Inverse association of testosterone and sex hormone binding globulin levels with leukocyte count in middle-aged and elderly men

Abstract

Objective: The inverse associations of testosterone and sex hormone-binding globulin (SHBG) levels with cardiometabolic diseases are well established and are increasingly viewed as inflammatory diseases. This study aimed to examine the associations of testosterone and SHBG levels with leukocyte count in 451 Korean men aged ≥50 years.

Methods: Serum testosterone and SHBG levels were categorized into tertiles. High leukocyte count was defined as ≥7340 cells/μl, which corresponded to the 75th percentile of the current sample. The odds ratios (ORs) and 95% confidence intervals (95% CIs) for high leukocyte count were calculated across testosterone and SHBG tertiles using multiple logistic regression analysis.

Results: The mean leukocyte counts significantly decreased with increasing testosterone and SHBG tertiles. The ORs (95% CIs) of high leukocyte count for the first tertile of testosterone and SHBG were 3.27 (1.34–7.95) and 2.38 (1.05–5.96), respectively, compared with the referent third tertile, after adjusting for age, smoking status, alcohol drinking, regular exercise, body mass index, blood pressure, fasting plasma glucose, triglyceride, and high-density lipoprotein (HDL) cholesterol level.

Conclusion: We found inversely graded associations of low testosterone and SHBG levels with leukocyte count. These findings suggest that low testosterone and SHBG levels may be interpreted as a state of low-grade inflammation.

Keywords:

• Testosterone
• sex hormone-binding globulin
• leukocyte count
• inflammation

1. Introduction

A low testosterone level has been traditionally considered to be a marker of male hypogonadism and is also related to quality of life issues [1–4], but recent epidemiological evidence has highlighted the significance of low testosterone level in male patients with cardiometabolic diseases such as coronary heart disease, metabolic syndrome, and type 2 diabetes through cross-sectional and longitudinal studies [5–12], which are closely related to subclinical atherosclerosis or autonomic imbalances [13,14]. Sex hormone-binding globulin (SHBG), produced mainly from the liver, is the primary transport glycoprotein for sex steroid hormones and modulates their bioavailability in peripheral target tissues. A growing body of previous studies documented the potential metabolic significance of SHBG in addition to controlling free sex hormone levels. A low SHBG level has been inversely associated with increased risk of type 2 diabetes and metabolic syndrome, independent of sex hormones in both sexes [5–9,15,16].

Although, multifactorial mechanisms may be interrelated in the development of cardiometabolic diseases, chronic low-grade inflammation is as a crucial contributing factor in the pathogenesis [17–19]. Leukocyte count, a nonspecific inflammation marker, is widely used in standard clinical practice. Recently, leukocyte count has become a useful predictor of cardiometabolic diseases such as coronary heart disease, ischemic stroke, and metabolic syndrome [20–22].

The inverse associations of testosterone and SHBG levels with cardiometabolic diseases are well established, which are increasingly viewed as inflammatory diseases. However, limited data exist linking testosterone and SHBG levels to inflammatory markers. Therefore, we examined the associations of testosterone and SHBG levels with leukocyte count in middle-aged and elderly men.

2. Materials and methods

2.1. Study participants

We retrospectively reviewed the medical records of 569 participants aged ≥50 years who underwent a medical examination at the Health Promotion Center of Gangnam Severance Hospital in Seoul, Korea, between November 2012 and July 2013. The individuals voluntarily visited the health promotion center to regularly monitor their health condition. Informed consent was obtained from each participant. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Yonsei University College of Medicine, Seoul, Korea. We also excluded those who met at least one of the following criteria: a history of exogenous testosterone therapy; a history of ischemic heart disease, stroke, cancer, thyroid, respiratory, renal, hepatobiliary, or rheumatologic disease; hepatic enzyme levels higher than two times the upper limit of the reference range or a leukocyte count ≥10,000 (cells l⁻¹); and missing data or not fasting for 12 h prior to testing. After these exclusions, 451 participants were included in the final analysis.

