der Medizinischen Hochschule Hannover (Direktor Prof. Dr. M.P. Manns)
Age-related changes in serum concentrations of
SHBG, testosterone, estrogens and IGF-I in men:
results from cross-sectional investigation on healthy subjects and calculations from previously reported studies
Dissertation
zur Erlangung des Doktorgrades der Medizin an der Medizinischen Hochschule Hannover
vorgelegt von Vitali Gorenoi aus St. Petersburg (ehem. Leningrad)
Hannover, 2002.
Angenommen vom Senat der Medizinischen Hochschule Hannover am 24.06.2003
Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover Rektor: Professor Dr. Horst v. der Hardt
Betreuer: Professor Dr. Ernst-Georg Brabant Referent: Professor Dr. Wolf-Rüdiger Külpmann Korreferent: Professor Dr. Christian-Georg Stief
Tag der mündlichen Prüfung: 24.06.2003 Promotionsausschussmitglieder:
Professor Dr. Michael Peter Manns Professor Dr. Marion Haubitz Professor Dr. Arnold Ganser
my parents my children my wife
CONTENTS:
Summary ... 8
Introduction ... 10
The role of androgens and estrogens in men ... 10
Physiology of androgens and estrogens in men... 11
Serum blood fractions of androgens and estrogens and their measurements ... 14
Changes of androgens and estrogens during ageing in men... 16
Somatotropic axis and its changes during ageing in men ... 19
Research questions ... 21
Subjects and Methods ... 22
Study subjects... 22
Measurements ... 23
Calculations ... 24
Comparison of calculations with measured and with fixed albumin values... 26
Comparison of measured and calculated free testosterone... 27
Analysis of our cross-sectional investigation ... 28
Analysis of the data from previously reported studies... 29
Results I Analysis of our investigation on healthy subjects... 30
Sex Hormone Binding Globulin ... 30
Testosterone ... 32
Estrogens ... 35
Insulin-like growth factor I ... 38
Results II Calculations from previously reported studies... 41
Description of the studies... 41
Cross-sectional studies on SHBG and sex-hormone changes with age... 41
Longitudinal studies on SHBG and sex-hormone changes with age ... 46
Cross-sectional studies on IGF-1 changes with age ... 48
Sex Hormone Binding Globulin ... 50
Total Testosterone ... 51
Free Testosterone ... 52
Bioavailable Testosterone ... 53
Total Estradiol ... 54
Bioavailable Estradiol ... 55
Estrone... 56
Discussion ... 58
Methodological issues... 58
Sex-Hormone Binding Globulin... 62
Mean levels and effect of age... 62
The cause of changes and biological regulation of SHBG serum levels ... 63
Testosterone ... 66
Mean levels and effect of age... 66
Effect of SHBG, BMI and other factors ... 69
The cause and the clinical importance of testosterone changes with age ... 70
Estrogens ... 72
Mean levels and effect of age... 72
Effect of SHBG, BMI and other factors ... 75
The cause and the clinical importance of changes in estrogens with age ... 76
Insulin-like-growth-factor-I ... 78
Mean levels and effect of age... 78
Effect of BMI, sex-steroids and some other factors ... 79
The cause and the clinical importance of IGF-I changes with age ... 81
Conclusions ... 83
References ... 84
Anhang ... 93
Danksagungen... 93
Lebenslauf ... 94
Liste der wissenschaftlichen Veröffentlichungen ... 95
Erklärung nach § 2 Abs. 2. Nrn. 5 und 6 ... 96
FIGURES:
Fig. 1. The steroid biosynthetic pathways leading to testosterone and estradiol production. ... 12
Fig. 2. Schematic representation of the interrelationship between bioavailable (free and albumin(A)- bound) and SHBG-bound fractions of testosterone(T) and estradiol(E2). ... 14
Fig. 3. Exclusion for BMI in the study. ... 22
Fig. 4. Calculated with measured vs. with fixed albumin free testosterone. ... 26
Fig. 5. Measured free testosterone vs. calculated free testosterone. ... 27
Fig. 6. Ratio of measured to calculated free testosterone vs. SHBG. ... 27
Fig. 7. SHBG distribution and its 5th, 25th, 50th, 75th and 95th percentiles (n=394). ... 30
Fig. 8. Total testosterone and its 5th, 25th, 50th, 75th and 95th percentiles (n=512). ... 32
Fig. 9. Free testosterone and its 5th, 25th, 50th, 75th and 95th percentiles (n=380). ... 33
Fig. 10. Bioavailable testosterone and its 5th, 25th, 50th, 75th and 95th percentiles (n=380). ... 33
Fig. 11. Total estradiol distribution and its 5th, 25th, 50th, 75th and 95th percentiles (n=504)... 35
Fig. 12. Bioavailable estradiol and its 5th, 25th, 50th, 75th and 95th percentiles (n=375). ... 36
Fig. 13. Estrone distribution and its 5th, 25th, 50th, 75th and 95th percentiles (n=391). ... 36
Fig. 14. Distribution of IGF-I and its 5th, 25th, 50th, 75th and 95th percentiles (n=478). ... 38
Fig. 15. Prevalence of men in our study with serum levels of testosterone or estrogen fractions below values determined in 20-29-year-old pesrons... 40
Fig. 16. Prevalence of men in our study with serum levels of IGF-I alone or simultaneously with sex- hormone fractions below values determined in 20-29-year-old persons... 40
Fig. 17. Trend of SHBG levels with age (for simplicity 95%CI not shown). ... 50
Fig. 18. Trend of total testosterone levels with age (for simplicity 95%CI not shown). ... 51
Fig. 19. Trend of free testosterone levels with age (for simplicity 95%CI not shown). ... 52
Fig. 20. Trend of bioavailable testosterone levels with age (for simplicity 95%CI not shown). ... 53
Fig. 21. Trend of total estradiol levels with age (for simplicity 95%CI not shown). ... 54
Fig. 22. Trend of bioavailable estradiol levels with age (for simplicity 95%CI not shown). ... 55
Fig. 23. Trend of estrone levels with age (for simplicity 95%CI not shown)... 56
Fig. 24. Trend of IGF-I levels with age (for simplicity 95%CI not shown). ... 57
Fig. 23. Schematic representation of SHBG changes with age in men and women... 63
Fig. 24. Conclusions: average changes of total and bioavailable testosterone serum levels and remaining hormone concentrations in healthy adult men with age. ... 68
Fig. 25. Conclusions: average changes of total and bioavailable estradiol serum levels and remaining hormone concentrations in healthy adult men with age. ... 74
Fig. 26. Conclusions: average changes of Insulin-like factor I serum levels and remaining hormone concentrations in healthy adult men with age. ... 79
TABLES:
Table 1. Reported data on changes of sex-hormones fractions with age. ... 18
Table 2. Reported data on SHBG changes with age. ... 19
Table 3. Reported data on IGF-I changes with age. ... 20
Table 4. RIA’s used in the study. ... 23
Table 5. Median, mean ± SD and ranges of SHBG serum levels (n=394)... 30
Table 6. The association of SHBG level with age, BMI, IGF-1 and the ratio baE2/baT... 31
Table 7. Median, mean ± SD and ranges of total testosterone levels (n=512). ... 32
Table 8. Median, mean ± SD and ranges of free testosterone levels (n=380). ... 33
Table 9. Median, mean ± SD and ranges of bioavailable testosterone levels (n=380). ... 33
Table 10. Median, mean ± SD and ranges of total estradiol levels (n=512). ... 35
Table 11. Median, mean ± SD and ranges of bioavailable estradiol levels (n=375). ... 36
Table 12. Median, mean ± SD and ranges of estrone levels (n=391)... 36
Table 13. Median, mean ± SD and ranges of IGF-I serum levels (n=478). ... 38
