Disertasi German

Aus der Abteilung fr Gastroenterologie, Hepatologie und Endokrinologie
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.

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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-Rdiger Klpmann
Korreferent: Professor Dr. Christian-Georg Stief

Tag der mndlichen Prfung: 24.06.2003

Promotionsausschussmitglieder:
Professor Dr. Michael Peter Manns
Professor Dr. Marion Haubitz
Professor Dr. Arnold Ganser

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my parents
my children
my wife

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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
Insulin-like growth factor-I................................................................................................ 57
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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
Effect of SHBG, BMI and other factors ............................................................................. 69
The cause and the clinical importance of testosterone changes with age ......................... 70
Estrogens.............................................................................................................................. 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
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 Verffentlichungen............................................................. 95
Erklrung nach 2 Abs. 2. Nrn. 5 und 6 .......................................................................... 96

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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 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=394). ............................. 30
Fig. 8. Total testosterone and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=512). .............................. 32
Fig. 9. Free testosterone and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=380). ............................... 33
Fig. 10. Bioavailable testosterone and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=380). ................ 33
Fig. 11. Total estradiol distribution and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=504)............... 35
Fig. 12. Bioavailable estradiol and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=375). ..................... 36
Fig. 13. Estrone distribution and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=391). ......................... 36
Fig. 14. Distribution of IGF-I and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
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
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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. RIAs 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

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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 (meanse) were 91% for
SHBG, -101% for total testosterone, -171% for bioavailable testosterone, -171% for free
testosterone, -71% for total estradiol, -121% for bioavailable estradiol, -41% for estrone
and -101% 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
estradiol and IGF-I increased in our study with advancing age.
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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.
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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 ER and ER
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);
however, estrogen receptors were also found in muscle and hair follicles (Sharpe 1998,
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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
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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 17-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 17-hydroxysteroid dehydrogenase
determines the amount of the active estradiol metabolised to biologically inactive estrone
(Rommerts 1998), which again could be converted to 17-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.
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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 ER and ER (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 ER or ER 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 5-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).

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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
A
E2 E2
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
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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 (Sdergard 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 10
4
10
5

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 (Sdergard 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 (Sdergard 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
__________________________________________________________________________________________
_______________________________________________________________________________________ 16
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 (Sdergard 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 (Sdergard 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
increased prevalence of impotence (Kaufman and Vermeulen 1998).
__________________________________________________________________________________________
_______________________________________________________________________________________ 17
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
__________________________________________________________________________________________
_______________________________________________________________________________________ 18
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 =-0,40,1%/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
Vermeulen et al. 1996 age-trend/year =-1,2%/yr between 25-100 yrs
Albumin-bound testosterone
Bioavailable testosterone
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
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
(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
relation between this capacity and bound to SHBG hormone fraction. As is the case for
__________________________________________________________________________________________
_______________________________________________________________________________________ 19
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.
Barrett-Connor and Khaw 1987 increase after 40 yrs (age-trend not reported)
Gray et al. 1991 age-trend/year =1,20,1%/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 1940s (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-
__________________________________________________________________________________________
_______________________________________________________________________________________ 20
receptors, mostly to IGF-Rs 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.
Yamamoto et al. 1991 Decrease 2,36g/l/yr between 21-80 yrs
Juul et al. 1994 Decrease (IGF-I)= -0,11 g/l/yr between 20-80 yrs
Landin-Wilhelmsen et al. 1994 Decrease 2,1 g/l/yr between 25-64 yrs
Nystrm et al. 1997 Decrease 3,281g/l/yr between 20-69 yrs
OConnor et al. 1998 Decrease 1,972 ng/ml/yr between 20-90 yrs
Hilding et al. 1999 Decrease lg(IGF-I)=-0,00647g/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)

__________________________________________________________________________________________
_______________________________________________________________________________________ 21
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).

__________________________________________________________________________________________
_______________________________________________________________________________________ 22
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)
B
M
I

(
k
g
/
m
)
3 mean SD
Exclusion for obesity
3 mean SD

Fig. 3. Exclusion for BMI in the study.

It must be noted that age-adjusted BMIs of our subjects were grossly similar to the BMIs
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, Nystrm 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).
__________________________________________________________________________________________
_______________________________________________________________________________________ 23
It is methodologically important that although BMI is probably the most useful scale to define
obesity (Bjrntorp 1997), no consensus about the appropriateness of the different cut-off
points has been reached (Molarius and Seidell 1998). Body mass index (weight/height), 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/m, 289 men (56%)
between 20,0-24,9 kg/m, 195 men (38%) between 25,0-29,9 kg/m and 14 men (3%) with a
BMI of up 30 kg/m. The mean BMI was 24,6 2,6 kg/m.

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 4C. 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 RIAs (Tabl. 4).
Table 4. RIAs 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/ml3,47=pmol/l; SHBG: ng/L11,765=nmol/l; estradiol
and estrone: pg/ml3,7=pmol/l; IGF-I: ng/ml/7,649=nmol/l.
__________________________________________________________________________________________
_______________________________________________________________________________________ 24
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 (Sdergard 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 2
1 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.
n n
si
shbg
si
alb
, - number of binding sites for steroid Si on each molecule of SHBG or albumin.
K K
si
shbg
si
alb
, - 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. (Sdergard et al. 1982).
As in all cases of hormone binding to albumin the term K Si
si
alb
is much smaller than unity,
it can be omitted from the denominator (Sdergard et al. 1982). Thus, in general:

Si
n K shbg Si
K S K S K Sn
n K Alb Si Si
si
shbg
Si
shbg
S
shbg
S
shbg
Sn
shbg Si
alb
Si
alb
=

+ + + +
+ +
1 1 2
1 2
...
;

Five ligands: testosterone, estradiol, 5a-dihydrotestosterone, 5-androstene-3,17-diol, 5a-
androstane-3a,17-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 (Sdergard et al. 1982).
__________________________________________________________________________________________
_______________________________________________________________________________________ 25
SHBG binding capacity, SHBG n shbg
si
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 ( K n K
t
app
t
alb
t
alb
= ,
K n K
e
app
e
alb
e
alb
= ) can be determined experimentally.
Thus, after transformation we have the following second degree equations:

N n K Alb
t
alb
t
alb
= ; M n K Alb
e
alb
e
alb
= L K t
t
shbg
=
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:

TBA, EBA - 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
( 58 7 2 10
4
, , /

mol 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 mol L =

6 2 10
4
, / ) concentrations, where:
N K Alb M K Alb
t
app
e
app
( ) ( ) 22 .
e
E L b b
K M
e
shbg
=
+ +
+
( )
( )
1
1
2
b
K SHBG E L M
K M
e
shbg
e
shbg
=
+ + +
+
( ) ( )( )
( )
1 1
2 1
t
T a a
K N
t
shbg
=
+
+
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
K
e
shbg
'
( ( ) )
=
+ + + 1 1
2
T BA
T a a N
K
t
shbg
'
( )
=
+ +
2
1
K m
t
shbg
=

9 8 10
8 1
, K m
e
shbg
=

5 0 10
8 1
,
K n K K n K m
t
app
t
alb
t
alb
e
app
e
alb
e
alb
( ) ( ) ,

3 6 10
4 1
__________________________________________________________________________________________
_______________________________________________________________________________________ 26
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 (Sdergard 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
( 58 7 2 10
4
, , /

mol 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 mol L =

6 2 10
4
, / ) 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 = 1,0767x - 14,537
R
2
= 0,8727
0
200
400
600
800
1000
1200
1400
0 200 400 600 800 1000 1200
calculated with fixed albumin free testosterone (pmol/l)
c
a
l
c
u
l
a
t
e
d

w
i
t
h

m
e
a
s
u
r
e
d

a
l
b
u
m
i
n

f
r
e
e

t
e
s
t
o
s
t
e
r
o
n
e

(
p
m
o
l
/
l
)

Fig. 4. Calculated with measured vs. with fixed albumin free testosterone.
__________________________________________________________________________________________
_______________________________________________________________________________________ 27
Comparison of measured and calculated free testosterone.
Free testosterone measured in our study by a-ligand radioimmunoassay represented only 12%
of calculated (from total testosterone and SHBG) free testosterone levels and was much lower
than any levels obtained by other methods (Fig. 5). The ratio of measured to calculated free
testosterone as well as a FAI, free androgen index, were subjects of SHBG variability (Fig. 6;
the data for FAI not represented). If calculated free testosterone is indeed a reliable parameter
of free testosterone fraction, measured by a-ligand radioimmunoassay free testosterone and
free androgen index are probably not reliable indices of free testosterone activity (Vermeulen
et al. 1999). The data for a-ligand radioimmunoassay free testosterone and FAI will, therefore,
further not be represented.

y = 0,1195x + 20,261
R
2
= 0,4866
0
20
40
60
80
100
120
140
160
180
0 200 400 600 800 1000 1200
calculated free testosterone (pmol/l)
a
-
l
i
g
a
n
d

f
r
e
e

t
e
s
t
o
s
t
e
r
o
n
e

(
p
m
o
l
/
l
)

