The Hypogonadism In Males study estimated the prevalence of hypogonadism in men aged ⩾45 years. A sub-analysis of patients not receiving testosterone (T) therapy was conducted. Blood draw times were 0800–1000 and 1000–1200 hours. Total T (TT) was not influenced by draw time for any age group; however, significantly greater free T (FT) and bioavailable T (BAT) values were observed in the overall population for earlier draw times. Sex hormone-binding globulin (SHBG) values were significantly lower in men aged 45–64 years at the earlier draw time. In men aged ⩾75 years, no significant differences in TT, FT, BAT or SHBG were observed on the basis of draw time. Early morning draw time may not be critical for capturing TT concentrations in men ⩾45 years; however, when measuring FT or BAT, an early morning draw time may be preferable for men aged <75 years.
It has been well established that testosterone (T) exhibits circadian variation over a 24-h period, with peak concentrations at approximately 0600 hours and trough concentrations at approximately 2000 hours.1, 2 Observed peak-to-trough T concentration variation is estimated to be approximately 3.47 nmol/l (100 ng/dl) in young men. To consistently capture peak T concentrations for evaluation, an early morning blood draw time generally has been accepted as standard practice in younger men. Previous studies have shown differences in diurnal variations in T concentration.1 However, advancing age is associated with a decrease in diurnal variation.1, 3 Therefore, a precise blood draw time may be less critical for capturing consistent measures in an ageing population.
The Hypogonadism In Males (HIM) study, a cross-sectional, epidemiologic survey, was performed to determine the prevalence of hypogonadism and its associated signs and symptoms in men aged ⩾45 years.4 Patients in the HIM study were considered to be hypogonadal if they had total T (TT) <10.41 nmol/l (<300 ng/dl), or if they were currently receiving treatment for hypogonadism. The main findings of the HIM study indicated that the crude prevalence rate of hypogonadism in the overall study population was 38.7%; this rate was 36.3% among men not receiving T treatment. Similar trends in prevalence were observed when analyses were based on free T (FT) or bioavailable T (BAT) values in these men.
The present analysis of data from the HIM study was undertaken to determine whether the precise time of blood draw affected concentrations of TT, BAT, FT or sex hormone-binding globulin (SHBG), and whether patient age influenced expected diurnal variations in T concentration.
Materials and methods
Before the start of the study, written approval for the protocol and for the patients' informed consent form was obtained from a central institutional review board (IRB). This study was conducted in accordance with the Code of Federal Regulations (21 CFR, parts 11, 50, 56, 312 and 314), following the ethical principles of the Declaration of Helsinki. All patients provided voluntary written informed consent before participating in any study-related procedures. The first patient was enrolled on November 17, 2003, and the last patient completed the study on January 25, 2004.
Methods for the HIM study have been described previously and are summarized here.4 Men aged ⩾45 years from 95 primary care centers who visited their centers during a 2-week period (regardless of the reason for the visit) were recruited to participate. After written informed consent was obtained, patient characteristics (comorbid conditions, demographics and reason for the visit) were documented on a case report form.
A single blood sample was drawn from each patient between 0800 and 1200 hours. For this analysis, samples drawn between 0800 and 1000 hours were compared with those drawn between 1000 and 1200 hours. Only patients who currently were not being treated with T for hypogonadism were included.
Blood samples were assayed for TT, FT, BAT and SHBG. All assays were performed by a central laboratory (Esoterix Endocrinology, Calabasas Hills, CA, USA). SHBG was evaluated with radioimmunoassay (RIA). TT was determined by RIA after T had been extracted from human serum. FT was determined by equilibrium dialysis and scintillation counting with a radiolabeled tracer to determine the percentage of T in free form. BAT was determined through ammonium sulfate precipitation of the SHBG-bound fraction of T, followed by scintillation counting of a radioactive tracer to determine the percentage of TT in bioavailable form. FT percentage values and BAT percentage values were then multiplied by TT to derive FT and BAT concentrations, respectively.