Table 14. Association between serum IGF-I and sex-hormones fractions in male donors... 39
Table 15. Identified cross-sectional studies reporting changes of sex-hormones with age. ... 41
Table 16. Parameters measured in the identified cross-sectional studies. ... 41
Table 17. Subjects and blood storage time in the identified studies. ... 46
Table 18. Longitudinal studies reporting changes of total testosterone with age... 46
Table 19. Cross-sectional studies describing changes of IGF-I levels with age... 48
Table 20. Calculation of age-trends for SHBG. ... 50
Table 21. Calculation of age-trends for total testosterone. ... 51
Table 22. Calculation of age-trends for free testosterone... 52
Table 23. Calculation of age-trends for bioavailable testosterone. ... 53
Table 24. Calculation of age-trends for total estradiol... 54
Table 25. Calculation of age-trends for bioavailable estradiol. ... 55
Table 26. Calculation of age-trends for estrone serum levels... 56
Table 27. Calculation of age-trends for IGF-I serum levels. ... 57
Table 28. The percentage of average changes and remaining hormone levels in blood serum in healthy adult men at the age of 65, 75 or 85 years compared to 25-year-old persons. ... 83
SUMMARY
Many of the clinical features of ageing are reminiscent of the clinical changes seen in hypogonadism and insufficiency of the somatotropic axis in young men. Therefore, at least some of these changes in ageing men may be causally related to changes of the activity of the gonadotropic and somatotropic axes.
Several studies concerning changes in serum concentrations of testosterone, estrogens and IGF-I with ageing in adult men have been reported. However, the data are inconsistent: age- intervals and sample-sizes were often small and only few reports on healthy subjects are available. In addition, different measures of age-related changes, i.e. slopes and age-trends units, have been given, making their results hardly comparable.
Possible effects of age on the gonadal and somatotropic axes are becoming more complex by subdividing of the total persisting in blood hormones to the hormone fractions which are thought to be bioavailable or not bioavailable to the peripheral tissues. Changes in these fractions with age are not parallel to changes in the total serum levels, but their measurement is not a routine procedure.
The primary aim of our study was to estimate the mean age-related trends in serum gonadal and somatotropic activity in adult men. Thus, firstly, we measured total serum concentrations of testosterone, estradiol, estrone and IGF-I as well as of SHBG and albumin in 514 male blood donors aged 20-72 years and calculated from these measurements free and bioavailable testosterone as well as bioavailable estradiol fractions. Secondly, we selected from Medline relevant studies reporting changes of these parameters with age and calculated for these studies mean age-trends of SHBG, IGF-I and sex-hormone fractions. All studies were analysed with regard to their validity to estimate the mean age-trends of the hormones changes. However, as the studies showed very heterogeneous findings, no statistical combination was employed. In addition, we studied interrelations between BMI, SHBG, IGF- I and different sex-hormone fractions.
In our cross-sectional investigation on 20-72-year-old blood donors SHBG serum levels increased, whereas serum concentrations of all hormone fractions decreased with advancing age. Age-trends of SHBG and hormonal parameters pro decade (mean±se) were 9±1% for SHBG, -10±1% for total testosterone, -17±1% for bioavailable testosterone, -17±1% for free testosterone, -7±1% for total estradiol, -12±1% for bioavailable estradiol, -4±1% for estrone and -10±1% for IGF-I serum levels. BMI, SHBG, IGF-I and sex-hormone fractions were interrelated; however, the magnitude of these interrelations was small. Only BMI exerted a meaningful age-adjusted effects on SHBG levels. Of all sex-steroid fractions bioavailable estradiol was found to be the best predictor of serum IGF-I concentrations. The prevalence of men with low serum levels of total testosterone, bioavailable testosterone, bioavailable
Ten cross-sectional and two longitudinal relevant studies reporting changes of SHBG and sex- hormone fractions in adult men with age as well as eight cross-sectional studies of IGF-I were identified. No study was ideal to describe the proper effect of age on these hormones;
however, hormone changes in healthy men reflect these effect most likely better than trends measured in the general population. After analysing the studies and the calculated age-trends it was concluded that exponential function appear to be the most appropriate to describe age- related changes and that in healthy adult men, whose hormone changes reflect the proper effect of age probably better than trends in the general population, serum SHBG increases with age on average between 8-14% pro decade, whereas total testosterone decreases on average between 4-10%, bioavailable testosterone between 10-17%, free testosterone between 10-17%, total estradiol between 7-0%, bioavailable estradiol between 5-12%, estrone between 7-0% and IGF-I between 10-15% pro decade. In the general population, most likely due to a higher rate of diseases in old age, the mean decrease of testosterone fractions and IGF-I is accentuated; however, the mean decrease of estradiol fractions is diminished, probably because of the increase of adipose tissue. Changes with age in estrone serum levels, especially in the general population, remain to be further investigated.
Our study provides new data on changes of serum levels of SHBG, IGF-I and sex-hormone fractions with age in healthy adult men and describes interrelations between these parameters.
It is also the first which estimates the mean age-trends in serum SHBG, sex-steroid and IGF-I activity in adult male persons, summarising results from the most relevant reported investigations. Using these age-trends the average percentage of the hormone changes in healthy adult men may be estimated as well as the percentage of the remaining hormone levels. The results of our study may be important for better understanding of the endocrinology of ageing and for definition of possible therapeutic strategies concerning the supplementation of the gonadal and somatotropic activity in the elderly.
INTRODUCTION
The role of androgens and estrogens in men
Ancient Indians, Greeks, and Egyptians believed that extracts of animal testis could promote virility, potency, and vigour in men. In modern times, the castration and testis-transplantation experiments, conducted initially by John Hunter and later by Adolf Berthold, established the link between secretions of the testis and some of the sexually dimorphic features. Brown- Sequard recognised the association between changes in testicular function and the loss of vigour in older men; he claimed to have rejuvenated himself by injecting the extracts of guinea pig testis. Later, when testosterone was discovered and chemically synthesised, this hormone was given an exclusively role in generating and maintaining male phenotypes in mammals (Bhasin et al. 1998 for review).
