Original Article SERUM TESTOSTERONE AND LH IN HEALTHY MEN BOYCE et al. Are published normal ranges of serum testosterone too high? Results of a cross-sectional survey of serum testosterone and luteinizing hormone in healthy men MALCOLM J. BOYCE, KATHY J. BAISLEY, ELIZABETH V. CLARK and STEVEN J. WARRINGTON Hammersmith Medicines Research, Central Middlesex Hospital, London, UK Accepted for publication 13 May 2004 OBJECTIVE To derive normal ranges of serum testosterone and luteinizing hormone (LH) concentrations in healthy men, and thus evaluate whether testosterone replacement therapy is prescribed inappropriately. SUBJECTS AND METHODS The study comprised 266 healthy male volunteers (aged 18 75 years) who were defined as healthy by strict eligibility criteria. Subjects had a body mass index (BMI) of 18.6 32.2 kg/m 2, smoked 0 10 cigarettes/day, and had an alcohol intake 0 40 units/week (one unit = 8 g ethanol). We measured serum testosterone and LH concentrations in the morning (08.00 09.00 hours) and evening (20.00 21.00 hours). RESULTS Morning normal ranges of testosterone for men aged 40 years were 10.07 38.76 nmol/ L (2.90 11.18 mg/l), and for men age 40 years, 7.41 24.13 (2.14 6.96); the respective evening normal ranges were 6.69 31.51 (1.93 9.09) and 6.46 21.93 (1.86 6.33). Both morning and evening serum testosterone declined significantly with increasing age and BMI. LH was significantly higher in the morning than in the evening, but did not vary between the age groups or with BMI. The calculated normal ranges of LH were 0.9 7.0 IU/L (morning) and 0.7 6.8 IU/L (evening). CONCLUSIONS The lower limit of normal for serum testosterone was 3 4 nmol/l (0.86 1.15 mg/l) lower than that of published ranges. The results have important implications for the diagnosis of hypogonadism and use of testosterone replacement therapy. KEYWORDS testosterone, androgen replacement, normal ranges, LH, hypogonadism INTRODUCTION Testosterone and luteinizing hormone (LH) concentrations in adult men are measured mainly for the diagnosis of hypogonadism. However, there is no consensus among clinicians as to what testosterone concentrations are normal in different age groups [1 4]. Physicians tend to prescribe testosterone replacement for men, particularly older men, whose testosterone concentrations are near the lower end of the published normal range [2,3]. In several recent clinical pharmacology studies in healthy men, we measured testosterone and LH as screening tests before the study. Many results were below the published normal ranges [5,6], which suggested that those ranges were not appropriate. Therefore, we analysed serum testosterone and LH concentrations in healthy men who had volunteered for our clinical pharmacology studies, to establish normal ranges and to determine if separate ranges are needed for different age groups. We also examined whether body mass index (BMI), alcohol intake and smoking habit were determinants of testosterone or LH. SUBJECTS AND METHODS The selection criteria were: men aged 18 75 years; BMI 18 30 kg/m 2 ; smoking 10 cigarettes/day; alcohol intake 30 units/week (1 unit = 8 g ethanol); taking no prescription medicines; no current illness or history of chronic illness; diastolic blood pressure 95 mmhg; a negative urine test for drugs of abuse; and judged healthy on the basis of a medical examination, 12-lead electrocardiogram, vital signs and laboratory tests (haematology, biochemistry, urine analysis and serology, including hepatitis B and C, and HIV 1 and 2). The study protocol was approved by the local research ethics committee, and all subjects gave informed, written consent. A sample (10 ml) of venous blood was taken in the morning (08.00 09.00 hours) and evening (20.00 21.00 hours). Serum was stored at -20 C or below. Serum testosterone and LH were measured in the Analytical Unit, St George s Hospital, London, using a commercial, validated radio-immunoassay (DRG Instruments GmbH, Germany) and an automated immunoassay (Diagnostic Products Corporation, UK), respectively. The lower limits of quantification and intra and inter-assay coefficients of variation of the assays used were, respectively: testosterone 0.35, 5.8% and 9.6%, and LH 0.10, 4.2% and 6.5%. For the testosterone assay, a full calibration was constructed with every run, using six calibrators and a zero calibrator, supplied with the kit, and three quality controls (Bio-Rad Laboratories, UK). All calibrator, control and test samples were analysed in duplicate. The LH assay did not require a standard curve to be processed with each run; instead, stored master curves, generated by the manufacturer and adjusted in the laboratory for each kit, were used. Three quality controls (Bio-Rad) were used at the beginning, middle and end of each run; all controls and test samples were analysed in single samples. 2004 BJU INTERNATIONAL 94, 881 885 doi:10.1111/j.1464-410x.2004.05051.x 881
