimated by a colorimetric method using commercial kit (ProDia International, UAE) on the spectrophotometer. LDL-Ch level was calculated using the Friedewald formula except when TG exceeded 4.5 mM (Friedewald et al., 1972). Serum testosterone concentration was estimated using a commercial enzyme immunoassay kit (PATHOZYME TESTOSTERONE OD 497; OMEGA DIAGNOSTICS LTD, Omega House, UK; Star Fax 1000) according to the manufacturer’s instructions with the lowest detection limit of 0.06 ng ml 1 and coefficient variation of ≤10%. Serum hs-CRP was measured by a test kit based on turbidimetry (DIAgAM, Rue du Parc Industriel, 7822, Ghislenghien, Belgium). The analytical range of the test kit was 0.005–16.00 mg l 1, and the intra-assay and interassay coefficient of variations were 57 to ≤76, >76 to ≤101, >101 to ≤166 which included 26 (25.2%), 26 (25.2%), 28 (27.2%) and 23 (22.3%) patients respectively. The testosterone levels among the four groups did not show significant difference (P = 0.329), but hs-CRP levels showed a significant difference (P = 0.015). Leaman score ranged from 2.5 to 35 and the quartile categories were 2.5≤ to ≤10.5, >10.5 to ≤15.5, >15.5 to ≤20.5, >20.5 to ≤49.5, which included 26 (25.2%), 27 3
Serum hs-CRP concentration (mg l–1)
Testosterone and hs-CRP in atherosclerosis
C. M. Wickramatilake et al.
3.5
r = –0.608 P = 0.001
3.0 2.5 2.0 1.5 1.0 10
15
20
25
30
35
Serum total testosterone concentration (nM)
Fig. 2 Negative correlation between serum concentrations of total testosterone and hs-CRP in controls.
(26.2%), 27 (26.2%) and 23 (22.3%) patients respectively. The severity categories of Leaman score did not show significant difference with testosterone levels (P = 0.694), while hs-CRP levels showed a significant (P = 0.028) difference among the four severity groups. Discussion In the present study, we found that basal serum total testosterone levels in established angiographically documented coronary artery disease patients were significantly lower than that in the controls. These findings are consistent with several reported case–control studies (Dunajska et al., 2004; Rosano et al., 2007; Turhan et al., 2007; Hu et al., 2011) which reveal lower total testosterone levels in patients with proven CAD. There are other studies that show no significant differences in the total testosterone levels, but significant differences have been shown in relation to free testosterone (FT) and bioavailable testosterone (BT) (English et al., 2000; Yang et al., 2005) between cases and controls. Some studies have reported lower TT and FT levels in the CAD group compared to control group (Rosano et al., 2007; Turhan et al., 2007). Furthermore, a study has linked the presence of low testosterone level to the development of premature CAD (Turhan et al., 2007). Among the studies carried out on Western population (English et al., 2000; Dunajska et al., 2004; Rosano et al., 2007), two studies showed significant difference of TT levels between cases and controls (Dunajska et al., 2004; Rosano et al., 2007). Analysis between mean TT level of the case groups of these studies and our case group showed that there was a significant difference (English et al., 2000; Dunajska et al., 2004; Rosano et al., 2007), but only one study showed a significant difference of TT levels between control groups (Rosano et al., 2007). So,
4
this reflects the difference between Western population and Asians. Few studies carried out on Chinese men showed that there was a significant difference of TT levels between cases and controls (Cao et al., 2010; Hu et al., 2011), while others do not (Yang et al., 2005; Zhang et al., 2011). The mean TT testosterone levels of our case group and control group were significantly different compared to the TT level of respective groups of one of their study (Yang et al., 2005), whereas one showed no difference (Hu et al., 2011). Another study carried out on Iranians revealed that there was no significant difference of TT concentration between cases and controls (Davoodig et al., 2007); however, when TT levels of our study groups were compared with their TT levels of the respective groups, there were significant differences. Therefore, it implies that even among the non-Western population, there may be variations in the TT levels in coronary artery disease patients as well as in control subjects according to the ethnicity. It further demonstrates the importance of this study conducted on Sri Lankans. Our study showed a high odds ratio for low testosterone as a risk factor for CAD, reflecting an increased risk. Results also support the hypothesis that hypotestosteronaemia is likely to increase the risk of CAD, for consideration as an independent risk factor. The latter is supported by two studies, which have shown low testosterone as an independent predictor of CAD (Turhan et al., 2007; Hu et al., 2011). In our study, the prevalence of risk factors among cases was higher than in the controls. Recent studies have shown that low endogenous testosterone was not only related to cardiovascular disease, but also to its risk factors, including hypertension, obesity, dyslipidaemia, diabetes mellitus and metabolic syndrome (Phillips et al., 1993; Laaksonen et al., 2004). These findings strongly suggest that low testosterone level may be involved in the pathogenesis of CAD through the mediation of the traditional risk factors. The serum lipid and plasma glucose levels are also significantly higher in the CAD patient group than in the control group. Therefore, it is possible to state that the lower levels of testosterone among our cases are associated with a higher prevalence of CAD risk factors. In this case–control study, significant differences in serum testosterone and the lipid concentrations were observed consistently, between case and control groups, which remained unchanged following adjustments for covariates. It was shown that there was a significant positive correlation between the serum levels of HDL-Ch and testosterone, while a significant negative correlation was elicited between concentrations of LDL-Ch and testosterone in patients with established coronary artery disease.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