Recently, due to the findings of widespread distribution of estrogen receptors ERα and ERβ in reproductive and other tissues of the male and local expression of aromatase, found at the majority of sites at which androgen receptors AR are expressed, a new understanding of the role of estrogens and androgens in men has been established. In a wider context, this concept coincides with the growing awareness that estrogens and androgens (precursors of estrogens) have pervasive effects throughout the body, that either one or the other hormone (or the balance between the two) plays a fundamental role not only in reproductive function and sexual activity but also in muscle activity, bone growth and development and in some other functions (Sharpe 1998).
In reproductive system androgens are essential for the development and maintenance of the specific tissues such as testis, prostate, epididymis, seminal vesicles and penis (Rommerts 1990, 1998) and estrogens appear to be important in the regulation of fluid resorption from the efferent ducts and in the structural and functional development of the Wolffian excurrent duct system, as well as that of the prostate (Sharpe 1998). Testosterone and estradiol (as well as dihydrotestosterone) exert a negative modulation of LH secretion: testosterone probably acts at the hypothalamus by decreasing pulse frequency without a change in pulse amplitude, whereas estradiol probably acts at the pituitary by decreasing pulse amplitude without changing pulse frequency (De Kretser et al. 1995). Testosterone has a psychotropic effect on sexuality, aggression, activity level, performance, cognition, emotion and personality characteristics (Hubert 1990, Christiansen 1998) whereas estrogens were also found to play a role in brain masculinization and sexual behaviour (Sharpe 1998). Aspects of pubertal development in boys (growth of the long bones, their mineralization and epiphyseal closure) and maintenance of the bone mass attributed to the actions of androgens are now recognised as being mediated in part by estrogens (Sharpe 1998, Finkelstein 1998, Riggs et al. 1998);
Androgens are also essential for such characteristic of male properties such as increased muscle strength, hair growth (Rommerts 1990, 1998, Bhasin et al. 1998, Randall 1998);
Faustini-Fustini et al. 1999). Many other tissues (or organs) such as prostate, liver, kidney, skin are targets of androgens, and such tissues (or organs) as heart, kidney, liver, thymus/immune system, gut are targets for estrogens in the male (Handelsman 1995, Sharpe 1998, Faustini-Fustini et al. 1999).
Androgens (Kaufman and Vermeulen 1998) and estrogens (Sharpe 1998) appear to be associated with several major diseases, such as osteoporosis, cardiovascular diseases and reproductive organ cancers; these disorders are of an increasing importance in Western societies (Lamberts et al. 1997, Ybarra et al. 1996, Eastell et al. 1998). Androgens and estrogens are also associated with frailty, impaired sense of well-being and sexual dysfunction, which have emerged as important quality-of-life issues of ageing men; and especially loss of muscle strength is the limiting factor by the increasing number of the healthy oldest elderly that determines their chances for living an independent life until death (Lamberts et al. 1997). Thus the activity of androgens and estrogens in male could have consequences that reach far beyond the reproductive system.
Physiology of androgens and estrogens in men
A number of naturally occurring sex-steroids exist with the capacity to exhibit androgen or estrogen activity. They have the same precursors in the steroidogenic pathways (Fig. 1) which mostly expressed in Leydig cells of males testis, but also in the adrenal cortex and in some other tissues showing many similarities in different cell types (Kretser et al. 1995; Rommerts 1990, 1998). The biosynthesis of biologically active sex-steroids is a process of formation of biologically inactive pregnenolone from cholesterol and its stepwise degradation. These reactions take place in sex-steroid producing cells inside the mitochondria and the endoplasmatic reticulum, respectively (Rommerts 1990, 1998).
The major circulating androgen in the human male is testosterone, a major product of the Leydig cells with the most potent androgenic action. More than 95% of serum testosterone is secreted by the testis, which produces approximately 7 (3-10) mg of testosterone daily (De Kretser et al. 1995, Handelsman 1995). The metabolic steps required for the conversion of cholesterol into androgens take place in approximately 500 million Leydig cells which constitute only a few percent of the total testicular volume (Rommerts 1990, 1998).
Testosterone circulate in blood in nanomolar concentrations.
Although after puberty greater than 95% of circulating testosterone is derived from testicular secretion, the remainder arise from metabolic conversion of precursors of low intrinsic androgen potency such as dehydroepiandrosterone (DHEA), androstenedione and others, predominantly secreted by the adrenal cortex (Handelsman 1995). Some of these weak androgens also leak out in small amounts of the Leydig cells, as far as the total capacity of the pregnenolone-converting enzyme system is insufficient to convert all available pregnenolone
into testosterone (Rommerts 1990, 1998, De Kretser et al. 1995). Androgens with low androgen potency constitute a reservoir of precursors for extragonadal conversion to bioactive sex steroids in extragonadal tissues, including liver, kidney, muscle, and adipose tissue (Handelsman 1995). These weak androgens also persist in blood in nanomolar concentrations.
Fig. 1. The steroid biosynthetic pathways leading to testosterone and estradiol production.
(from Kretser et al. 1995)
With regard to estrogens, the testis produces approximately 20-25% of the total daily production of 17ß-estradiol, and the remainder (both estradiol and estrone) being derived by conversion of both testicular and adrenal androgens, such as androstenedione and DHEA, by the enzyme aromatase, which is expressed in many tissues including testes, fat tissue, liver, brain, hair, follicles and the brain. (De Kretser et al. 1995). The relative contribution of locally produced and circulating estrogens to estrogen action at its various target sites remains a highly unexplored issue (Sharpe 1998). When a target cell is estrogen-dependent, its aromatase activity and the supply of androgen substrate are of major importance for determining the rate of synthesis of estrogens. However, which kind of estrogen is produced in each tissue is quite tissue specific, depending on the source of sex-steroid, presented to the aromatase (Faustini-Fustini et al. 1998). The activity of 17ß-hydroxysteroid dehydrogenase determines the amount of the active estradiol metabolised to biologically inactive estrone (Rommerts 1998), which again could be converted to 17ß-estradiol in the tissues. Thus, although estradiol is the most important estrogen in human male, estrone appears to be a large and easily available reservoir for estradiol in the periphery. Estrogens circulate in blood in subnanomolar concentrations.
Effects of testosterone (and also of DHT and other androgens) are mediated by binding to the androgen receptor AR (Rommerts 1990, Handelsman 1995), and the effects of estrogens are mediated by binding to the estrogen receptors ERα and ERβ (Sharpe 1998, Faustini-Fustini et al. 1999), which reside in the nucleus of the cells. In the majority of cases androgen receptors AR and estrogen receptors ERα or ERβ are expressed in the same tissues, such as reproductive, muscle, brain, bone, skin and others, with a notable exception of germ cells.