Fig. 5. Measured free testosterone vs. calculated free testosterone.
y = 0,0022x + 0,0799
R
2
= 0,5273
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0 20 40 60 80 100 120 140 160
SHBG (nmol/l)
R
a
t
i
o

a
-
l
i
g
a
n

t
o

c
a
l
c
u
l
a
t
e
d

f
r
e
e

t
e
s
t
o
s
t
e
r
o
n
e

Fig. 6. Ratio of measured to calculated free testosterone vs. SHBG.
__________________________________________________________________________________________
_______________________________________________________________________________________ 28
Analysis of our cross-sectional investigation
The normality of distribution of serum hormone levels was checked by a Kolmogorov-
Smirnov test. In most cases the tabulated distribution was mildly skewed toward the high
values and in all cases ln-transformation increased the normality of distribution of the data.
All of the assessed associations were visually inspected before the analysis. Spearman
correlation coefficient was used without any assumption about the normality of distribution of
the variables, and Pearson correlation coefficients for simple correlation and partial
correlation were used on ln-transformed hormone levels (Gray et al. 1991).
To allow the maximum flexibility in describing age trends, subjects were stratified into 10
year-interval groups. Medians and means of serum hormone concentrations as well as
standard deviations and variation coefficients SD% were calculated. The 5
th
, 25
th
, 50
th
, 75
th

and 95
th
percentiles for age-stratified groups were calculated and plotted, but, because of the
limited number of men over 50 years, the last age group plotted, except of SHBG, covered all
men over 50 years (mean age 57,4 years).
To reveal the differences of the means between stratified age-groups, ANOVA-model with
Post-Hoc Bonferroni test on ln-transformed data was used for the analysis. Logarithmically
transformed data showed in contrast to non-transformed data the homogeneity of variances.
Simple and multivariate linear regression models on ln-logarithmically transformed hormone
data was employed to evaluate possible effects of age, BMI and other parameter on serum
hormone concentrations. The distribution of the ln-transformed data about fitted regression
models was virtually normal in many cases and in no case the distribution of residuals was
severely asymmetrical, as was shown by Durbin-Watson statistic.
The age-related changes in serum hormone concentrations were analysed using a typical log-
linear regression model based on the following general equation (Belanger et al. 1994):
Ln[hormone parameter] = A +B*age:
The percent rate of change between the ages x1 and x2, its standard error and pooled residual
variability were estimated as followed (Gray et al. 1991):
% / ( ) ( )
( )
change x x e
b x x
2 1 1 100%
2 1
=

[ ]
SE change x x e
se b x x
(% / ( )) ( )
( )
2 1 1 100%
2 1
=

CV e
sd
% ( )
#
= 1 100%
where b and se are b-coefficient of the regression and its error and sd# - is the residual SD
about the fitted regression model for ln(y).
Some other functions (inverse, quadrate and cubic) were also tested for the best-fitting model.
However, as any of them lead to any substantial increase in the explained total variability of
the variables studied; therefore, equations of these functions will not be represented.
__________________________________________________________________________________________
_______________________________________________________________________________________ 29
Analysis of the data from previously reported studies
To compare our study with existed data and to summarise research evidence about age-related
changes of SHBG, testosterone and estrogen fractions as well as of IGF-I in adult men,
relevant studies reporting these parameters and their changes with age were selected from
Medline. The search strategy was a combination of index terms testosterone, estrogens,
sex-hormone-binding-globulin and insulin-like growth factor I, restricted to aging,
human and (adult or middle-aged), all index-terms in MESH.
Studies were included in the analysis if the following criterias were met: 1) persons in more
than 30 years age-intervals were investigated, 2) data for about 30 and more subjects pro age-
decade were reported (for IGF-I for more than 10 subjects pro decade as only few studies with
stronger criterias were available) and 3) such parameters as mean age-trends, mean hormone
changes for age-interval studied or average (mean or median) hormone values for more than
three decades of age were given.
All original data from the papers were transformed in mol-units. Coefficients of variation
(SD%) and 95%CI of mean levels for age-groups were calculated (if possible) and plotted. In
the case that mean (or median) free or bioavailable testosterone (or estradiol) concentrations
for age-groups were not reported, these levels were calculated from the reported average
SHBG and total testosterone (and estradiol) concentrations (s. above). For some studies the
average IGF-I levels for age-groups were estimated from the regression equations.
Plots were visually inspected and the conclusion about the appropriateness of the exponential
model of age-related changes has been made. B-coefficients of exponential regression and the
percentage of hormone changes per decade were calculated from the regression of the mean
or median hormone concentrations, when given or estimated, or from hormone changes pro
age-intervals reported by the authors:
b = Ln(percent of changes/100+1)/age-interval.
As the distribution of investigated parameters is skewed, regression from the mean
concentrations can sometimes be misleading. However, analysing the data from studies,
where both mean and median data were available, only small differences in regression slopes
of these two models were found (s. Discussion).
As studies showed very heterogeneous findings, no statistical combination was employed.
The final conclusions were made after analysing the studies with regard to their validity to
estimate the mean age-trends of the hormones changes.

Computations were performed using program Microsoft Excel and statistical software
package SPSS (Chicago, IL, USA). All statistical tests were performed at a 5% two-tailed
level of confidence.
__________________________________________________________________________________________
_______________________________________________________________________________________ 30
RESULTS I Analysis of our investigation on healthy subjects
Sex Hormone Binding Globulin
The distribution of SHBG serum levels was mildly positively skewed and logarithmical
transformation of the data increased the normality of distribution. Similar pattern of SHBG
distribution was also seen in all decades of age.
SHBG serum levels of our subjects were weakly but significantly positively associated with
age (Rspearman=0,235; p<0,001; R(lnSHBG)=0,243; p<0,001). A slight age-related increase
in mean and median levels was accompanied by an increase in all percentiles plotted (Fig.7,
Tabl. 5). However, in ANOVA-model on ln-transformed data this increase was significant
only after the 5
th
decade of age (Post Hoc test Bonferroni, p<0,05). SHBG levels varied
widely in all investigated age-groups: SD%=34-41%.
0
50
100
150
20 30 40 50 60 70
Age (years)
S
H
B
G

(
n
m
o
l
/
L
)

Fig. 7. SHBG distribution and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
percentiles (n=394).
Table 5. Median, mean SD and ranges of SHBG serum levels (n=394).
Age group (N) 20-29 (80) 30-39 (124) 40-49 (101) 50-59 (58) 60-72 (31)
Median (nmol/l) 44,2 A 43,0 A 47,5 A 51,0 AB 57,8 B
Mean SD (nmol/l) 45,4 17,0 45,9 18,7 51,5 20,6 52,5 18,1 66,6 27,3
Range (nmol/l) 18,3114,1 12,9119,5 14,6-108,6 21,1-109,2 36,5-147,8
Log-transformed data with the same letter do not differ at p<0,05 (Bonferroni).

The increase of SHBG level with age could be approximated by an exponential function. The
mean age-trend of SHBG se, calculated from b- regression coefficient for age and its se
(0,0084 0,001), was 9 1% pro decade (p<0,001 for model). However, age in this model
could explain only a small percentage of SHBG total variability (R=0,06) and calculated
from the residual sd about the fitted regression model pooled residual variability was
CV%=48%.
Rspearman = 0,235
Age-trend = 91%/decade
__________________________________________________________________________________________
_______________________________________________________________________________________ 31
Apart from linear regression model of age on ln-transformed SHBG levels (exponential
changes of SHBG with age) some other functions (quadratic, cubic and inverse) were also
tested for best-fitted regression model. But no one of these functions could meaningfully
increase the percentage of explained SHBG variability.

SHBG level was weakly but significantly associated with BMI (R(lnSHBG)=-0,161; p<0,001;
Rspearman=-0,134, p<0,001), with IGF-1 (R(lnSHBG)=-0,216, p<0,001; Rspearman=-0,248,
p<0,001), but not with baE2/baT ratio (p>0,05) (Tabl. 6).
In multiple stepwise linear regression model of age, BMI, IGF-I and baE2/baT ratio on SHBG
BMI was the second independent variable after age included in the model and increased the
percentage of explained SHBG total variability (R=0,14 vs. R=0,06; p<0,001 for model).
SHBG in this model showed a stronger partial association with BMI (Rpartial=-0,294,
p<0,001), as well as with age (Rpartial=0,343; p<0,001) in comparison to unadjusted
correlations.
Inclusion of IGF-1 in multiple regression model had a significant but not meaningful
(R=0,15 vs. R=0,14; p<0,001 for model) effect on the percentage of explained SHBG total
variability and the adjusted association of IGF-I with SHBG was very weak (Rpartial=-0,105,
p<0,05). The baE2/baT ratio had no significant impact on SHBG variability in this model
(p>0,05).
Table 6. The association of SHBG level with age, BMI, IGF-1 and the ratio baE2/baT.
Age BMI IGF-1 baE/baT R
R (lnSHBG) 0,243*** -0,161** -0,216*** NS -
Rspearman 0,235*** -0,134** -0,248*** NS -
Stepwise linear regression
R (lnSHBG) 0,243*** - - - 0,06***
Rpartial (lnSHBG) 0,343*** -0,294*** - - 0,14***
Rpartial (lnSHBG) 0,270*** -0,289*** -0,105* NS 0,15***
*p<0,05, **p<0,01; ***p<0,001; NS - non-significant.
__________________________________________________________________________________________
_______________________________________________________________________________________ 32
Testosterone
The distribution of serum levels of free, bioavailable and total testosterone fractions was
mildly positively skewed and logarithmical transformation of the data increased the normality
of distribution. Similar pattern of distribution was seen in most decades of age.
Serum levels of total, free and bioavailable testosterone fractions of our subjects were
moderately and significantly negatively associated with age (Rspearman=-0,414, -0,498 and -
0,503; R(ln)=-0,410, -0,521 and -0,511, respectively; p<0,001 in all cases). The age-related
decrease in mean and median levels was accompanied by decline in all percentiles plotted
(Fig. 8-10, Tabl. 7-9). In ANOVA-model on ln-transformed data this decrease was significant
practically between all decades of age (Post Hoc test Bonferroni, p<0,05). The variability
(SD%) of total, free and bioavailable testosterone was 26-28%, 27-43% and 34-38%,
respectively.