2.2. Data collection

Each participant completed a questionnaire about his lifestyle and medical history. Self-reported cigarette smoking, alcohol consumption, and physical activity were determined from the questionnaires. Smoking status was categorized as a nonsmoker, ex-smoker, and current smoker. Alcohol drinking was defined as consumption ≥ twice per week. Regular exercise was defined as exercise ≥ three times per week. Body mass and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, in light indoor clothing without shoes. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters (kg/m²). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in the patient’s right arm with a standard mercury sphygmomanometer (Baumanometer, W.A. Baum Co Inc., Copiague, NY). All blood samples were obtained from the antecubital vein after a 12 h overnight fast. Fasting plasma glucose, triglycerides, high-density lipoprotein (HDL) cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured by enzymatic methods using a chemistry analyzer (Hitachi 7600, Hitachi Co., Tokyo, Japan). Leukocyte count was quantified using an automated blood cell counter (ADVIA 120, Bayer, NY). Testosterone and SHBG concentrations were measured using an electrochemiluminescence assay with a Modular Analytics E170 system (Roche Diagnostic Systems, Basel, Switzerland).

The modified National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) was used to define metabolic syndrome. Because waist circumference was not measured, we defined obesity as a BMI ≥25 kg/m², as suggested by the position statement of the American College of Endocrinology [23]. Therefore, metabolic syndrome was defined by the presence of three or more of the following risk factors: obesity with BMI ≥25 kg/m², elevated systolic blood pressure ≥130 mmHg, elevated diastolic blood pressure ≥85 mmHg or use of anti-hypertensive medication, high fasting plasma glucose ≥6.1 mmol/l or use of anti-diabetes medication, high triglyceride ≥1.70 mmol/l, and low HDL cholesterol <1.04 mmol/l.

2.3. Statistical analysis

Normal distribution was evaluated with determination of skewness using a Kolmogorov-Smirnov test. Serum triglyceride, AST, and ALT levels have skewed distributions, so these variables were expressed as median (interquartile range, IQR) in descriptive analysis and log-transformed prior to simple correlation and multiple logistic regression analysis. Testosterone and SHBG levels were categorized into tertiles as follows: T1: ≤13.9, T2: 14–18.4, and T3: ≥18.5 nmol/l for testosterone and T1: ≤33.4, T2: 33.5–36.5, and T3: ≥36.6 nmol/l for SHBG. The clinical characteristics of the study population according to testosterone and SHBG tertiles were compared independently using a one-way analysis of variance (ANOVA) or Kruskal-Wallis test for continuous variables according to the normality of distributions and chi-square test for categorical variables. Continuous data are presented as mean (standard deviation, SD) or median (interquartile range, IQR), and categorical data are presented as frequencies. High leukocyte count was defined as ≥7340 cells/μl, which corresponded to the 75th percentile of the current sample. The odds ratios (ORs) and 95% confidence intervals (95% CIs) for high leukocyte count were calculated after adjusting for confounding variables across testosterone and SHBG tertiles using multiple logistic regression analysis. All analyses were conducted using SAS statistical software (version 9.4; SAS Institute Inc., Cary, NC). All statistical tests were two-sided and statistical significance was determined at p < .05.

3. Results

Table 1 shows the clinical characteristics of the study population according to testosterone and SHBG tertiles. The mean BMI and the median triglycerides and ALT levels were lowest, whereas HDL cholesterol level was highest in the first tertile of testosterone and SHBG. The proportion of regular exercise was highest in the third tertile of SHBG level. The prevalence of metabolic syndrome decreased proportionally with increasing testosterone and SHBG tertiles.

Table 1. Clinical characteristics according to the serum testosterone and sex hormone binding globulin (SHBG) tertiles.^a