Due to this coincidence and to the coincidence that gene aromatase is also expressed at many of the same tissues, it can be envisaged that the local balance between estrogen and androgen action could be finally regulated (Sharpe 1998). The affinity constants for androgen and estrogen receptors in tissues are in (sub)nanomolar ranges (Rommerts 1998).
A small proportion of circulating testosterone is metabolised to biologically active metabolites in certain target tissues to modulate biological effects, whereas most of these androgens as well as estradiol (which is also a metabolite of testosterone) are converted to inactive metabolites for urinary and/or biliary excretion. One of the important active metabolites is 5a-dihydrotestosterone (DHT). About 4 per cent of circulating testosterone is converted by 5a-reductase to this steroid, a more active androgen receptor agonist. This occurs efficiently within the stroma of the prostate and to a lesser extent in other tissues, such as skin and liver. Another active metabolites are 5ß-androgenic metabolites, which stimulate the production of heme in bone marrow and liver (Rommerts 1998). Some metabolites excreted as free steroids, whereas others are conjugated to sulphate or to glucuronide group.
The majority of metabolic reactions takes place in liver, kidney, muscle, and adipose tissue (Handelsman 1995), but the prostate and the skin also contribute significantly to the metabolism of androgens (Rommerts 1990). Alterations in the rate of degradation of sex- steroids are as important as changes in the rate of synthesis (Rommerts 1998). Concentrations in blood for the most of the sex-steroid metabolites are also in nanomolar ranges; however, for sulfate-conjugated substrates of androsterone, estrone and estradiol in submicromolar, and for dehydrosterone-sulfate in micromolar (Belanger et al. 1994).
Testicular testosterone secretion is principally governed by luteinizing hormone (LH) acting on the rate-limiting step, the conversion of cholesterol to pregnenolone within Leydig cell mitochondria (Handelsman 1995). Driven by brief bursts of hypothalamic secretion of GnRH into the pituitary portal bloodstream, pituitary gonadotropes secrete LH episodically in pulses of high amplitude at about hourly intervals with little intervening interpulse basal LH secretion, so that circulating LH levels are distinctly pulsatile (Handelsman 1995).
Testosterone participates in a negative testicular feedback cycle through its inhibition of hypothalamic GnRH and, consequently, pituitary gonadotropin secretion. Such negative feedback involves both testosterone effects on androgen receptors and aromatisation to estradiol within the hypothalamus (Handelsman 1995).
Serum blood fractions of androgens and estrogens and their measurements A number of parameters of testosterone and estradiol activity in blood serum has recently been discussed in the literature. For the better evaluation of hormone measurements, it is important to understand what reflects each parameter and which parameter reflects the situation in the target tissues and the steady-state of the feedback regulation more accurately.
Sex-steroids circulate in plasma as bound to protein, and, in small percentage, as unbound (free) fractions (Fig. 2). This unbound fraction persist in blood only in a very small percentage of about 2-3 per cent of the total hormone level: in subnanomolar concentrations for free testosterone and in picomolar concentrations for free estradiol, which is lower than the affinity constant for estrogen receptor, persisting in (sub)nanomolar range (Rommerts 1998).
Fig. 2. Schematic representation of the interrelationship between bioavailable (free and albumin(A)-bound) and SHBG-bound fractions of testosterone(T) and estradiol(E2).
Binding proteins in body fluids can act as a storage for steroids which have a high rate of metabolism during passage of blood through the liver (Rommerts 1998), and the free and loosely protein-bound hormone fractions (commonly referred to as the bioavailable fractions) constituting a large and accessible buffer for the readily diffusible free steroids (Handelsman 1995).
E1 T
T E2 E2 A
SHBG
T E2
bioavailable fraction
aromatisation
(testis, periphery)
pregnenolon, DHEA androstenedion, etc.
estradiol precursor aromatisation
(testis, periphery) A
synthesis (testis, periphery)
conversion (periphery) inactive
fraction
inactive fraction SHBG
bioavailable fraction
Such loosely protein-bound fractions are albumin-bound hormones. Albumin, a non-specific binding protein, has low-affinity, high-capacity binding sites, and there is only a weak competition between testosterone, estradiol or any of androgen metabolites for these binding sites (Södergard et al. 1982). The concentration of the albumin-bound testosterone and estradiol in blood is linearly related to their unbound fraction and is about 20-22 fold higher.
Albumin-bound fractions constitute about 30-50 per cent of the total hormone levels and persists in blood in nanomolar range for albumin-bound testosterone and in subnanomolar range for albumin-bound estradiol fractions.
Some of the circulating hormones are specifically bound to sex hormone-binding globulin (SHBG). Circulating SHBG is secreted by the liver and persists in blood in nanomolar concentrations. It is structurally identical to the testicular androgen-binding protein and has a single high-affinity androgen-binding site. Its affinity to sex-steroids is more than 104–105 higher than the affinity to sex-steroids of albumin. Testosterone, estradiol, DHT, androstenediol and androstanediol compete for the same binding sites on SHBG, but have different affinities (Södergard et al. 1982). SHBG binds testosterone to a higher degree than it does estradiol, acting as an amplifier of estradiol action (Knochenhauer et al. 1998). The concentrations of SHBG-bound fractions of testosterone and estradiol are about 50-70 per cent of the total hormone level and are in nanomolar range for SHBG-bound testosterone and in subnanomolar range for SHBG-bound estradiol fraction. The binding of sex-steroids to other proteins in blood serum is very small (Södergard et al. 1982).
“Total” concentrations of sex-steroids are the concentrations of their free plus protein-bound fractions. Total levels of testosterone and estradiol are SHBG-dependent, related non-linearly to the levels of their unbound or albumin-bound fractions and appear not to be a reliable parameter of these sex-steroids. The same applies to some other commonly used parameters such as free testosterone index or free estradiol index, calculated as the quotients of total testosterone or total estradiol to SHBG (Vermeulen et al. 1999).
A number of measurements for sex-hormones are available from clinical laboratories. The measurement of serum steroids are usually performed by radioimmunoassay and measures
“total” (free plus protein bound) fractions. “Free” or dialyzable testosterone measurements are estimates of the fraction of testosterone in blood that is not bound to protein. These assays require determination of the percentage of unbound testosterone by a dialysis procedure, estimation of total testosterone, and the calculation of free testosterone. Kits are available for determination of free testosterone without dialysis and are used to provide a free testosterone measurement by many laboratories. Unfortunately, these measurements are often inaccurate, especially when testosterone levels are low and SHBG levels are elevated. Obtained by such assays values substantially differ from values obtained using a variety of other methods (Rosner 1997). Recently it has been shown that these direct measurements (by an analog ligand immunoassay procedure) are likely not to be reliable parameters of free testosterone
activity (Vermeulen et al. 1999). Free testosterone as well as free estradiol can also be calculated if total testosterone, SHBG, albumin, and total estradiol (only for free estradiol) concentrations are known (Södergard et al. 1982, see methods). Another measurement of testosterone and estradiol commonly made is that of “bioavailable” or non-SHBG bound fractions. This measurement takes into account that SHBG precipitates at a lower concentration of ammonium sulfate (of about 50%) than albumin. Bioavailable fractions of testosterone and estradiol can also be calculated if testosterone, SHBG, albumin, and estradiol (only for bioavailable estradiol) levels are available (Södergard et al. 1982). The calculation of free and non-specifically bound testosterone fractions was demonstrated to be a simple and a reliable method to measure these hormone concentrations (Vermeulen et al. 1999).