Fig. 8. Total testosterone and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
Table 7. Median, mean SD and ranges of total testosterone levels (n=512).
Age group (N) 20-29 (111) 30-39 (169) 40-49 (126) 50-59 (69) 60-72 (37)
Median (nmol/l) 21,9 A 20,2 B 17,8 C 16,7 CD 15,2 D
Mean SD (nmol/l) 22,4 6,0 20,5 5,8 18,1 4,6 16,8 4,4 15,3 4,0
Range (nmol/l) 11,3-41,5 9,0-44,8 8,6-34,4 7,3-28,1 7,6-26,4

The age-related decrease of total, free and bioavailable testosterone fractions could be well
approximated by the exponential functions. The mean age-trends ses, calculated from b-
regression coefficients for age and their ses (-0,0104 0,001, -0,0188 0,001 and 0,0187
0,001), were -10 1%, -17 1% and -17 1% pro decade for these testosterone fractions,
respectively (p<0,001 for all models).
Rspearman = -0,414
Age-trend = -10%/decade
0
10
20
30
40
20 30 40 50 60 70
Age (years)
t
o
t
a
l

t
e
s
t
o
s
t
e
r
o
n
e

(
n
m
o
l
/
L
)
__________________________________________________________________________________________
_______________________________________________________________________________________ 33

Fig. 9. Free testosterone and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
Table 8. Median, mean SD and ranges of free testosterone levels (n=380).
Age group (N) 20-29 (76) 30-39 (118) 40-49 (99) 50-59 (57) 60-72 (30)
Median (pmol/l) 414,9 A 345,9 A 302,8 B 271,9 B 187,4 C
Mean SD (pmol/l) 446,2 182,0 396,9 169,2 308,3 96,5 270,7 74,1 212,5 77,7
Range (nmol/l) 165,1-1207,1 122,5-1125,7 113,5-591,2 127,5-418,1 114,0-396,9

Fig. 10. Bioavailable testosterone and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
Table 9. Median, mean SD and ranges of bioavailable testosterone levels (n=380).
Age group (N) 20-29 (76) 30-39 (118) 40-49 (99) 50-59 (57) over 60 (30)
Median (nmol/l) 9,19 A 7,77 B 6,47 C 5,66 CD 4,24 D
Mean SD (nmol/l) 9,78 3,74 8,44 3,14 6,86 2,32 5,85 1,96 4,66 1,65
Range (nmol/l) 4,17-23,35 3,04-18,25 2,4-15,47 1,84-10,22 2,42-8,07
Rspearman = -0,503
Rspearman = -0,498
0
200
400
600
800
20 30 40 50 60 70
Age (years)
f
r
e
e

t
e
s
t
o
s
t
e
r
o
n
e

(
p
m
o
l
/
L
)
0
5
10
15
20
20 30 40 50 60 70
Age (years)
b
i
o
a
v
a
i
l
a
b
l
e

t
e
s
t
o
s
t
e
r
o
n
e

(
n
m
o
l
/
L
)
__________________________________________________________________________________________
_______________________________________________________________________________________ 34
In these models, however, age could explain only a small percentage of total, free and
bioavailable testosterone total variability (R=0,17, 0,27 and 0,26, respectively) and calculated
from the residual sds about the fitted regression models pooled residual variabilities were
CV%= 31%, 43% and 44%, respectively for these testosterone fractions.
Apart from linear regression models of age on ln-transformed levels of all testosterone
fractions (exponential changes of untransformed levels with age) some other functions
(quadratic, cubic and inverse) were also tested for best-fitted regression models. But no one of
these functions could meaningfully increase the percentage of explained variability for any
testosterone fraction.

The prevalence of persons with low total, free or bioavailable testosterone levels increased
with age and 9%, 13% and 26% of the persons between the ages of 50-70 had total, free or
bioavailable testosterone levels, respectively, below the lowest values (11 nmol/l, 0,16 nmol/l
and 4 nmol/l, respectively) seen in young adults, aged between 20-29 years (Fig. 15).

Total testosterone was positively albeit weakly associated with SHBG (Rspearman=0,176,
p<0,01 R(lnTotalTestosterone)=0,210, p<0,01). This association increased when age was
taken into account (Rpartial=0,320, p<0,001) and linear regression model of age and SHBG
on total testosterone level increased the percentage of explained total testosterone variability.

Total testosterone inversely correlated with BMI (Rspearman=-0,243, p<0,01; r(ln)=-0,248,
p<0,01), but there was no significant association of free and only marginally significant of
bioavailable testosterone fractions with body-mass-index (Rspearman=-0,071, p=0,169;
R(ln)=-0,059, p=0,255 and Rspearman=-0,115, p=0,025; R(ln)=-0,102, p=0,047,
respectively). Inclusion of BMI in multiple linear regression analysis of age on total, free or
bioavailable testosterone fractions increased significantly but not meaningfully the percentage
of explained variability of these fractions (R=0,18 vs.R=0,17; R=0,29 vs. R=0,27 and
R=0,27 vs. R=0,26, respectively) compared to univariate regression models.
Adjusted for age in these models associations of total, free and bioavailable testosterone with
BMI (Rpartial= -0,097, 0,198 and 0,136, respectively; p<0,05) and adjusted for BMI
associations of these fractions with age (Rpartial= -0,349, -0,546 and 0,517, respectively;
p<0,001) remained similar to the associations in unadjusted regression models.

__________________________________________________________________________________________
_______________________________________________________________________________________ 35
Estrogens
The distribution of serum levels of total and bioavailable estradiol fractions and distribution
of serum estrone levels was mildly positively skewed and logarithmical transformation of the
data increased the normality of distribution. Such pattern of distribution of these variables was
only seen in a few decades of age.
Serum levels of total and bioavailable estradiol fractions of our subjects were moderately and
significantly negatively associated with age (Rspearman=-0,349, -0,412; R(ln)=-0,364, -
0,438, respectively; p<0,001 in both cases). An age-related decrease in mean and median
levels was also seen in all percentiles plotted (Fig. 11-12; Tables 10-11). In ANOVA-model
this decrease was significant between each two decades of age (Post Hoc test Bonferroni,
p<0,05). Estrone was only marginally associated with age (Rspearman=-0,098, p=0,054;
R(ln)=-0,107, p=0,034; Fig. 13, Tabl. 12). The variability (SD%) of total estradiol,
bioavailable estradiol and estrone was 19-23%, 25-35% and 37-42%, respectively.

Fig. 11. Total estradiol distribution and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
Table 10. Median, mean SD and ranges of total estradiol levels (n=512).
Age group (N) 20-29 (109) 30-39 (162) 40-49 (127) 50-59 (69) 60-79 (37)
Median (nmol/l) 98,8 A 92,8 B 90,7 B 80,3 C 78,0 C
Mean SD (nmol/l) 102,8 23,4 94,5 18,2 90,8 18,4 80,9 16,1 78,2 16,2
Range (nmol/l) 51,0-190,2 45,9-140,7 48,2-176,1 51,7-122,5 48,8-112,8

The age-related decrease of total, bioavailable estradiol fractions and estrone could be
approximated by the exponential functions. The mean age-trends ses, calculated from b-
regression coefficients for age and their ses (-0,0070 0,001, -0,0132 0,001 and 0,0038
0,002), were -7 1%, -12 1% and -4 2% pro decade for these estrogen fractions,
respectively (p<0,001 for both estradiol fractions, p=0,034 for estrone).
Rspearman = -0,349
40
70
100
130
160
20 30 40 50 60 70
Age (years)
t
o
t
a
l

e
s
t
r
a
d
i
o
l

(
p
m
o
l
/
L
)
__________________________________________________________________________________________
_______________________________________________________________________________________ 36

Fig. 12. Bioavailable estradiol and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
Table 11. Median, mean SD and ranges of bioavailable estradiol levels (n=375).
Age group (N) 20-29 (74) 30-39 (115) 40-49 (99) 50-59 (57) 60-72 (30)
Median (nmol/l) 55,3 A 53,4 AB 48,5 BC 41,3 CD 32,4 D
Mean SD (nmol/l) 61,6 21,5 55,3 17,2 49,6 13,6 42,4 12,6 34,5 8,7
Range (nmol/l) 27,5-134,0 20,1-102,4 18,6-93,8 11,4-81,7 14,2-51,8