BOYCE ET AL. TABLE 1 The demographics of the study subjects Variable Value Mean (SD) [range] Age, years 33.6 (14.7) [18 75] BMI, kg/m 2 24.6 (2.9) [18.6 32.2] Alcohol, units/week 8.0 (7.0) [0 40] Ethnic group, n European 243 Afro-Caribbean 12 Asian 11 Smoking status Smoker 73 Non-smoker 193 Mean (SD) [range] Cigarettes per day* 6.5 (3.1) [1 10] *includes only those subjects who smoke cigarettes. TABLE 2 Back-transformed mean (95% CI) serum testosterone and LH concentrations for all 226 subjects. Sample sizes for morning and evening were the same for testosterone and LH Testosterone, nmol/l LH, IU/L Variable N Morning N Evening Morning Evening Age group, years 20 33 21.97 (19.75 24.36) 33 16.03 (13.90 18.37) 2.5 (2.1 3.0) 2.0 (1.6 2.4) 21 30 132 21.43 (20.22 22.69) 132 16.30 (15.27 17.37) 2.7 (2.4 2.9) 2.5 (2.3 2.8) 31 40 33 20.12 (17.42 23.08) 33 14.98 (12.79 17.40) 3.0 (2.6 3.5) 2.1 (1.7 2.6) 41 50 21 14.61 (12.47 16.97) 18 12.47 (10.70 14.44) 2.5 (1.8 3.4) 2.7 (2.0 3.6) 51 60 24 14.56 (12.92 16.34) 12 12.56 (10.73 14.59) 2.7 (2.2 3.3) 2.8 (1.9 3.9) 61 23 13.37 (11.80 15.07) 19 12.91 (10.80 15.28) 3.5 (2.7 4.7) 3.4 (2.4 4.7) BMI, kg/m 2 <23 85 21.79 (20.44 23.20) 83 17.03 (15.74 18.39) 2.7 (2.4 3.0) 2.5 (2.3 2.8) 23 26.9 116 19.60 (18.24 21.02) 109 15.11 (13.99 16.29) 2.9 (2.6 3.1) 2.5 (2.2 2.7) 27 65 15.83 (14.41 17.35) 55 13.33 (12.11 14.63) 2.6 (2.3 3.0) 2.4 (2.0 2.9) Non-smoker 193 19.31 (18.28 20.37) 177 15.27 (14.39 16.20) 2.9 (2.7 3.1) 2.6 (2.4 2.8) Smoker 73 19.24 (17.72 20.83) 70 15.43 (14.17 16.75) 2.4 (2.1 2.6) 2.2 (1.9 2.4) Before statistical analysis, the concentration data were transformed using a power transformation, so that the data conformed to a normal distribution, as assessed by the Shapiro-Wilk test. A mixed-effect ANOVA was used to examine relationships between concentrations of each hormone and age, time of sample, BMI, cigarette smoking and alcohol intake. Subject was included in the ANOVA model as a random effect; all other factors were fixed effects. Cigarette smoking and alcohol intake were categorical yes/no variables. If age or BMI were statistically significant as a continuous covariate, we made it a categorical variable, to examine differences between groups. The Wald test was used to assess the significance of each term in the model, and differences in residual log likelihood to compare the fit of nested models. We reduced the model by removing terms that were neither significant predictors of the hormone concentration, nor contributed to model fit. If age or time was significantly associated with hormone concentration separate ranges were calculated for each age group or time, as appropriate. The normal range was taken as that including 95% of subjects, calculated as the mean ± 1.96 SDs of the transformed data, then back-transformed [7,8]. RESULTS Morning and evening samples were obtained from 266 men, and their demographic data are shown in Table 1, with mean serum hormone concentrations are in Table 2. Age had a significant inverse association with serum testosterone concentrations (P < 0.001), with mean testosterone decreasing by ª0.75% each year. When age was included in the final model as a six-level factor (Table 2) the mean testosterone level did not differ significantly among the three younger groups (P 0.31) or among the three older groups (P 0.77; Table 3). However, mean testosterone concentrations in each of three younger groups were significantly higher than those in the three older groups (P 0.01). The mean morning testosterone concentrations were significantly higher than those in the evening (P < 0.001; Table 3). There was also a significant interaction between age and time, indicating that the effect of time on testosterone concentration varied with the subject s age. Among subjects aged 61 years, the mean testosterone concentrations did not differ between morning and evening (P = 0.51; Table 3). In contrast, mean testosterone levels were significantly higher in the morning than in evening in the other five age groups (P 0.03). Age had a significant positive association with serum LH (P = 0.005). Mean LH concentrations increased with age by ª0.1% each year. However, when age was included in the final model as a six-level categorical variable, mean LH did not differ significantly among the age groups (Table 3). The mean LH concentration was significantly higher in the morning than in the evening (P = 0.01; Table 3). There was no interaction between age of subject and time of sample. BMI had a significant inverse association with serum testosterone, independent of age and time, with testosterone decreasing by ª2.5% for every one unit increase in BMI (P < 0.001). When BMI was included in the final model as a three-level categorical variable (Table 3) the mean testosterone level was significantly lower in subjects with a BMI of 27 kg/m 2 than in those with a BMI of 23 26.9 and <23 kg/m 2 (P 0.01; Table 3). There was no significant interaction between age and BMI, or sample time and BMI. BMI was not significantly associated with serum LH concentrations. Neither alcohol intake nor cigarette smoking was associated with serum testosterone. Cigarette smoking, but not alcohol intake, was significantly associated with serum LH concentrations; subjects who smoked had significantly lower mean LH concentrations than those who did not (P = 0.01; Table 3). There was no interaction between cigarette smoking and age, or time of sample. The derived normal ranges are presented in Table 4; because subjects aged >40 years had significantly lower mean testosterone levels than younger men, at both times of day, we 882 2004 BJU INTERNATIONAL