As 95% of circulating testosterone in men is produced by the testes, serum testosterone fractions may be used as markers of testicular secretion; but which serum testosterone fraction reflects the testosterone activity in the target tissues more accurately has not been fully investigated. However, recently there has been good evidence that although free and non- specifically bound (bioavailable) fraction of testosterone in plasma reflects only partially the hormone available at the cellular level in specific tissues (where partial dissociation of the specific steroid-protein complex may occur and inactive prohormones may be converted intracellularly to active hormones), bioavailable testosterone fraction reflects the clinical situation more precisely than the total hormone levels in plasma (Vermeulen et al. 1999).
The most circulating estradiol in the male blood derives from the testis and adipose tissue and consequently reflects their activity. Whether and to what extent any blood fraction of estradiol or their combination with estrone reflects estrogen activity in the target tissues also remains unclear. However, recently there has been some evidence that the bioavailable estradiol fraction could be a good indicator of such estrogen activity (Riggs et al. 1998, Khosla et al.
1998).
Changes of androgens and estrogens during ageing in men
Many of the clinical features of ageing are reminiscent of the clinical changes seen in hypogonadism in young men. In healthy men ageing is usually accompanied by decrease in conception rate as well as by noticeable declines in sexual interest and activity and in erectile capacity. Equally important, there are decreases in lean body, bone, and in muscle mass; a redistribution of body fat; and a reduction in sexual hair, with a consequence loss of strength and virile appearance (Blackman 1995). The hypogonadism in young men is also accompanied by decrease in general well-being, mood changes, decrease of energy and virility, decrease in sexual pilosity and skin thickness, decrease in muscular mass and strength, increase in upper and central body fat, decrease in bone density and increase in prevalence of osteoporotic fractures, decrease in libido and sexual drive, and a markedly
However, an “andropause”, as defined as the male equivalent of the menopause, which in women signals the end of the reproductive life and a near total cessation of gonadal sex steroid production, does not exist. Indeed, ageing in healthy men is normally not accompanied by abrupt or drastic alterations of gonadal function, and androgen production as well as fertility can be largely preserved until very old age (Kaufman and Vermeulen 1998).
Whether ageing is associated with decrease of sex-hormones has long been a highly controversial issue. The early reports of decreased serum testosterone levels in elderly men were followed by several studies that failed to confirm these changes, but the later studies which included exclusively healthy ambulatory young and elderly men confirmed the age- associated decline in serum testosterone concentrations (Vermeulen 1991 for review). Also a meta-analysis of studies of testosterone in ageing men revealed a highly significant inverse relation between total serum testosterone and age (Gray et al. 1991). Recent studies in the nineties, performed on a larger populations, confirmed age-related changes of total testosterone and showed a higher decrease of free and non-specifically bound testosterone fractions (Vermeulen et al. 1996, Khosla et al. 1998, Ferrini and Barret-Connor 1998). The decrease of testosterone with age has also been documented in a few longitudinal studies (Morley et al. 1997, Zmuda et al. 1997, Harman et al. 2001, Feldman et al. 2002).
Even more controversial data are present for age-related changes in serum estrogens concentrations. Some early studies observed an increase in total estradiol (Blackman et al.
1995 for review), another showed no changes (Gray et al. 1991, Vermeulen et al. 1996) or decrease in estrogens levels (Simon et al. 1992, Belanger et al. 1994). The recent studies in male confirmed a decline in total estradiol with age and showed a higher decline in bioavailable estradiol concentrations (Khosla et al. 1998, Ferrini and Barret-Connor 1998).
Despite the number of studies reporting age-related changes in male sex-steroids serum levels (Tabl. 1), the data are quantitatively hardly comparable and there is no common measure of the magnitude of these changes. Most authors present the degree of hormone changes for age- intervals of their study, some give age-trends pro year or decade, another report only mean or median levels of the hormone fractions for stratified age-groups.
Likewise, as estimation of bioavailable testosterone and estradiol fractions is not a routine procedure, the data on changes of these fractions with age are scarce. Nevertheless, free and bioavailable testosterone and estradiol fractions can be estimated over calculations, when total concentrations of these hormones and SHBG are reported.
Most of the reported studies were performed on population-based collectives. However, today it is generally accepted that the age-associated decline in testosterone levels may often be accentuated by intercurrent diseases, and some chronic diseases can induce more longstanding decrease in testosterone level: testosterone tend to be decreased in elderly men with impaired glucose tolerance, noninsulin-dependent diabetes mellitus and obesity, in men with coronary atherosclerosis, with chronic liver disease, etc; and chronic use of some drugs such as
glucocorticoids and neuroleptics can also induce a marked suppression of testosterone levels (Kaufman and Vermeulen 1998, for review). However, data about testosterone and estrogen changes in explicitly healthy subjects over the wide range of ages are rather limited.
Table 1. Reported data on changes of sex-hormones fractions with age.