Fig. 13. Estrone distribution and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
Table 12. Median, mean SD and ranges of estrone levels (n=391).
Age group (N) 20-29 (79) 30-39 (123) 40-49 (100) 50-59 (58) 60-72 (31)
Median (nmol/l) 134,7 A 126,5 A 119,3 A 119,7 A 116,2 A
Mean SD (nmol/l) 144,1 56,2 134,4 55,1 138,3 58,7 122,6 45,2 123,7 51,6
Range (nmol/l) 35,2-333,4 26,3-328,2 47,0-373,0 30,7-240,1 35,2-239,0
Rspearman = -0,412
Age-trend = -12/decade
0
30
60
90
120
20 30 40 50 60 70
Age (years)
b
i
o
a
v
a
i
l
a
b
l
e

e
s
t
r
a
d
i
o
l

(
p
m
o
l
/
L
)
0
100
200
300
20 30 40 50 60 70
Age (years)
e
s
t
r
o
n
e

(
p
m
o
l
/
L
)
__________________________________________________________________________________________
_______________________________________________________________________________________ 37
In these models, however, age could explain only a small percentage of total and bioavailable
estradiol and estrone total variability (R= 0,13, 0,19 and 0,01, respectively) and calculated
from the residual sds about the fitted regression models pooled residual variabilities were
CV%= 23%, 37% and 52%, respectively for these estrogen fractions.
Apart from linear regression models of age on ln-transformed levels of estrogen fractions
(exponential changes of untransformed levels with age) some other functions (quadratic,
cubic and inverse) were also tested for best-fitted regression models. But no one of these
functions could meaningfully increase the percentage of explained variability of any estrogen
fraction.

The prevalence of persons with low bioavailable but not total estradiol or estrone levels
increased with age and 16% of the persons between the ages of 50-70 had bioavailable
estradiol levels below the lowest values (28pmol/l) seen in young adults, aged between 20-29
years (Fig. 15).
All estradiol fractions and estrone were age-independently associated with all testosterone
fractions (Rpartial: TT/E2= 0,384; TT/BAE= 0,169; BAT/E2= 0,355; BAT/BAE= 0,720;
TT/E1=0,191; p<0,001 in all cases; BAT/E1=0,128; p<0,05). There was also an age-
independent association between all estrogen fractions (Rpartial: E2/E1=0,551; E2/BAE=
0,754; E1/BAE=0,435; p<0,001 in all cases).

No any significant unadjusted association of serum total, bioavailable estradiol or estrone
levels with BMI was found (p>0,05). Inclusion of BMI in multiple linear regression analysis
of age on total estradiol and estrone levels increased significantly but not meaningfully the
percentage of explained variability of these fractions (R=0,15 vs. R=0,13; R=0,03 vs.
R=0,01, respectively) compared to univariate regression models. Adjusted for age in these
models BMI was weakly but significantly associated with total estradiol and estrone levels
(Rpartial= 0,148 and 0,153, respectively; p<0,001 both); adjusting for BMI increased the
association of these hormone fractions with age (Rpartial= -0,389 and -0,161, respectively;
p<0,001 both).
Inclusion of BMI in multiple linear regression model of age on bioavailable estradiol levels
increased the percentage of explained bioavailable estradiol total variability (R=0,25 vs.
R=0,19). In this model bioavailable estradiol was more associated with BMI (Rpartial=0,279,
p<0,001) as well as with age (Rpartial=-0,503, p<0,001) compared to univariate regression.
Calculated from the b-coefficient for age for this regression model (adjusted for BMI) the
mean age-trend of bioavailable estradiol was -16%/decade.
__________________________________________________________________________________________
_______________________________________________________________________________________ 38
Insulin-like growth factor I
The distribution of IGF-I serum levels was slightly positively skewed and logarithmical
transformation of the data increased the normality of distribution. Such pattern of distribution
of IGF-I serum levels was seen in all decades of age.
IGF-I serum levels of our subjects were moderately and significantly negatively associated
with age (Rspearman=-0,470; p<0,001; R(lnIGF-I)=-0,463; p<0,001). An age-related
decrease in mean and median levels was accompanied by parallel decline in all percentiles
plotted (Fig. 14, Tabl 13). In ANOVA-model this decrease was significant practically
between all decades of age (Post Hoc test Bonferroni, p<0,05). IGF-I levels varied moderately
in all investigated age-groups: SD%=23-27%.
0
10
20
30
40
50
20 30 40 50 60 70
Age (years)
I
G
F
-
1

(
n
m
o
l
/
L
)

Fig. 14. Distribution of IGF-I and its 5
th
, 25
th
, 50
th
, 75
th
and 95
th
Table 13. Median, mean SD and ranges of IGF-I serum levels (n=478).
Age group (N) 20-29 (104) 30-39 (153) 40-49 (119) 50-59 (67) 60-72 (35)
Median (nmol/l) 28,6 A 23,9 B 22,4 BC 20,1 CD 18,6 D
Mean SD (nmol/l) 29,3 6,7 24,3 6,0 22,6 5,4 20,7 4,8 19,6 5,4
Range (nmol/l) 16,2-52,8 11,6-51,9 11,4-44,5 12,4-32,8 11,2-32,8

The decrease of IGF-I level with age could be approximated by the exponential function. The
mean age-trend of IGF-I se, calculated from b- regression coefficient for age and its se (-
0,0108 0,001), was -10 1% pro decade (p<0,001 for model). Age in this model could
explain a small percentage of IGF-I total variability (R=0,22) and calculated from the
residual sd about the fitted regression model pooled residual variability was CV%=27%. The
age-related decrease of IGF-I level could also be approximated by the linear function with b-
regression coefficient for age and its se 2,60,2 nmol/l/decade (p<0,001 for model). Age in
this model could also explain only a small percentage of IGF-I total variability (R=0,21).
Rspearman = -0,470
__________________________________________________________________________________________
_______________________________________________________________________________________ 39
Apart from linear regression model of age on untransformed and ln-transformed IGF-I serum
levels (linear and exponential changes of IGF-I with age) some other functions (quadratic,
cubic and inverse) were also tested for best-fitted regression model. However, no one of these
functions could meaningfully increase the percentage of explained IGF-I or ln(IGF-I) total
variability (maximal change in R=0,02).

IGF-I was positively correlated with height (Rspearman=0,177; p<0,001; r(ln(IGF-I)=0,169,
p<0,001) and inversely correlated with BMI (Rspearman=-0,125, p<0,001; r(ln(IGF-I))=-
0,126, p<0,001), but these associations disappeared when age was allowed for in multiple
regression. Only age remained a significant predictor in this model.

Bioavailable testosterone and estradiol fractions were closely associated with IGF-I compared
to total hormone levels and these interrelations remained closer than associations of total
hormones also after adjustment for age and body-mass-index (Tabl. 14). The multiple linear
regression analysis included age, BMI, total as well as bioavailable testosterone and estradiol
fractions revealed that only age and bioavailable estradiol remained independent predictors of
IGF-I serum levels. However, only a small increase in percentage of explained total IGF-I
variability (R = 0,24 vs. 0,22) was observed in this model compared to the univariate analysis
between IGF-I serum levels and age.
Table 14. Association between serum IGF-I and sex-hormones fractions in male donors.
n Simple /Age /Age/BMI
Total testosterone 477 0,209*** NS NS
Bioavailable testosterone 368 0,349*** 0,152** 0,144**
Total estradiol 469 0,262*** 0,108* 0,099*
Bioavailable estradiol 363 0,324*** 0,150** 0,140**
Rpearson and Rpartial for multiple regression. All data are ln-transformed. Estrone no significant association
*p<0,05, **p<0,01; ***p<0,001; NS - non-significant.

The prevalence of persons with low IGF-I levels increased with age and 19% of the persons
between the ages of 50-70 had IGF-I levels below the lowest values (16 nmol/l) seen in young
adults, aged between 20-29 years (Fig. 16).
The prevalence of subjects with simultaneously low IGF-I and other hormone fraction also
increased with age: 3%, 5%, 8% and 2% of the persons between the ages of 50-70 had low
IGF-I simultaneously with total or free testosterone or bioavailable testosterone or
bioavailable estradiol, respectively (below the lowest values seen in young adults, aged 20-29
years). In addition, 2% of the persons had simultaneously low IGF-I with other two hormone
parameters: bioavailable testosterone and bioavailable estradiol levels (Fig. 16).