C. M. Wickramatilake et al.
There are several ways by which testosterone may affect lipid metabolism and atherosclerosis. One proposed mechanism is that testosterone acts through modulation of lipid factors, supported by findings of a favourable relationship with HDL-Ch. Our finding of a potentially antiatherogenic effect of natural endogenous testosterone within the physiological range by modulating the HDL cholesterol level is in agreement with earlier reports of studies carried out on healthy men (Van Pottelbergh et al., 2003; Bataille et al., 2005). Hence, it appears that testosterone levels within the physiological range have a beneficial effect on the lipid profile and on the prevention of atherosclerosis. Serum hs-CRP level was significantly higher in patients compared to controls, supporting hs-CRP as a marker of vascular inflammation and a risk factor of CAD (Ridker et al., 2000; : Ballantyne et al., 2004). The inverse relationship between serum total testosterone and hs-CRP may indicate that low levels of endogenous testosterone may encourage low-grade chronic inflammation creating an atherogenic environment. Reports on the association of testosterone and the hs-CRP are restricted to few, all showing negative correlation but with variable strength of relationships (Yang et al., 2005; Davoodig et al., 2007; Hu et al., 2011). Therefore, the negative correlation existing between these two variables supports the notion that lower testosterone status may induce an inflammation contributing to atherosclerosis. In our study, the severity of CAD assessed by the vessel score, Gensini score and the Leaman score did not show significant association with the serum total testosterone concentrations. These observations are consistent with several previous reports where the degree of CAD assessed by coronary angiogram was not correlated with serum testosterone levels (English et al., 2000; Dunajska et al., 2004). Yet, there are studies that have found significant negative correlation between angiographically assessed CAD severity and the serum testosterone level (Rosano et al., 2007; Hu et al., 2011). Moreover, there is limited evidence on the association of hs-CRP with the atherosclerotic burden. These studies show variable associations between C-reactive protein concentration and the extent of atherosclerosis. Some studies show that serum hs-CRP concentrations are correlated with the severity of peripheral arterial disease assessed by ankle brachial pressure index (Vainas et al., 2005; Owens et al., 2007), while others show a correlation with the extent and severity of CAD (Zebrack et al., 2002, 2003). In contrast, there are studies suggesting that serum concentration of hs-CRP is not associated with the severity of CAD assessed angiographically (Sukhija et al., 2007; Niccoli et al., 2008). Our study showed a significant positive correlation between hs-CRP and © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
Testosterone and hs-CRP in atherosclerosis
the CAD severity assessed by the three types of scoring systems with a significant difference among the severity categories. In conclusion, total testosterone levels were low, while hs-CRP levels were high in patients with coronary artery disease compared to controls. The hs-CRP level was negatively correlated with that of total testosterone level. The low levels of total testosterone, high levels of hs-CRP and abnormal lipid profile appeared to be risk factors in atherogenesis. The positive correlation between testosterone and the HDL-Ch levels reflected a protective role for testosterone in CAD. Testosterone level did not show a correlation with the severity of CAD, while hs-CRP level did show a significant positive correlation with the severity. Due to financial constraints and limited facilities, additional assays such as sex hormone binding globulin and free and bio-available testosterone could not be performed. Acknowledgements We wish to acknowledge the University Grants Commission, Sri Lanka, for the financial assistance provided for the project, Mrs. DABN Amerasekara (Statistician, Applied Statistic Association, Sri Lanka), Senior Lecturer, Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Sri Lanka, for the statistical advice provided, and Mrs. Kawmadhi Abeywaradana, Technical Officer, Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Sri Lanka, for the support offered in the biochemical assays. Conflict of interest The authors declare that they have no conflict of interest concerning this article. References Ballantyne CM, Hoogeveen RC, Bang H, Coresh J, Folsom AR, Heiss G, Sharrett AR (2004) Lipoprotein-associated phospholipase A2, high- sensitivity C-reactive protein, and risk for incident coronary heart disease in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study. Circulation 109:837–842. Bataille V, Perret B, Evans A, Amouyel P, Arveiler D, Ducimetiere P, Bard JM, Ferrieres J (2005) Sex hormonebinding globulin is a major determinant of the lipid profile: the PRIME study. Atherosclerosis 179:369–373. Cao J, Zou H, Zhu BP, Wang H, Li J, Ding Y, Li XY (2010) Sex hormones and androgen receptor: risk factors of coronary heart disease in elderly men. Chin Med Sci J 25:44–49.
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