Hormones/Studies Reported data
Total testosterone
Barrett-Connor and Khaw 1987 decrease between 30-79 yrs (age-trend not reported) Gray et al. 1991 age-trend/year =-0,4±0,1%/yr between 40-70 yrs
Simon et al. 1992 decrease between 20-59 yrs, from young yrs (age-trend not reported) Belanger et al. 1994 age-trend/decade=-16,0%/decade between 40-80 yrs
Vermeulen et al. 1996 stable till 55 yr, afterward age-trend/yr= -0,85%/yr till 100 yrs Khosla et al. 1998 29,5% decrease between 25-85 yrs
Ferrini and Barrett-Connor 1998 1,9 pg/ml/yr (6,6 pmol/l/yr) between 24-90 yrs, NS Fatayerji and Eastell 1999 26% decrease between 20-79 yrs
Feldman et al. 2002 age-trend/year =-0,8%/yr between 50-80 yrs
Zmuda et al. 1997 (longitudinal) decrease 41ng/dl between 41-61 yrs (-0,121 nmol/l/yr) or 0,2%/yr Morley et al. 1997 (longitudinal) decrease -110ng/dL/10yrs (-0,382 nmol/l/yr) between 61-87 yrs Harman et al 2001 (longitudinal) decrease –0,110 (-0,124) nmol/l/yr between 22-91 yrs
Feldman et al. 2002 (longitudinal) age-trend/year =-1,6 (95%CI: -2,0;-1,3) %/yr between 40-80 yrs Free testosterone
Gray et al. 1991 age-trend/year =-1,2±0,2%/yr between 40-70 yrs Vermeulen et al. 1996 age-trend/year =-1,2%/yr between 25-100 yrs Feldman et al. 2002 age-trend/year =-1,7%/yr between 50-80 yrs
Feldman et al. 2002 (longitudinal) age-trend/year =-2,8 (95%CI: -3,2;-2,3) %/yr between 40-80 yrs Albumin-bound testosterone
Gray et al. 1991 age-trend/year =-1,0±0,2%/yr between 40-70 yrs Feldman et al. 2002 age-trend/year =-2,0%/yr between 50-80 yrs
Feldman et al. 2002 (longitudinal) age-trend/year =-2,5 (95%CI: -3,0;-2,1) %/yr between 40-80 yrs Bioavailable testosterone
Khosla et al. 1998 64,1% decrease between 25-85 yrs
Ferrini and Barrett-Connor 1998 decrease 18,5 pg/ml/yr (64 pmol/l/yr) between 24-90 yrs Total estradiol
Barrett-Connor and Khaw 1987 decrease between 30-79 yrs (age-trend not reported) Gray et al. 1991 age-trend/year NS between 40-70 yrs
Simon et al. 1992 decrease between 20-59 yrs, from young yrs (age-trend not reported) Belanger et al. 1994 age-trend/decade: NS between 40-80 yrs
Vermeulen et al. 1996 stable between 25-100 yrs
Khosla et al. 1998 11,5% decrease between 25-85 yrs, NS Fatayerji and Eastell 1999 33% decrease between 20-79 yrs
Ferrini and Barrett-Connor 1998 decrease 0,03 pg/ml/yr (0,11 pmol/l/yr) between 24-90 yrs, NS Bioavailable estradiol
Khosla et al. 1998 47,1% decrease between 25-85 yrs
Ferrini and Barrett-Connor 1998 decrease 0,12 pg/ml/yr (0,44 pmol/l/yr) between 24-90 yrs Estrone
Barrett-Connor and Khaw 1987 increase between 30-79 yrs (age-trend not reported) Gray et al. 1991 age-trend/year: NS between 40-70 yrs
Simon et al. 1992 stable between 20-59 yrs
Belanger et al. 1994 age-trend/decade= -11,3%/decade between 40-80 yrs Khosla et al. 1998 -11,5% decrease between 25-85 yrs, NS
Feldman et al. 2002 age-trend/year =-0,8%/yr between 50-80 yrs
Feldman et al. 2002 (longitudinal) age-trend/year =-3,6 (95%CI: -4,0;-3,1) %/yr between 40-80 yrs (from the large, with >30 subjects/decade, cross-sectional and all longitudinal studies)
The differences in changes of total and non-SHBG-bound fractions are due to the age-related increases of sex-hormone-binding globulin (SHBG) capacity and due to the non-linear
testosterone, serum SHBG negatively correlates with obesity, insulin levels, activity of somatotropic axis and the presence of some chronic diseases (Kaufman and Vermeulen 1998).
The data on age-associated variability of sex-hormone-binding globulin capacity may be used for the estimation (calculations) of free and bioavailable testosterone as well as estradiol fractions. However, these data in the presented literature are also scarce, quantitatively hardly comparable and especially changes of SHBG levels with age in healthy subject have been investigated only insufficiently (Tabl. 2).
Table 2. Reported data on SHBG changes with age.
Hormones/Studies Reported data
Barrett-Connor and Khaw 1987 increase after 40 yrs (age-trend not reported) Gray et al. 1991 age-trend/year =1,2±0,1%/yr between 40-70 yrs Vermeulen et al. 1996 early (before 40 yrs) increase (age-trend not reported) Khosla et al. 1998 124,3% increase between 25-85 yrs
Fatayerji and Eastell 1999 62% increase between 20-79 yrs
Feldman et al. 2002 age-trend/year =1,6%/yr between 50-80 yrs
Harman et al 2001 (longitudinal) curvilinear increase, higher rate in the older than in younger men Feldman et al. 2002 (longitudinal) age-trend/year =1,3 (95%CI: 0,8; 1,6) %/yr between 40-80 yrs (from the large, with >30 subjects/decade, cross-sectional and longitudinal studies)
Somatotropic axis and its changes during ageing in men
Although the first suggestion of a connection of growth with a specific function of the Pituitary Gland was made by Pierre Marie in 1886 on acromegaly, the need for primate growth hormone in primates was not recognised until the late 1940’s (Tattersall 1996). The role of intermediate substances as growth-promoting factors was found in 1957 in experiments on cartilage of a hypophysectomised rat (Daughaday 1995). In 1958 the first successful therapeutic use of growth hormone in a human pituitary dwarf was reached.
Growth hormone (GH) is essential for skeletal, muscular and visceral growth in humans, and is a major anabolic hormone that exerts important stimulatory effects on protein synthesis, especially in the liver, spleen, kidneys, thymus, and red blood cells, on lipolysis and on mineral metabolism (Daughaday 1995, Blackman et al. 1995). GH in plasma is bound with high affinity to growth-hormone binding protein and exerts its effects by binding to growth- hormone receptors, mostly presented in the liver, but also in other tissues (Daughaday 1995).
Most of the peripheral tissue effects of GH are mediated by insulin-like growth factor I (IGF- I), also known as somatomedin C, which stimulate mitogenesis and promote the differentiation of several cell types (Daughaday 1995). IGF-I exerts its effects by way of endocrine, paracrine, and autocrine mechanisms. Most circulating IGF-I is generated in the liver by the action of GH, and regulates GH secretion by negative feedback at the hypothalamic and pituitary levels (Blackman et al. 1995).
IGF-I is bound to IGF-binding proteins, predominantly (about three quarters) to IGF-binding protein 3 (IGFBP-3) as a ternary complex, and its effects are mediated by binding to IGF-
receptors, mostly to IGF-R’s Type I (Daughaday 1995). Total IGF-I persists in blood in nanomolar concentrations and free IGF-I only accounted for about 1% of the total IGF-I levels (Yu et al. 1999). Some diseases such as malnutrition, malabsorbtion, hypothyreosis, liver diseases, diabetes mellitus, adiposity affect serum total and free IGF-I concentrations.
Whereas the direct measurement of GH is complicated because of ultradian pattern of GH- secretion, there is a strong positive relation between baseline serum IGF-I levels and spontaneous 24-hour GH secretion in young adults. There is also a strong (fast age- independent) positive correlation of IGF-1 with IGFBP-3 level (Blackman et al. 1995) and, therefore, IGF-I appears to be a reliable parameter of somatotropic activity in blood serum.
RIA is still the predominant technique for measurements of total IGF-I concentrations (Blum and Breier 1994).