__________________________________________________________________________________________
_______________________________________________________________________________________ 40
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.
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.
7%
0% 0% 0% 0% 0%
11%
0%
2%
1% 1% 1%
16%
2% 2%
4%
0% 0%
23%
6%
10%
17%
7% 7%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
IGF-I IGF-I and total
testosterone
IGF-I and free
testosterone
IGF-I and bioavailable
testosterone
estradiol
testosterone and
bioavailable estradiol
30-39 years 40-49 years 50-59 years 60-72 yrs
3%
2%
6%
1%
3%
1%
3%
6%
10%
1%
5%
0%
7%
4%
19%
0%
11%
2%
14%
30%
40%
3%
27%
0%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
total testosterone free testosterone bioavailable
testosterone
total estradiol bioavailable estradiol estrone
30-39 years 40-49 years 50-59 years 60-72 yrs
__________________________________________________________________________________________
_______________________________________________________________________________________ 41
RESULTS II Calculations from previously reported studies
Description of the studies
Cross-sectional studies on SHBG and sex-hormone changes with age
Identified cross-sectional studies on changes of SHBG and sex-hormones with age (studies
with age-interval over 30 years, with 30 or more subjects pro age-decade, inpatient persons
excluded) and their main characteristics are summarised in the tables 15-17. Although many
studies were performed on population-based samples, selection of relatively healthy subjects
in these studies because of strong inclusion criteria (especially in Rochester study) or of low
response rates was programmed or cannot be excluded, respectively (Tabl. 17). In two studies
reported storage time was very long; however, for many studies data on storage of SHBG and
hormones has not been reported by the authors.

Table 15. Identified cross-sectional studies reporting changes of sex-hormones with age.
Studies groups and subjects S-size age-range N/year References
Rancho Bernardo I, USA; CHD-free
Rancho Bernardo II, USA; pop-based
pop-based
590
810
856
30-79
24-90*
50-89
11,8
12,1
21,4
Barrett-Connor and Khaw 1987
Ferrini and Barrett-Connor 1998
Barrett-Connor et al. 1999 etc
Massachusetts I, USA; subgroup healthy
subgroup not healthy
pop-based, severe illness excluded
415
1294
1241
39-70
39-70
38-70
12,9
40,4
37,6
Gray et al. 1991
Gray et al. 1991
Field et al, 1994
Massachusetts II, USA; pop-based 1156 50-80 38,5 Feldman et al. 2002
Paris, France; occ-based 1408 20-59 35,2 Simon et al. 1992
Quebec, Canada; pop-based 2423 40-80 59,1 Belanger et al. 1994
Ghent, Belgium, healthy I
healthy II
healthy BMI 20-26
292
365
250
20-89
18->100
25-100
4,2
4,3
3,4
Vermeulen 1991
Vermeulen 1993
Vermeulen et al. 1996
Rochester, USA; pop-based/healthy 346 23-90 5,1 Khosla et al. 1998
Sheffield, UK; healthy 178 20-79 3,0 Fatayerji and Eastell 1999
Hannover, Germany, donors 514 20-72 9,7 OUR DATA
*- only 27 person under 50 years. CHD-coronary heart disease.

Table 16. Parameters measured in the identified cross-sectional studies.
Study groups and subjects SHBG TT FT BAT TE BAE E1
Rancho Bernardo II, USA pop-based
+
-
+
+
-
-
-
+
+
+
-
+
+
-
Massachusetts I, USA; pop-based + + + + + - +
Massachusetts II, USA; pop-based + + + + +
Paris, France; occ-based - + - - + - +
Quebec, Canada; pop-based - + - - + - +
Ghent, Belgium, healthy
healthy BMI 20-26
+
+
+
+
+
+
-
-
-
+
-
-
-
-
Rochester, USA; pop-based/healthy + + a + + + +
Sheffield, UK; healthy + + - - + - -
a - analog-ligand radioimmunoassay.

__________________________________________________________________________________________
_______________________________________________________________________________________ 42
In the Rancho Bernardo I study subjects were 82% of the residents in Rancho Bernardo,
California, USA; a Caucasian upper middle class retirement community; surveyed for heart
disease risk factors of a Lipid Research Clinic Prevalence study. In 1972-1974 plasma was
obtained between 07.00-11.00 h from 12-16h-fasted (95% persons) subjects and storaged at -
70C. Blood was thawed for sex-hormone and SHBG determination in 1985-1986 (storage
time 11-14 yrs). Measured parameters: SHBG (Precipitation by ammonium sulfate, Rosner
1972), total testosterone (RIA), total estradiol (RIA) and estrone (RIA).
In Barrett-Connor and Khaw 1987: analysis of 590 men aged 30-79 yrs without a personal
history of cardiovascular disease. Age-distribution of the subjects: 30-39, n=67; 40-49, n=38;
50-59, n=100; 60-69, n=222; 70-79, n=163. Represented data: mean levels and SDs for age-
groups of all subjects, as well as for subgroups: never smokers, former smokers and current
smokers. Age-trends: SHBG increases after 40 yrs; total testosterone and total estradiol
decrease but estrone increases between 30-79 yrs of ages (age-trends not reported).

In the Rancho Bernardo II study subjects were 82% of the surviving community-dwelling
residents of Rancho Bernardo I study, attended a clinic visit to study diabetes. In 1984-1987
blood was obtained between 7.30-11.00 h from 12h-fasted subjects and storaged at -70C;
Hormone measurements were performed in 1992-1993 (storage time 5-9 yrs). Measured
parameters: total testosterone (RIA), bioavailable testosterone (ammonium sulphate
precipitation, Tremblay and Dube 1974), total estradiol (RIA) and bioavailable estradiol
(ammonium sulphate precipitation, Tremblay and Dube 1974).
In Ferrini and Barrett-Connor 1998: analysis of 810 men aged 24-90 yrs (only 27 persons
below 50), whose data on BMI, waist/hip ratio, current smoking, alcohol use and caffeine
intake were completed. Analyses were adjusted for specimen storage time. Represented data:
Age-trends and figures but any mean hormone concentrations. Age-trends: total and
bioavailable testosterone decreased 1,9 pg/ml/yr (NS) and 18,5 pg/ml/yr (p<0,001) between
24-90 yrs, respectively; total and bioavailable estradiol decreased 0,03 pg/ml/yr (NS) and 0,12
pg/ml/yr (p<0,001) between 24-90 yrs, respectively.
In Barrett-Connor et al. 1999: analysis of 856 ambulatory men aged 50-89 yrs, who were not
using sex hormone therapy, whose sex hormones were measured, and who completed the
Beck Depression Inventory (81% of all age-eligible participants). Age-distribution of the
subjects: 50-59, n=152; 60-69, n=221; 70-79, n=328; 80-89, n=155. Represented data: Mean
levels and SDs for age-decades. Age-trends: not reported.

In the Massachusetts I study subjects were respondents in the Massachusetts Male Aging
Study, aged 39-70 yrs, randomly sampled from cities and towns in the area of Boston, MA,
USA, between 1986 and 1989. Of the 2300 men invited to participate, 1709 completed the
__________________________________________________________________________________________
_______________________________________________________________________________________ 43
interview and measurements (74%). All participants were carefully screened to assess medical
factors known or suspected to alter endocrine function. Blood samples were drawn within 2h
of the subjects awakening and 30 min thereafter and then storaged at -70C (maximally 4
yrs). Measured parameters: SHBG (Filtration assay, Longcope et al. 1987), total testosterone
(RIA), free testosterone (centrifugal ultrafiltration, Longcope et al. 1987), albumin-bound
testosterone (centrifugal ultrafiltration, Hammond et al. 1982), total estradiol (RIA, Longcope
et al. 1986) and estrone (RIA, Longcope et al. 1986).
In Gray et al. 1991: analysis of two subjects subgroups: healthy (non-obese within 20% of
ideal body weight; no self-reported excess of alcohol consumption >600ml ethanol/week; no
self-reported chronic illness: cancer, CHD, hypertension, diabetes, or ulcer; no self-reported
prostatic hypertrophy or history of prostate surgery; and not currently taking medication,
n=415) vs. not healthy (remaining men, n=1294). Healthy subjects were significantly younger
than not healthy: 51,28,4 vs. 55,78,5. Represented data: age-trends/year, benchmark
median concentrations and percentage of difference between groups. Age-trends: SHBG
increases 1,20,1%/yr between 40-70 yrs; testosterone fractions decrease with ages as
follows: total testosterone =-0,40,1%/yr, free testosterone =-1,20,2%/yr and albumin-bound
testosterone =-1,00,2%/yr between 40-70 yrs of ages; total estradiol and estrone remain
stable between 40-70 yrs.
In Field et al. 1994: analysis of 1241 men, whose data were completed; excluded were men
with self-reported histories of cancer, with high energy intakes outside the range of 600-4200
cal/day, also men using tobacco solely in the form of cigars, cigarillos and pipes smokers.
Represented data: geometric mean serum levels and 95%CI by age-strata, adjusted for BMI
and smoking. Age-trends: not reported.

In the Massachusetts II study, Feldman et al. 2002, subjects were eligible cohort of
Massachusetts I study, aged 50-80 yrs; 1156 men were reinterviewed between 1995 and 1997.
The retention rate was 76% of those still living. Health status and current treatment were
ascertained by prompted self-report. Men taking androgens, estrogen, or bromocriptine were
not eligible for the study. Nonfasting blood samples were drawn within 4h of the subjects
awakening and storaged at -70C (maximally 5 yrs). Measured parameters: SHBG (RIA),
total testosterone (RIA), free testosterone (centrifugal ultrafiltration, Longcope et al. 1987),
albumin-bound testosterone (centrifugal ultrafiltration, Hammond et al. 1982) and estrone
(RIA, Longcope et al. 1986). Represented data: age-trends/year and mean levels for all
subjects. Age-trends: SHBG increases 1,6%/yr between 50-80 yrs; testosterone fractions
decrease with ages as follows: total testosterone =-0,8%/yr, free testosterone =-1,7%/yr,
albumin-bound testosterone =-2,0%/yr and estrone =-0,8%/yr between 50-80 yrs of age.