The present analysis for T therapy-naïve patients enrolled in the HIM study (n=2085) was conducted to study the effects of blood draw timing on measured hormone concentrations and to determine whether advancing age influenced the diurnal rhythm of T concentrations. Hormone concentrations (including TT, FT, BAT and SHBG) were stratified by draw time in various age groups (45–54, 55–64, 65–74, 75–84 and >84 years).
Data are reported as mean±s.d. The Mann–Whitney U-test was used to compare values between groups with blood sampled between 0800 and 1000 hours and blood sampled between 1000 and 1200 hours.
Medical histories for patients enrolled in the HIM study who were not currently taking T are summarized in Table 1, stratified by age group. The incidence of prostatic disease/disorder was higher in the older age groups, and obesity (body mass index >30 kg/m2) was largely represented in the younger age groups.
In the overall population, there was no significant difference in the mean TT concentrations obtained between 0800 and 1000 hours and those obtained between 1000 and 1200 hours (Table 2). However, the early and late morning concentrations were significantly different for BAT, FT and SHBG. The mean BAT concentration was 11.5% higher (P<0.001) and the mean FT concentration was 9.3% higher in the early morning (P<0.001) compared with the late morning. In contrast, the mean SHBG concentration was 11.3% higher in the late morning than in the early morning (P<0.001).
When results are considered by patient age (Table 3), no statistical differences in TT concentrations between early morning and late morning draw groups were noted for any age group. Slightly but significantly (P⩽0.042) greater values of FT and BAT were observed for the earlier draw time (before 1000 hours) in all men aged 45–74 years. For men aged 45–64 years, SHBG values obtained from samples drawn before 1000 hours were significantly (P⩽0.011) lower than values obtained between 1000 and 1200 hours. For men aged ⩾65 years, no significant differences in SHBG concentrations were seen in samples drawn before 1000 hours versus those drawn after 1000 hours.
As expected, TT decreased with advancing age (Table 3 and Figure 1), from a total group mean (±s.d.) of 13.0±5.3 nmol/l (376±154 ng/dl) in men aged 45–54 years (n=724) to 10.5±5.1 nmol/l (303±146 ng/dl) in men aged >84 years (n=31). The mean concentration of FT in men aged >84 years was approximately half that observed in the youngest age group (45–64 years); mean BAT decreased to approximately one-third. Mean SHBG increased with age, from 51±28 to 89±31 nmol/l.
A previous analysis of data from the HIM study, in which patients were stratified by age, found a higher prevalence of hypogonadism in the older age groups; hypogonadism was identified in 34% of men aged 45–54 years, 40% of men aged 55–64 years, 40% of men aged 65–74 years, 46% of men aged 75–84 years and 50% of men aged >84 years.4 Findings from this analysis are consistent with those from previous studies showing that TT concentration decreases with advancing age.5, 6 Furthermore, age-related changes in FT, BAT and SHBG in younger versus older men were observed in this study (Table 3 and Figure 1), again similar to the findings of previous studies.5, 6
The Endocrine Society's Clinical Practice Guidelines include a suggestion that morning measurement of T be used as the initial test for the diagnosis of androgen deficiency in men.7 However, this recommendation has been challenged on the basis of a lack of evidence that morning levels are more accurate in diagnosing androgen deficiency than those measured in the afternoon.8 A morning blood draw may be especially unnecessary in older men, considering that diurnal variations in T concentrations decrease with age. The present study showed no effect of draw time on TT concentrations in any age group in the HIM study population, suggesting that early morning draw times may not be necessary when evaluating TT. This finding is consistent with that of Luboshitzky et al.,9 who showed that middle-aged men have an attenuated diurnal variation compared with younger men. This attenuation might be attributable, in part, to a decrease in hypothalamic-pituitary function and decreased testicular Leydig cell function associated with advancing age.10 Although draw time did not affect TT values, it did have a significant effect on FT and BAT values for men 45–74 years of age.
While currently approved T replacement therapies only provide for dosing based on TT levels, some guidelines recommend that FT/BAT be evaluated in lieu of TT values in patients with unusually high or low SHBG concentrations.11 As SHBG increases with age and decreases with obesity, it has been suggested that FT/BAT may be more clinically relevant than TT in elderly and obese patients.4 Our data suggest that when FT or BAT is evaluated, it may be important to obtain an early morning blood draw. Men whose FT or BAT values are close to the lower limit of the normal range may be misclassified as hypogonadal on the basis of late morning blood draws.