	Testosterone tertiles (nmol/l)				SHBG tertiles (nmol/l)
	T1: ≤13.9(n = 149)	T2: 14–18.4(n = 150)	T3: ≥18.5 (n = 152)	p value	T1: ≤33.4(n = 151)	T2: 33.5–36.5(n = 150)	T3: ≥36.6(n = 150)	p value
Age (year)	57.2 (6.2)	58.1 (7.0)	57.7 (6.8)	.684	55.2 (5.3)	57.0 (5.6)	60.9 (7.6)	<.001
BMI (kg/m^2)	25.1 (2.7)	24.3 (2.5)	23.7 (2.2)	<.001	25.2 (2.5)	24.5 (2.2)	23.4 (2.6)	<.001
Systolic BP (mm Hg)	123.9 (15.0)	125.3 (14.8)	123.5 (14.3)	.639	124.5 (14.6)	123.0 (13.9)	125.1 (15.6)	.587
Diastolic BP (mm Hg)	79.3 (9.3)	79.1 (9.1)	78.0 (9.1)	.564	80.7 (8.7)	78.1 (8.7)	78.6 (9.9)	.462
Fasting plasma glucose (mmol/l)	5.69 (1.46)	5.71 (1.41)	5.43 (1.00)	.254	5.80 (1.41)	5.61 (1.49)	5.41 (0.94)	.116
Triglycerides (mmol/l)	1.49 (0.99–2.19)	1.37 (0.96–2.05)	1.12 (0.88–1.59)	.008	1.70 (1.03–2.48)	1.32 (1.01–1.85)	1.03 (0.77–1.52)	<.001
HDL cholesterol (mmol/l)	1.19 (0.20)	1.22 (0.28)	1.32 (0.35)	.004	1.16 (0.23)	1.23 (0.30)	1.31 (0.31)	<.001
AST (U/l)	22 (20–27)	24 (20–28)	23 (19–27)	.927	22 (19–28)	22 (19–26)	23 (20–28)	.353
ALT (U/l)	26 (19–35)	24 (20–31)	22 (17–28)	.048	27 (21–40)	23 (18–29)	21 (17–28)	<.001
Current smoking (%)	22.8	28.9	29.2	.296	29.4	27.9	25.6	.975
Alcohol drinking^b (%)	16.5	18.4	20.0	.756	18.3	23.8	15.5	.357
Regular exercise^c (%)	48.1	48.6	59.4	.105	38.9	53.9	56.3	.041
Metabolic syndrome^d (%)	38.9	34.2	18.8	<.001	43.2	29.3	17.9	<.001

BMI: body mass index; BP: blood pressure; HDL: high density lipoprotein; AST: aspartate aminotransferase; ALT: alanine aminotransferase.

^aData are expressed as the mean (SD), median (interquartile range, IQR), or percentage.

^bAlcohol drinking ≥ twice/week.

^cRegular exercise ≥ three times/week.

^dMetabolic syndrome is defined as having three or more of the following conditions: BMI ≥ kg/m²; systolic BP ≥130 mm Hg, diastolic BP ≥85 mmHg or on anti-hypertensive medication; fasting plasma glucose ≥6.1 mmol/l or on anti-diabetes medication; triglyceride ≥1.70 mmol/l; and HDL cholesterol <1.04 mmol/l.

Figure 1 shows the mean leukocyte counts according to testosterone and SHBG tertiles. The mean leukocyte counts significantly decreased with increasing testosterone and SHBG tertiles: 6679, 6045, and 5868 cells/μl, respectively for testosterone tertile and 6634, 6385, and 5921 cells/μl, respectively for SHBG tertiles (All p < .001).

Figure 1. Mean leukocyte count according to testosterone and SHBG tertiles (p values were calculated using ANOVA test).

Table 2 shows the results of multiple logistic regression analysis to assess the odds for predicting high leukocyte count in terms of testosterone and SHBG tertiles. Compared with the referent third tertile, the ORs (95% CIs) of high leukocyte count for the first tertile of testosterone and SHBG levels were 3.27 (1.34–7.95) and 2.38 (1.05–5.96), respectively, after adjusting for age, smoking status, alcohol drinking, regular exercise, BMI, blood pressure, fasting plasma glucose, triglyceride, and HDL cholesterol level.

Table 2. Odds ratios and 95% confidence intervals for high leukocyte count according to testosterone and SHBG tertiles.