__________________________________________________________________________________________
_______________________________________________________________________________________ 44
In the Paris study (Telecom Study), Simon et al. 1992, subjects were France Telecom
employees, Paris, France. Examined were 1408 consecutive healthy white men aged 20-60
yrs, who voluntarily attended a centre for preventive medicine. Most of the subjects were
sedentary office workers. Known diabetic patients were excluded. Blood was drawn between
08.00-09.00 and storaged at 20 C, on average for 2 days. Age-distribution of the subjects:
20-29, n=444; 30-39, n=445; 40-49, n=235; 50-59, n=284. Measured parameters: total
testosterone, total estradiol and estrone (all RIAs after column chromatography). Represented
data: means and SDs for age-decades. Age-trends: total testosterone and total estradiol
decrease between 20-59 yrs from young ages (age-trends not reported), estrone remains stable
between 20-59 yrs.

In the Quebec study, Belanger et al. 1994, subjects were respondents in the Laval University Prostate
Cancer Detection Program, aged 40-80 yrs, randomly selected from the electoral rolls of Quebec City
and vicinity, Canada. Between 1989 and 1992, 7245 patients were examined. None of these patients
took medication known to affect the pituitary-adrenal and pituitary-testicular axis. Blood samples were
drawn between 07.30-09.30 h. No data about storage conditions and duration are reported.
Measurements were done in randomly selected 2423 patients (due to the costs of analysis). Age-
distribution of the subjects: 40-45, n=33; 45,1-50, n=142; 50,1-55, n=496; 55,1-60, n=496; 60,1-65,
n=515; 65,1-70, n=413; 70,1-75, n=268; 75,1-80, n=60. Measured parameters: total testosterone, total
estradiol and estrone (all RIAs after column chromatography). Represented data: age-trends/decade of
ages, benchmark median concentrations and SEMs at 40, 50, 60, 70, and 80 yrs. Age-trends: total
testosterone decreases =-16,0%/decade and estrone decreases =-11,3%/decade between 40-80 yrs of
ages; total estradiol remain stable between 40-80 yrs.

In the Ghent study, Belgium, no data about subjects selection and inclusion are presented. It is also
unclear regarding the inequality of the represented in the publications collectives. No data about
storage conditions and duration are reported.
In Vermeulen 1991: analysis of 292 healthy subjects aged 20-89 yrs. Age-distribution of the subjects:
20-30, n=70; 40-59, n=54; 60-69, n=41; 70-79, n=51; 80-89, n=76. Measured parameters: total and
free testosterone (methods not reported). Represented data: hormone mean levels and SDs for age-
groups. Age-trends: total and free testosterone decrease with age (age-trends not reported).
In Vermeulen 1993: analysis of 365 healthy subjects aged 18-over 100 yrs. Age-distribution of the
subjects: 18-29, n=105; 30-49, n=30; 50-59, n=24; 60-69, n=63; 70-79, n=63; 80-89, n=60; 90-100,
n=10; over 100, n=10. Measured parameters: SHBG, total and free testosterone (methods not
reported). Represented data: hormone mean levels and SDs for age-groups. Age-trends: SHBG
increases but total and free testosterone decrease with age (age-trends not reported).
__________________________________________________________________________________________
_______________________________________________________________________________________ 45
In Vermeulen et al. 1996: analysis of 250 healthy non-obese subjects (BMI=20-26 kg/m) aged 25-100
yrs, living in semiindustrial area. None was taking any medication or reported alcohol consumption.
Health condition was evaluated by clinical examination and routine blood biochemistry. (The
percentage of smokers was probably higher in elderly age-groups). All blood samples were obtained
between 08.00-10.00 h after an overnight fast. No data about blood storage are present. Age-
distribution of the subjects: 25-34, n=45; 35-44, n=22; 45-54, n=23; 55-64, n=43; 65-74, n=47; 75-
84,n=48; 85-100, n=21. Measured parameters: SHBG (equilibrium dialysis, Vermeulen et al. 1971),
total testosterone (RIA), free testosterone (not reported, probably equilibrium dialysis,) and total
estradiol (RIA). Represented data: hormone mean levels and SDs for age-groups. Age-trends: SHBG
increases early, before 40 yrs of age (age-trend not reported); total testosterone remains stable till the
age of 55 yr, decreases afterwards with age-trend =-0,85%/yr till 100 yrs of age; free testosterone
decrease with age-trend =1,2%/yr; total estradiol remain stable over the whole life-span.

In the Rochester study, Khosla et al. 1998, subjects were recruited from an age-stratified random
sample of Rochester, MN, USA, including both free-living and institutionalised individuals. Of the
approached 1138, ineligible were 239 men (demented, debilitated or died, etc). Of the 899 eligible
participated 348 men, aged 23-90 yrs: only 2 men were excluded. Fasting state serum samples were
obtained between 08.00-09.00 h and storaged at -70C (duration not reported). Measured parameters:
SHBG (RIA), total testosterone (RIA), bioavailable testosterone (ammonium sulphate precipitation,
Tremblay and Dube 1974), total estradiol (RIA), bioavailable estradiol (ammonium sulphate
precipitation, Tremblay and Dube 1974) and free testosterone (a-ligand RIA). Represented data:
hormone-changes between 25-85 yrs and figures. Mean levels and SDs for age-decades were received
by personal contact with the first author. Age-distribution of the subjects: nearly 50 person pro decade.
Age-trends: SHBG increased by 124,3% between 25-85 yrs, whereas hormone fractions decreased
between 25-85 yrs as follows: total testosterone 29,5%, bioavailable testosterone 64,1%, total estradiol
11,5% and bioavailable estradiol 47,1%.

In the Sheffield study, UK, Fatayerji and Eastell 1999, subjects were 178 men aged 20-79 yrs. Letters,
inviting to participate in the study, were sent to men on a local general practitioners register, until
nearly 30 men pro decade were recruited (uptake rate 20%, of which 70% were eligible). All
volunteers were healthy Caucasian men, taking no medication to affect bone or calcium metabolism.
Blood samples were collected between 09.00 and 10.00 h after an overnight fast and storaged at -80C
(duration not reported). Measured parameters: SHBG, total testosterone and total estradiol (all RIAs).
Represented data: mean levels for age-decades and increase between 20-79 yrs. Age-trends: SHBG
increased by 62% between 20-79 yrs, whereas total testosterone decreased by 26% and total estradiol
decreased by 33% between 20-79 yrs of age.

__________________________________________________________________________________________
_______________________________________________________________________________________ 46
Table 17. Subjects and blood storage time in the identified studies.
Studies groups and subjects Subjects detailed Storage
Rancho Bernardo II, USA; pop-based
CHD-free from 82% of population
80% with complete data from 81% of population
11-14 yrs
5-9 yrs
healthy subjects from 74% of population
not healthy subjects from 74% of population
max 4yrs
Massachusetts II, USA; pop-based 76% of still living of 74% baseline population max 5 yrs
Paris, France; occ-based healthy "Telecom" workers 2 days
Quebec, Canada; pop-based respondents from population (% not reported) max 4yrs
Ghent, Belgium, healthy I
healthy II
healthy BMI 20-26
healthy (no data about involvement)
healthy (no data about involvement)
healthy with BMI 20-26
not reported
not reported
not reported
Rochester, USA; pop-based/healthy 38% from population without severe diseases not reported
Sheffield, UK; healthy healthy, 70% of respondents not reported
Baltimore, USA; pop-based 52% of the population studied max 33 yrs
Massachusetts, USA; pop-based 76% of still living of 74% baseline population max 4-5 yrs

Longitudinal studies on SHBG and sex-hormone changes with age
All identified longitudinal studies (also with small sample-sizes) on changes of sex-hormones
with age are shown in Table 18. However, only two of these studies were relevant (see
inclusion criteria). Although studies were performed on population-based samples, selection
of relatively healthy subjects in these studies because of low response rates cannot be
excluded (Tabl. 17). Only serum total testosterone was measured in all studies.
Table 18. Longitudinal studies reporting changes of total testosterone with age.
Studies (or study groups) and subjects S-size age-range follow-up References
Pittsburgh, USA, pop-based 66 41-61 13 yrs Zmuda et al. 1997
New Mexico, USA, pop-based 77 61-87 15 yrs Morley et al. 1997
Baltimore, USA; pop-based
pop-based (mixed-effects model)
16
782
55-90
22-91
15 yrs
10 yrs
Carter et al. 1995
Harman et al 2001
Massachusetts, USA; pop-based 1156 40-80 7-10 yrs Feldman et al. 2002

In the Baltimore Study, Harman et al. 2001, all men were participants in the Baltimore Longitudinal
Study on Aging, a largely middle-class, 87% Caucasian population. Included were 890 subjects whose
serum samples of adequate volume were available. These men were younger (but not significantly)
than the remaining 821 subjects, who were not included in the study. Sera from each subjects most
recent and previous 3 visits and from visits closest to 10, 15, 20, 25, and 30 yrs from the most recent
one were retrieved (mean 4 samples pro subject). Blood samples were obtained in the morning
between 07.00-09.30 h, after an overnight fast. Before 1992, samples were stored at 20C, samples
collected after 1992 were kept at 80C (duration up to 33 yrs, mean 10yrs). Measured parameters:
SHBG and total testosterone (both RIAs). Preliminary analysis of data from 3565 samples strongly
suggested the presence of a date (i.e. storage time)-artefact. All values of total testosterone were
corrected to adjust for constant and systematic underestimates. Mixed-effects model, which combined
__________________________________________________________________________________________
_______________________________________________________________________________________ 47
individual regression equations and grouped average effects, was created for the analysis. Represented
data: figures, best-fitted lines and age-trend for total testosterone. Age-trends: SHBG increases with
age curvilinear, at a slightly greater rate in the older than in younger men; total testosterone decreases -
0,110 (-0,124) nmol/l/yr between 22-91 yrs of age.