As GH can also stimulate local IGF-I production (Corpas et al. 1993 for review), measured serum IGF-I levels may not be an accurate index of somatotropic activity in the tissues.
However, this parameter remains to be the best available index of the activity of GH/IGF-I axis.
The fact that ageing in humans is accompanied by a generalised decrease in protein synthesis and a concomitant reduction in muscle and bone mass, lean body mass, and increase in fat mass, signs known to accompany growth-hormone insufficiency (Corpas et al. 1993), suggests that GH (IGF-I) secretion or action might decrease with advancing age. The association of human ageing with IGF-I serum levels (GH secretion) has been evaluated by some researchers, who showed that serum IGF-I concentration in men decreases with age.
However, the sample-sizes of these studies were very small. Moreover, most of them were population-based; thus, age-related decrease of IGF-I can be pronounced in these studies (Blackman et al. 1995).
Likewise, comparable to sex-steroids, data on IGF-I changes with age in the literature are scarce and quantitatively hardly comparable and there is no common measure of the magnitude of age-related changes (Tabl. 3). The authors presented different measures of age- related IGF-I variability: degree of IGF-I changes in investigated age-intervals, equations for linear regression model for untransformed, logarithmically or square-root-transformed data.
Table 3. Reported data on IGF-I changes with age.
Hormones/Studies Reported data
Yamamoto et al. 1991 Decrease –2,36µg/l/yr between 21-80 yrs
Juul et al. 1994 Decrease √(IGF-I)= -0,11 µg/l/yr between 20-80 yrs Landin-Wilhelmsen et al. 1994 Decrease –2,1 µg/l/yr between 25-64 yrs
Nyström et al. 1997 Decrease –3,281µg/l/yr between 20-69 yrs O’Connor et al. 1998 Decrease –1,972 ng/ml/yr between 20-90 yrs
Hilding et al. 1999 Decrease lg(IGF-I)=-0,00647µg/l/yr between 20-96 yrs Fetayerji and Eastell 1999 Decrease 54% between 20-79 yrs, lg-quadratic- function (from the large, with >10 subjects/decade, cross-sectional studies; no longitudinal studies reported)
GH/IGF-I axis as well as sex hormones undergo some of the largest changes among the hormonal systems in human during life. Activity of both systems dramatically increases during puberty and markedly decreases during ageing (Pfeilschifter et al. 1996 for review).
There is an increasing evidence that the activity of the GH/IGF-I system and of sex hormones in men may be in fact closely interrelated: Such associations as the strong association between IGF-I and SHBG (Pfeilschifter et al. 1996, Vermeulen et al. 1996), and the association between IGF-I and bioavailable, but not total testosterone, were recently reported (Pfeilschifter et al. 1996).
However, reported data concerning sample-size, investigated hormone parameters (Vermeulen et al. 1996) or investigated ages and the absence of diseases (Pfeilschifter et al.
1996) are limited, and there is no data about interrelations between IGF-I and bioavailable estradiol levels.
Research questions
The primary aim of our study was to obtain the new cross-sectional data on changes in serum concentrations of SHBG, total, free and bioavailable testosterone fractions, total and bioavailable estradiol fractions as well as estrone and IGF-I in healthy adult men with age and to summarise research evidence from the literature about the magnitude of the age-related changes of these hormones (and hormone fractions) in adult male persons.
The additional purpose of the study was to examine the interrelations between SHBG, IGF-I and different sex-hormone fractions as well as their association with BMI (body-mass-index).
SUBJECTS AND METHODS Study subjects
525 regular male blood donors, aged 20-72 years, were randomly recruited from the Department of Transfusion Medicine of the “Medizinische Hochschule Hannover” to participate in the study and after signing an informed consent were interviewed concerning their age, bodyweight- and height as well as their health status.
According to the general requirements to the blood donors all subjects included had no acute infection disease or severe internistic disorders. They also took no medication, known or suspected to influence the somatotropic or the gonadal axes.
As it is known, that BMI (body-mass-index) associated with the highest mortality rate is in the extreme BMI values for each age period (Blackman et al. 1995), subjects with BMI beyond 3 mean SD of the mean age-specific BMI levels (n=11) were considered as not healthy and excluded from the investigation (Fig. 3).
10 15 20 25 30 35 40 45
10 20 30 40 50 60 70 80
age (years)
BMI (kg/m²)
3 mean SD
Exclusion for obesity
3 mean SD
Fig. 3. Exclusion for BMI in the study.
It must be noted that age-adjusted BMI’s of our subjects were grossly similar to the BMI’s observed in many west populations (WHO 1995) and in healthy men in the studies concerning changes of sex-steroids and IGF-I with age (Vermeulen 1991, Nyström et al. 1997), although in some studies BMI levels were lower (Vermeulen et al. 1996) or higher (Fatayerji and Eastell 1999). Also height and weight of our subjects were similar to these parameters observed in Germany and in another west populations (WHO 1995, Greil 1998).
It is methodologically important that although BMI is probably the most useful scale to define obesity (Björntorp 1997), no consensus about the appropriateness of the different cut-off points has been reached (Molarius and Seidell 1998). Body mass index (weight/height²), a measure of relative weight, does not distinguish fat from muscle in people with the same height and weight. However, major changes in body composition occur, causing a redistribution of body fat mass and a lean body mass even without a concomitant alteration in body weight. Fat constitutes 18,3% of weight in young vs. 26,2% of weight in old people (Blackman et al. 1995). Thus, the real effects of body composition could be underestimated by using body mass index.
Finally, 514 subjects between the ages of 20 to 72 years were recruited: 111 men aged 20-29 years, 169 men aged 30-39 years, 127 men aged 40-49 years, 70 men aged 50-59 years and 37 men over 60 years. There were 16 men (3%) with a BMI below 20 kg/m², 289 men (56%) between 20,0-24,9 kg/m², 195 men (38%) between 25,0-29,9 kg/m² and 14 men (3%) with a BMI of up 30 kg/m². The mean BMI was 24,6 ± 2,6 kg/m².
Measurements
The blood donors had their regular morning meal. Blood samples were drawn from the antecubital vein into heparinized tubes between 09.00-12.00 h in the morning. The blood was centrifuged at 11,500 rev./min for 20 min at 4°C. The plasma was separated and stored in a polypropylene tubes at -20C° until it was thawed for hormone determinations (maximally 2 years). Serum samples were assayed by commercially available RIA’s (Tabl. 4).
Table 4. RIA’s used in the study.
assay producer detection
limits
intra assay (%)
inter assay (%)
n
Total Testosterone DSL, Germany 0,28 nmol/l 8,1 9,1 512
a-ligand Free Testosterone DSL, Germany 0,62 pmol/l 3,7 7,9 510 Sex-Hormone Binding Globulin DSL, Germany 3 nmol/l 3,7 11,5 394
Total Estradiol DSL, Germany 2,2 pmol/l 3,8 3,7 504
Estrone DSL, Germany 4,4 pmol/l 5,6 11,1 391
Insulin-like Growth Factor I Mediagnost, Germany 2,6 pmol/l - 7,4 478
In addition, serum albumin was determined by an in-house RIA using a monoclonal mouse antihuman-albumin-antibody (Clone 1C8, Hy-Test; Germany, CV 9,3%; n=500). The mean serum albumin concentration in our study did not change with age (5,93 ± 1,44 ×10-4 mol/L) and was a little lower than reference intervals (Campion et al. 1988).