	Testosterone tertiles (nmol/l)				SHBG tertiles (nmol/l)
	T1: ≤13.9	T2: 14–18.4	T3: ≥18.5	p for trend	T1: ≤33.4	T2: 33.5–36.5	T3: ≥36.6	p for trend
Model 1^a	3.59 (1.57–8.21)	3.02 (1.32–6.95)	1.00 (ref.)	.004	2.87 (1.28–6.43)	1.69 (0.74–3.82)	1.00 (ref.)	.005
Model 2^b	3.73 (1.59–8.74)	3.09 (1.33–7.16)	1.00 (ref.)	.004	2.98 (1.29–6.89)	1.74 (0.75–4.00)	1.00 (ref.)	.004
Model 3^c	3.27 (1.34–7.95)	2.55 (1.07–6.09)	1.00 (ref.)	.018	2.38 (1.05–5.96)	1.70 (0.70–4.11)	1.00 (ref.)	.029

^aModel 1: adjusted for age, smoking status, alcohol drinking, and regular exercise.

^bModel 2: adjusted for age, smoking status, alcohol drinking, regular exercise, and BMI.

^cModel 3: adjusted for age, smoking status, alcohol drinking, regular exercise, BMI, blood pressure, fasting plasma glucose, triglyceride, and HDL cholesterol level.

4. Discussion

In this cross-sectional study, we found that testosterone and SHBG levels were independently and inversely associated with leukocyte count after adjusting for potential confounding variables in middle-aged and elderly men. Our findings suggest that the known inverse association between lower testosterone and SHBG levels and increased cardiometabolic risk in men might be partly explained by chronic low-grade inflammation. To our knowledge, there was only one previous study in general population that examined the relationship between sex hormones and inflammation, albeit metabolic alterations among male patients with prostate cancer [24]. According to a study of 1986 non-diabetic middle-aged Finnish men by Laaksonen et al. [25], the age- and BMI-adjusted mean C-reactive protein concentrations were highest in the first tertile of testosterone, SHBG, and dehydroepiandosterone sulfate (DHEA) using analysis of covariance (ANCOVA) test. However, the study was not primarily designed to explore the association of sex hormone and SHBG levels with CRP concentration [25], so the results were not fully adjusted for confounding variables. We believe that the present study is the first to primarily demonstrate the inverse associations between testosterone and SHBG levels and leukocyte count, a useful biomarker of systemic inflammation.

The following potential mechanisms could explain the inverse relationship between testosterone and SHBG levels and leukocyte count. Visceral adipose tissue, accompanied by low testosterone and SHBG levels, secretes a number of adipocytokines such as tumor necrosis factor-α and interleukin-6, which cause a chronic low-grade inflammatory state in men [26,27]. In the present study, low testosterone and SHBG levels showed inverse associations with metabolic syndrome. From hormonal aspects, a low testosterone level could be a consequence of insulin resistance by decreasing skeletal muscles and increasing visceral adiposity [28], which are closely related to chronic low-grade inflammation. Moreover, increased visceral adipose tissue stimulates aromatase enzyme activity, which accelerates conversion from testosterone to estrogen and leads to a reduced testosterone level and promotes fat accumulation in the liver, consequently decreasing hepatic synthesis of SHBG from the liver [29,30].

Some limitations should be considered in the interpretation of this study. First, it was of a cross-sectional design, suggesting that caution should be used in causal and temporal interpretations. Although a significant inverse relationship between testosterone and SHBG levels and leukocyte counts existed in this study, it cannot be concluded whether testosterone and SHBG level is a risk factor actively involved in inflammatory process or just a consequence of inflammation. However, several prospective studies suggest that testosterone replacement therapy in hypogonadal males could improve subclinical inflammation [31–33]. Second, because the study participants were volunteers visiting for health promotion screenings in a single hospital and appeared to be slightly healthier than most community-based cohorts, the study population may not be representative of the general population. Third, only one measurement of leukocyte and testosterone level from a baseline examination was included in the analyses. To rule out an acute or brief episode of inflammation, we excluded participants with leukocyte count higher than the upper limit of the reference range and to minimize the diurnal variation in testosterone level, all blood samples were drawn early in the morning [34]. A large sale, long-term prospective research is warranted to elucidate these inverse correlations between testosterone and SHBG levels and inflammation.

In summary, we found inversely graded associations of low testosterone and SHBG levels with leukocyte count. These findings suggest that low testosterone and SHBG levels may be interpreted as a state of low-grade inflammation.

Disclosure statement

No potential conflict of interest was reported by the authors.