In the Massachusetts study, Feldman et al. 2002, subjects were 1156 men, aged 40-80 yrs, of the
eligible cohort of Massachusetts I study, who were reinterviewed between 1995 and 1997. The
retention rate was 76% of those still living. The median interval for reinterview was 8,9 yr, the range
was 7,1-10,4 yrs. The rate of follow-up was significantly higher for men who were Caucasian,
married, employed at baseline, or more highly educated. The reinterviewed men had been slightly
younger at baseline, more physically active and more often apparently healthy, but did not differ in
baseline serum testosterone, DHT, or SHBG levels. Men taking androgens, estrogen, or bromocriptine
were not eligible for the study. Nonfasting blood samples were drawn within 2h (at baseline) and 4h
(at follow-up) of the subjects awakening and storaged at -70C (maximally 4-5 yrs). Measured
parameters: SHBG (baseline: Filtration assay, Longcope et al. 1987; follow-up: RIA), total
testosterone (RIA), free testosterone (centrifugal ultrafiltration, Longcope et al. 1987), albumin-bound
testosterone (centrifugal ultrafiltration, Hammond et al. 1982) and estrone (RIA, Longcope et al.
1986). The mixed and the repeated measures models were used for the regression analysis.
Represented data: age-trends/year and mean levels for all subjects. Age-trends: SHBG increases 1,3
(95%CI: 0,8; 1,6) %/yr between 50-80 yrs; testosterone fractions decrease with ages as followed: total
testosterone =-1,6 (95%CI: -2,0; -1,3) %/yr, free testosterone =-2,8 (95%CI: -3,2; -2,3) %/yr, albumin-
bound testosterone =-2,5 (95%CI: -3,0; -2,1) %/yr and estrone =-3,6 (95%CI: -4,0; -3,1) %/yr between
50-80 yrs of age.

__________________________________________________________________________________________
_______________________________________________________________________________________ 48
Cross-sectional studies on IGF-1 changes with age
Identified studies on changes of IGF-I with age (studies with age-interval over 30 years, with
10 or more subjects pro age-decade, inpatient persons excluded) are shown in Table 19.
Although many studies were performed on population-based samples, selection of relatively
healthy subjects in these studies because of strong inclusion criteria or of low response rates
was programmed or cannot be excluded, respectively.

Table 19. Cross-sectional studies describing changes of IGF-I levels with age.
Study groups and subjects S-size age-range N/year References
Izumo, Japan, pop-based 103 21-80 1,7 Jamomoto et al. 1991
Copenhagen, Denmark, occ-based 78 20-80 1,3 Juul et al. 1994
Gteborg, Sweden, pop-based 197 25-64 4,9 Landin-Wilhelmsen et al. 1994
Linkping, Sweden, pop-based 101 20-70 2,0 Nystrm et al. 1997
Baltimore, USA, healthy 223 20-90 3,2 OConnor et al. 1998
Stockholm, Sweden, healthy 201 20-96 2,6 Hilding et al. 1999
Sheffield, UK, healthy 178 20-79 3,0 Fetayerji and Eastell 1999
Hannover, Germany, donors 514 20-72 9,7 OUR DATA

In the Izumo study, Japan, Jamomoto et al. 1991, 103 men aged 21-80 yrs were selected from a
population as a part of an ongoing epidemiological study. Blood samples were drawn after overnight
fasting and stored at 20C before RIA analysis (duration not reported). Age-distribution of the
subjects: 21-30, n=10; 31-40, n=20; 41-50, n=21; 51-60, n=24; 61-70, n=18; 71-80, n=10. Represented
data: mean levels and SDs for age-decades, regression line. Age-trends: linear decrease IGF-I=-
2,36*age+322,2 (g/l/yr) between 21-80 yrs.

In the Copenhagen study, Denmark, Juul et al. 1994, school teachers and staff were requested to
participate and 78 males between 20-80 yrs volunteered. Blood samples were drawn between 08.00-
13.00 h and serum was storaged at 20C up to 4 month before RIA analysis. Represented data: figure
and regression line. Age-trends: quadratic decrease IGF-I=-0,11*age+19,19 (g/l/yr) between 20-
80 yrs.

In the Gteborg study, Sweden, Landin-Wilhelmsen et al. 1994, more than 700 men aged 25-64 yr
were participants of the MONICA Project (Participation rate 71%). For the analysis 50 samples from
each age-group were selected at random. No data about blood collection and storage are reported.
Assay: RIA. A few samples could not be used, thus 197 men were included in the study. Age-
distribution of the subjects: 25-34, n=47; 34-44, n=49; 45-54, n=48; 55-64, n=53. Represented data:
mean levels and SDs for age-groups, regression line. Age-trends: linear decrease IGF-I=-
2,1*age+292,7 (g/l/yr) between 25-64 yrs.

__________________________________________________________________________________________
_______________________________________________________________________________________ 49
In the Linkping study, Sweden, Nystrm et al. 1997, 101 subjects aged 20-69 yrs were randomly
obtained from the population registry of the community of Linkping (participation rate 64%). All
subjects had normal renal and liver function. Blood was drawn at 08.00 h after an overnight fast and
storaged before RIA analysis (conditions and duration not reported). Age-distribution of the subjects:
20-29, n=21; 30-39, n=25; 40-49, n=18; 50-59, n=19; 60-69, n=18. Represented data: mean levels and
SDs for age-decades, regression line. Age-trends: linear decrease IGF-I=-3,281*age+366,221
(g/l/yr) between 20-69 yrs.

In the Baltimore study, USA, OConnor et al. 1998, subjects were a subset of 223 healthy 20-90-
year-old ambulatory Caucasian community dwelling volunteers, participants in the Baltimore
Longitudinal Study on Aging. Subjects were screened to exclude medications and diseases known to
interfere with GH/IGF-I axis function. Blood was drawn between 06.15 and 07.30 h after a 12 h fast.
Serum was stored at 70C before RIA analysis (duration not reported). Represented data: mean level
and SD for mean age, figure and regression line. Age-trends: linear decrease IGF-I=-
1,872*age+285,616 (ng/ml/yr) between 20-90 yrs.

In the Stockholm study, Sweden, Hilding et al. 1999, 201 men were apparently healthy subjects,
composed of blood donors and other healthy individuals, aged 20-96 yrs. None had diabetes or other
endocrine diseases or had received estrogen therapy. The elderly subjects were living independently at
home. Serum samples were taken in the morning after an overnight fast in all subjects, except for the
blood donors, who had their regular morning meal and storaged before RIA analysis (conditions and
duration not reported). Age-distribution of the subjects: 20-29, n=25; 30-39, n=30; 40-49, n=25; 50-
59, n=29; 60-69, n=39; 70-79, n=33, 80-89, n=27; 90-96, n=2. Represented data: geometric mean
levels 2SD for age-decades, regression line. Age-trends: exponential decrease lgIGF-I=-
0,00647*age+2,5661 (lg(g/l/yr)) between 20-96 yrs.

In the Sheffield study, UK; Fetayerji and Eastell 1999, were involved 178 healthy subjects, aged 20-
79 yrs (description s. above). Assay not reported, probably RIA. Age-trends: IGF-I decreased by 54%
between 20-79 yrs. Quadratic-curvilinear decrease of logarithmically-transformed data .

__________________________________________________________________________________________
_______________________________________________________________________________________ 50
Sex Hormone Binding Globulin
Results were inconsistent. The mean SHBG serum levels differed widely between the studies.
The calculated mean age-trends varied between 13-19%/decade in population-based studies;
in studies on healthy men between 8-14%/decade (Fig. 17, Tabl. 20).
Fig. 17. Trend of SHBG levels with age (for simplicity 95%CI not shown).