SERUM TESTOSTERONE AND LH IN HEALTHY MEN Variable N Mean (95% CI) Testosterone LH Age, years 20 33 17.75 (15.79 19.85) 2.2 (1.9 2.6) 21 30 132 18.44 (17.43 19.49) 2.5 (2.3 2.7) 31 40 33 17.30 (15.43 19.31) 2.4 (2.0 2.8) 41 50 21 13.73 (11.74 15.94) 2.5 (2.0 3.0) 51 60 24 13.86 (11.90 16.04) 2.6 (2.1 3.2) 61 23 13.44 (11.57 15.51) 3.3 (2.7 4.0) Time Morning 266 17.42 (16.55 18.33) 2.7 (2.5 2.9) Evening 247 14.02 (13.22 14.85) 2.5 (2.3 2.7) BMI, kg/m 2 <23 85 17.36 (16.01 18.78) 23 26.9 116 15.86 (14.79 16.98) 27 65 13.89 (12.69 15.17) Non smoker 193 2.8 (2.6 3.0) Smoker 73 2.4 (2.1 2.7) TABLE 3 Adjusted (model-based) means of testosterone and LH concentration, from mixed effects ANOVA with subject, age, sample time (both hormones), or BMI and age*time interaction (testosterone) or smoking status (LH) as factors TABLE 4 Calculated normal ranges of serum testosterone and LH (testosterone mg/l in parentheses) Hormone Time Age (years) N Lower Upper Testosterone morning 40 198 10.07 (2.90) 38.76 (11.18) nmol/l >40 68 7.41 (2.14) 24.13 (6.96) evening 40 198 6.69 (1.93) 31.51 (9.09) >40 49 6.46 (1.86) 21.93 (6.33) LH, IU/L morning 18 75 266 0.9 7.0 evening 18 75 247 0.7 6.8 consistent with those from other studies [15,16], confirming that diurnal variation in testosterone concentration decreases with age. The upper limit of the present calculated normal range for LH (7.0 IU/L) is lower than that of published ranges. Moreover, the present range is much narrower than published ranges, presumably because the present subjects were uniformly fit, with few extremes of body proportions, as judged by the BMI. LH is secreted in a pulsatile fashion, and fluctuates more than does testosterone; in clinical practice it is recommended that three blood samples be drawn ª20 min apart, and pooled before assay [5,17,18]. The present narrow normal range is therefore the more striking, as it was based on single samples at each time point. Ranges derived from pooled samples would be expected to be even narrower. Testosterone concentrations were inversely associated with BMI, even after adjusting for age and sample time. A decrease in morning testosterone concentrations with increasing BMI, independent of age, was reported by other authors, and low morning testosterone concentrations were reported in obese men [2,13,19,20]. However, in general, the present subjects were not obese; only five had a BMI of >30 kg/m 2 and the highest was 32.2 kg/m 2. derived normal ranges for men aged 40 and >40 years, and calculated separate normal ranges for each time of day. Although age was significantly associated with mean LH concentrations in the overall model, no between-group comparisons were statistically significant. Therefore, we felt it more appropriate to calculate one normal range for LH, rather than separate ones for each age group. However, because mean serum LH was significantly higher in the morning than in the evening, we calculated separate ranges for each time of day. DISCUSSION The present is the first study of its size to measure testosterone and LH concentrations in men aged 18 70 years who were defined as healthy by strict criteria. Previous large studies of testosterone concentrations have included men with known illness, who were taking prescription medication, or for whom good health was less strictly defined [4,9 11]. Published normal ranges of serum testosterone concentrations in healthy eugonadal men are typically 10.40 34.70 nmol/l (3.0 10.0 mg/l) [5,6,12]. Published normal ranges of serum LH concentrations in healthy men are more variable; the assay kit used in the present study stated a range of 1.4 11.0 IU/L, while other sources give ranges of 0.9 20.0 IU/L [5,6,12]. Normal ranges of both hormones in men are not usually adjusted for age nor for the time of day that the blood sample is taken. The present results show that the lower limit of normal for serum testosterone in healthy men may be at least 3 4 nmol/l (0.86 1.15 mg/l) lower than that of published ranges, particularly for samples taken in the evening, and for men aged >40 years. The decline of serum testosterone concentrations with age in healthy men is well documented [4,9,13,14]. The association