Although the differences in BAT and FT concentrations in the early morning versus the late morning were statistically significant, this finding is not necessarily clinically relevant. Thresholds for BAT and FT are not as clearly defined or accepted as the traditional threshold value for TT. However, we assume similarly recognized threshold values for BAT and FT will be available sometime in the future. If we apply the differences between early-morning and late-morning draw times for BAT and FT, that is 11.5 and 9.3%, respectively, to the evaluation of TT concentration at the threshold for determining gonadal status (10.41 nmol/l or 300 ng/dl), the differences in TT concentrations would be 1.21 nmol/l (35 ng/dl) and 0.97 nmol/l (28 ng/dl) higher, respectively, in the early morning. It seems unlikely that a clinical diagnosis would be changed because of this difference.
In the HIM study, the proportion of patients with concomitant conditions increased with advancing age; this may have an effect on the decreased circadian variation noted in TT concentrations. Factors such as good health and regular exercise may contribute to the maintenance of healthy diurnal rhythms and concentrations of T,11 whereas chronic illness is associated with decreased diurnal T variations6 and reduced T concentrations.12
The Massachusetts Male Aging Study (MMAS)6 investigated differences in T concentration with advancing age between apparently healthy patients (that is, those who had no medical history of chronic disease, were not receiving prescription medication that affected hormone levels, were not obese, had moderate drinking habits and were nonsmokers) and patients aged 40–79 years who had confounding factors thought to influence hormone levels, such as health status. In contrast, the HIM study evaluated T concentrations in men who were healthy, as well as those with concomitant conditions. Nonetheless, similar reductions in TT with older age were reported by both studies: data from the MMAS showed a 21% reduction in TT values in patients with advancing age compared with a 19% reduction in the HIM study. The incidence of concomitant conditions increased with age in the ‘apparently healthy’ MMAS population. Data from the MMAS indicate that the incidence of diabetes increased from 8% (131/1677) in 1989 to 13% (79/631) in 2004, and the incidence of hypertension increased from 31% (514/1677) in 1989 to 50% (313/631) in 2004.
The strength of the HIM study is the large sample size. However, the numbers of patients in the 75–84 and >84 year age groups were lower than the numbers in younger age groups. A possible limitation of this analysis is that it was conducted post hoc with the population not randomly assigned to one of the two draw times; however, draw time delineations were prespecified. Another limitation of this study is that there was only a single blood draw for each patient. The Endocrine Society guidelines for the diagnosis of androgen deficiency call for repeat measurement to confirm the initial finding.7 The lack of minority populations may represent another limitation; the population evaluated in the HIM study was 82% white and 14% black.
T concentration decreased and SHBG concentration increased with advancing age. No statistically or clinically relevant differences in TT concentration were observed in blood samples obtained between 0800 and 1000 hours versus those obtained between 1000 and 1200 hours in the middle-aged and older populations studied (groups ⩾45 years). These data suggest that advancing age may preclude the need for precise timing of early morning blood draws to ensure consistent TT concentration measurements. However, as significant draw time differences occurred in FT, BAT and SHBG concentrations, the timing of these evaluations may be more important for obtaining values representative of peak concentration.
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Solvay Pharmaceuticals Inc. provided financial support for this study and editorial assistance in the preparation of this paper. Dr Guay has served on advisory boards for Auxilium, Oscient, Solvay, Cellegy, Procter & Gamble and Indevus. He has participated in speakers' bureaus for Auxilium, Oscient and Solvay and has been the recipient of research grants from Auxilium, Solvay and Cellegy. Dr Miller and Ms McWhirter are employees of Solvay Pharmaceuticals Inc.
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Guay, A., Miller, M. & McWhirter, C. Does early morning versus late morning draw time influence apparent testosterone concentration in men aged ⩾45 years? Data from the Hypogonadism In Males study. Int J Impot Res 20, 162–167 (2008) doi:10.1038/sj.ijir.3901580
- sex hormone-binding globulin
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