Conversion factors: testosterone: pg/ml×3,47=pmol/l; SHBG: ng/L×11,765=nmol/l; estradiol and estrone: pg/ml×3,7=pmol/l; IGF-I: ng/ml/7,649=nmol/l.
Calculations
Free and bioavailable testosterone and estradiol levels were calculated, using the equation of distribution of one ligand between two binding proteins and the equation, derived from the law of mass action for the model: two or more ligands and two binding proteins, competition between the ligands for the same binding site(s) for one binding protein (Södergard et al.
1982, Eq.1-9):
Si= SiSHBG+SiAlb+ Si
∑
;SiSHBG n K shbg Si
K S K S K Sn
si shbg
si shbg
s shbg
s shbg
sn
= × × × shbg
+ × + × + + ×
1 1 1 2 2 ... ; competition between the ligands.
SiAlb n K Alb Si
K Si
si alb
si alb
si
= × alb× ×
+ ×
1 ; no competition between the ligands.
Where: Si – concentration of free steroid Si,
∑
Si - total concentration of steroid Si, SiSHBG, SiAlb – concentrations of steroid Si bound on SHBG or on albumin.shbg, Alb – concentrations of SHBG or albumin, respectively.
nsishbg,nsialb- number of binding sites for steroid Si on each molecule of SHBG or albumin.
Ksishbg,Ksialb- association constants for the binding of Si to SHBG or albumin.
These equations are based on the assumption that all binding sites of each protein are equivalent and independent and there is no competition between hormones for the binding to albumin. (Södergard et al. 1982).
As in all cases of hormone binding to albumin the term Ksialb×Si is much smaller than unity, it can be omitted from the denominator (Södergard et al. 1982). Thus, in general:
Si n K shbg Si
K siS K S K Sn n K Alb Si Si
shbg Si shbg
S shbg
S shbg
Sn
shbg Si
alb Si
∑
= 1+ × 1×+ ×× 2+ +× × + × alb × × +1 2 ... ;
Five ligands: testosterone, estradiol, 5a-dihydrotestosterone, 5-androstene-3ß,17ß-diol, 5a- androstane-3a,17ß-diol compete for the same binding sites on SHBG, but have different affinities.
As omitting androgen metabolites does not affect the calculations of free and bound testosterone as well as estradiol levels and additional omitting estradiol does not affect the calculations of free and bound testosterone fractions, these parameters can be left out from the equations, respectively (Södergard et al. 1982).
SHBG binding capacity, SHBG= nsishbg×shbg, can be measured by radioimmunoassay, which is likely a reliable measure of SHBG-binding sites in males (Vermeulen et al. 1999).
The albumin apparent association constants for estradiol and testosterone (Ktapp = ntalb× Ktalb, Keapp = nealb× Kealb) can be determined experimentally.
Thus, after transformation we have the following second degree equations:
N = ntalb× Ktalb× Alb; M = nealb× Kealb× Alb L= Ktshbg×t where: t, e – concentrations of free testosterone or estradiol;
T, E – total concentrations of testosterone or estradiol;
Hence, the bioavailable testosterone and estradiol fractions can be calculated:
T’BA, E’BA - concentrations of bioavailable fractions of testosterone or estradiol.
The experimentally derived values of the SHBG association constants for estradiol is 50% of those for testosterone and the albumin apparent association constants for estradiol and testosterone are comparable (Moll et al. 1981):
It was shown that albumin concentrations (Alb) within the physiological range of 40-50 g/L (5 8 7 2 10, − , × −4mol L/ ) does not significantly affect free testosterone values (Vermeulen et al. 1999). Therefore, calculations can be performed both with measured and with fixed albumin 43g/L (Alb= 6 2 10, × −4mol L/ ) concentrations, where:
N K( tapp× Alb)≈ M K( eapp× Alb)≈ 22 .
e E L b b
Keshbg M
= + + −
+
( )
( )
1 1
2
b K SHBG E L M
K M
e shbg
e
= − shbg+ + +
× +
( ) ( )( )
( )
1 1
2 1
t T a a
Ktshbg N
= + −
+
2
1
( )
a K SHBG T N
K N
t shbg
t
= shbg− + +
× +
( ) ( )
( )
1
2 1
T BA' = ×t (N +1); E BA' = ×e (M+1);
E BA E L b b M
Keshbg
' ( ( ) )
= + +1 2 − × +1
T BA T a a N
Ktshbg
' ( )
= + 2 − × +1
Ktshbg = 9 8 10, × 8m−1 Keshbg = 5 0 10, × 8m−1 Ktapp(ntalb× Ktalb)≅ Keapp(nealb × Kealb)≅ 3 6 10, × 4m−1
The calculations of free testosterone fraction from total testosterone and SHBG with this method were recently validated by some researches (Vermeulen et al. 1999), who had also shown that these calculations reliably reflect also the bioavailable testosterone fraction;
although due to the presence of lipids the relations between bioavailable and free fractions (N + ≅1 23 might be closer to 20. Hence, after the calculations of free testosterone, this ) factor (20) was used for calculations to obtain the values of bioavailable testosterone fraction.
Although calculations of free or bioavailable estradiol were concluded to be the alternative method to direct measurements of these fractions (Södergard et al. 1982), to our knowledge no validation of these calculations have been present in the recently available literature.
Comparison of calculations with measured and with fixed albumin values As it has been shown that albumin concentrations within the physiological range of 40-50 g/L ( 5 8 7 2 10, − , × −4mol L/ ) does not significantly affect free testosterone values (Vermeulen et al. 1999), we compared calculations performed with measured and with fixed albumin 43g/L (Alb= 6 2 10, × −4mol L/ ) concentrations.
Although albumin concentrations in our study varied widely and were on average in the low part of these ranges, there were high associations between calculated with measured and with fixed albumin concentrations of free testosterone (Rspearman=0,948; p<0,001; Fig. 4), of bioavailable testosterone (Rspearman=0,931; p<0,001) and of bioavailable estradiol (Rspearman=0,939; p<0,001) values.
y = 1 ,0 7 6 7 x - 1 4 ,5 3 7 R2 = 0 ,8 7 2 7
0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0
0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0
c a lc u la te d w ith fix e d a lb u m in fre e te s to s te ro n e (p m o l/l)
calculated with measured albumin free testosterone (pmol/l)
Fig. 4. Calculated with measured vs. with fixed albumin free testosterone.