with age was strongest in samples taken in the morning, when testosterone concentrations are generally at their highest [15,16]. That result is Serum LH concentrations had a statistically significant association with time of day, but the difference between the calculated morning and evening ranges was so slight as to be clinically irrelevant, so in practice one range could be used. When age was included in the model as a categorical variable, the mean LH concentrations did not differ significantly among the six age groups. However, overall, age had a small but statistically significant association with LH concentration; mean LH concentrations increased with age by ª0.1% per year. Other studies also reported an increase in serum LH with age. A study of men aged 61 87 years, who were followed for up to 14 years, found LH to increase with age [10]. A cross-sectional study of men aged 39 70 years showed a 1.2% per year increase in serum LH [19]. Alcohol intake was not significantly associated with either testosterone or LH concentrations. Whilst smokers had 2004 BJU INTERNATIONAL 883
BOYCE ET AL. significantly lower mean LH concentrations than did non-smokers, cigarette smoking was not associated with testosterone concentration. In contrast, Field et al. [13] found that cigarette smokers had significantly higher mean testosterone concentrations than did non-smokers. However, smokers in that study had an average of 25.7 cigarettes/ day, compared with 6.5/day in the present study. Thus, the association between testosterone and smoking may be detectable only in heavy smokers, who were excluded from the present study. Therefore, our calculated normal ranges should be used with caution in men who are heavy smokers. Although we had fewer samples from men aged >40 years than for those 40 years (49 vs 298) it is unlikely that more samples from men in the older group would significantly change the calculated normal ranges. However, a larger sample of older men might have allowed us to better characterize agerelated changes in testosterone and LH. We did not assay free or bioavailable testosterone, or sex hormone-binding globulin in this study. Those assays are more expensive and technically demanding than that for total testosterone, but provide a more accurate measurement of gonadal status. In clinical practice, testosterone-replacement therapy is often prescribed on the basis of measurements of only total testosterone; thus our aim was to evaluate the appropriateness of the reference ranges of total serum testosterone. Several different immunoassays of total testosterone have been approved by regulatory agencies. Whilst many of those assays have been rigorously validated and produce consistent results, with a normal range of 10.4 34.7 nmol/l (3.0 10.0 mg/l), some are more variable and have not been adequately validated for accuracy against a gold standard [21 23]. Furthermore, the population used to establish a normal range during validation of a particular assay may not be adequate or appropriate. Recently, Wang et al. [24] compared the performance of various immunoassays against a gold standard ; they concluded that most assays worked reasonably well over the normal adult male range, but that some automated assays lacked the accuracy and reliability to measure total testosterone concentrations of <3.47 nmol/l (1.0 mg/l). Such differences among assays might result in further problems in diagnosing testosterone deficiency, and should be considered when evaluating serum testosterone concentrations. For clinical purposes, testosterone deficiency has been defined as a testosterone concentration of <8.67 nmol/l (2.50 mg/l) [25,26], <10.40 nmol/l [11,27], and even <12.13 nmol/l [5]. All those values are well within the present calculated normal ranges, even for men aged 40 years. Although most authors caution against giving testosterone merely because of low serum testosterone concentrations, some recommend replacement therapy [1,27]. Furthermore, it seems that therapy is often patient-driven, and that many men with low testosterone levels are enthusiastic about replacement therapy [2,28]. If the published normal ranges for testosterone are indeed too high, as our findings suggest, clinicians are probably prescribing testosterone for men with concentrations well within the normal range. CONFLICT OF INTEREST None declared. REFERENCES 1 Bhasin S, Bagatell CJ, Bremner WJ et al. Therapeutic perspective: issues in testosterone replacement in older men. J Clin Endocrinol Metab 1998; 83: 3435 48 2 Tenover JL. Testosterone replacement therapy in older adult men. Int J Androl 1999; 22: 300 6 3 Smith KW, Feldman HA, McKinlay JB. Construction and field validation of a self-administered screener for testosterone deficiency (hypogonadism) in ageing men. Clin Endocrinol 2000; 53: 703 11 4 Feldman HA, Longcope C, Derby CA et al. 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