1. Rancho-Bernardo I, USA; CHD-free men (Barret-Conner and Khaw 1987, Mean levels, transformed).
Mt: Precipitation by ammonium sulfate (Rosner 1972). SD%=51-61%, r=0,29.
2. Massachusetts I, USA; a. healthy, b. not healthy (Gray et al. 1991, Benchmark median concentrations).
Mt: Filtration assay (Longcope et al. 1987). SD%=51-56%, r=0,20.
3. Ghent, Belgium. a. healthy (Vermeulen 1993, Mean levels), b. healthy BMI=20-26kg/m (Vermeulen et al.
1996, Mean levels). Mt: Equilibrium dialysis (Vermeulen et al. 1971). SD%=18-45%; r=0,34
4. Rochester, USA, pop-based/healthy (Khosla et al. 1998, Mean levels estimated from the figure).
Mt: RIA. SD%- no data; r=0,50;
5. Sheffield, UK, healthy (Fatayerji and Eastell 1999, Mean levels). Mt: RIA. SD%-no data; r=0,39.
6. Hannover, Germany, donors (Our data, Mean levels). Mt: IRMA. SD%=34-41%, r=0,24.

Table 20. Calculation of age-trends for SHBG.
Study groups and subjects Parameters used for calculations bse(10
-2
) %/decadese
Rancho Bernardo I, USA; CHD-free regression of the means 1,760,33 19,23,3%
age-trend/year =1,20,1%
age-trend/year =1,20,1%
1,20,1
1,20,1
131%
131%
Massachusetts II, USA; pop-based age-trend/year =1,6% 1,6 17%
healthy BMI 20-26
regression of the means
1,020,10
0,850,10
10,71,1%
8,81,0%
Rochester, USA; pop-based/healthy 124,3% increase between 25-85 yrs 1,34 14,4%
Sheffield, UK; healthy regression of the means 0,800,12 8,31,2%
OUR STUDY, donors regression from original data 0,840,10 8,81,0%
Massachusetts, USA; longitudinal age-trend/yr =1,3 (95%CI: 0,8; 1,6) 1,30,2 142%

0
10
20
30
40
50
60
70
80
20 30 40 50 60 70 80 90 100
age (years)
s
e
x

h
o
r
m
o
n
e

b
i
n
d
i
n
g

g
l
o
b
u
l
i
n

(
n
m
o
l
/
l
)
3b
3b
5
5
1
1
3a
3a
6
6
4
4
2a
2a
2b
2b
__________________________________________________________________________________________
_______________________________________________________________________________________ 51
Total Testosterone
Results were inconsistent. The mean total testosterone serum levels differed widely between
the studies. The calculated mean age-trends varied between 3-16%/decade in population-
based studies; in studies on healthy men between 4-10%/decade (Fig. 18, Tabl. 21).
Fig. 18. Trend of total testosterone levels with age (for simplicity 95%CI not shown).

1. Rancho-Bernardo I, USA; CHD-free men (Barret-Conner and Khaw 1987, Mean levels).
Mt: RIA. SD%=23-39%, r=-0,14.
Mt: RIA (Judd et al. 1974). SD%=53-66%, r=-0,10
3. Paris, France; occ-based (Simon et al. 1992, Mean levels). Mt: RIA. SD%=29-33%, r=-0,25.
4. Quebec, Canada; pop-based (Belanger et al. 1994, Benchmark median concentrations), Mt: RIA.
1996, Mean levels). Mt: RIA; SD%=28-47%, r=-0,44
6. Rochester, USA, pop-based/healthy (Khosla et al. 1998, personal correspondence, Mean levels).
Mt: RIA, SD%=28-64%, r=-0,25.
7. Sheffield, UK, healthy (Fatayerji and Eastell 1999, Mean levels). Mt: RIA
8. Rancho Bernardo II, USA; pop-based (Barrett-Connor et al. 1999, Mean levels). Mt: RIA. SD%=29-43%
9. Hannover, Germany, donors (Our data, Mean levels). Mt: RIA, SD%=26-28%, r=-0,41.
Table 21. Calculation of age-trends for total testosterone.
-2
) %/decadese
Rancho Bernardo I, USA; CHD-free regression of the means -0,340,23 -3,32,3%
age-trend/year =-0,40,1%
-0,40,1
-0,40,1
-41%
-41%
Massachusetts II, USA; pop-based age-trend/year =-0,8% -0,8 -8%
Paris, France; occ-based regression of the means -0,680,11 -6,51,1 %
Quebec, Canada; pop-based %/decade reported by authors -1,73 -16%
healthy BMI 20-26
-0,720,08
0,810,14
-6,90,8%
7,81,4%
Rochester, USA; pop-based/healthy 29,5% decrease between 25-85 yrs -0,58 -5,7%
Sheffield, UK; healthy regression of the means -0,530,09 -5,2%0,9%
OUR STUDY, donors regression from original data -1,040,10 -9,91,0%
Massachusetts, USA; longitudinal age-trend/yr =-1,6 (95%CI:-2,0; -1,3) -1,60,2 -152%
0
5
10
15
20
25
20 30 40 50 60 70 80 90 100
age (years)
t
o
t
a
l

t
e
s
t
o
s
t
e
r
o
n
e

(
n
m
o
l
/
l
)
1
1
2a
2b
2b
2a
3
3
4
4
5a
5a
5b
5b
6
6
7
7
9
9
8
8
__________________________________________________________________________________________
_______________________________________________________________________________________ 52
Free Testosterone
Results were inconsistent. The mean free testosterone serum levels differed widely between
based studies; in studies on healthy men between -11-17%/decade (Fig. 19, Tabl. 22).
Fig. 19. Trend of free testosterone levels with age (for simplicity 95%CI not shown).

Calculated from mean levels of SHBG and total testosterone.
2. Massachusetts I, USA; a. healthy, b. not healthy (Gray et al. 1991, Benchmark median concentrations). Mt:
centrifugal ultrafiltration (Longcope et al. 1987), assayed as percent of total testosterone. SD%=50-74%.
1996, Mean levels). Mt: not reported by authors, probably: equilibrium dialysis. SD%=12-43%
Calculated as 1/20 of mean levels of measured bioavailable testosterone. SD%=26-74%, r=-0,62
5. Sheffield, UK, healthy (Fatayerji and Eastell 1999, Mean levels).
Calculated from mean levels of SHBG and total testosterone.
6. Rancho Bernardo II, USA; pop-based (Barrett-Connor et al. 1999, Mean levels).
Calculated as 1/20 of mean levels of measured bioavailable testosterone. SD%=26-41%
7. Hannover, Germany, donors (Our data, Mean levels). Calculated from SHBG and total testosterone.
SD%=27-39%, r=-0,55
Table 22. Calculation of age-trends for free testosterone.
-2
) %/decadese
-1,20,2
-1,20,2
-112%
-112%
healthy BMI 20-26
-1,820,09
1,330,06
-16,60,9%
12,50,6%
Rochester, USA; pop-based/healthy from BAT decrease between 25-85 yrs -1,71 -15,7%
Sheffield, UK; healthy regression of the means -1,190,12 -11,21,2%
0
100
200
300
400
500
600
700
20 30 40 50 60 70 80 90 100
age (years)
f
r
e
e

t
e
s
t
o
s
t
e
r
o
n
e

(
n
m
o
l
/
l
)
1
1
2a
2b
2b
2a
3a
4
4
3a
3b
3b
5
5
7
7
6
6
__________________________________________________________________________________________
_______________________________________________________________________________________ 53
Bioavailable Testosterone
Results were inconsistent. The mean bioavailable testosterone levels differed widely between
based studies; in studies on healthy men between -10-17%/decade (Fig. 20, Tabl. 23).
Fig. 20. Trend of bioavailable testosterone levels with age (for simplicity 95%CI not shown).

Calculated as *20 calculated free testosterone.
Mt: centrifugal ultrafiltration (Hammond et al. 1982). Calculated as a sum of measured free and albumin-
bound testosterone. SD%=64-47%, r=-0,21
3. Ghent, Belgium. a. healthy (Vermeulen 1993, Mean levels); b. healthy BMI=20-26kg/m (Vermeulen et al.
1996, Mean levels). Calculated as *20 measured free testosterone. SD%=12-43%
Mt: ammonium sulphate precipitation, SD%=26-74%, r=-0,62
Calculated as *20 calculated free testosterone.
Mt: ammonium sulphate precipitation (Tremblay and Dube 1974). SD%=26-41%
7. Hannover, Germany, donors (Our data, Mean levels). Calculated as *20 calculated free testosterone.
SD%=27-39%, r=-0,55.
Table 23. Calculation of age-trends for bioavailable testosterone.
-2
) %/decadese
-1,00,2
-1,00,2
-102%
-102%
healthy BMI 20-26
regression of FT means
regression of FT means
-1,820,09
1,330,06
-16,60,9%
12,50,6%
Sheffield, UK; healthy regression of FT means -1,190,11 -11,21,1%
0
2
4
6
8
10
12
14
20 30 40 50 60 70 80 90 100
age (years)
b
i
o
a
v
a
i
l
a
b
l
e

t
e
s
t
o
s
t
e
r
o
n
e

(
n
m
o
l
/
l
)
1
1
2a
2b
2b
2a
3a
4
4
3a
3b
3b
5
5
7
7
6
6
__________________________________________________________________________________________
_______________________________________________________________________________________ 54
Total Estradiol
Results were inconsistent. The mean total estradiol serum levels differed widely between the
studies. The calculated mean age-trends varied between 0 and -3%/decade in population-
based studies; in studies on healthy men between 0 and -7%/decade (Fig. 21, Tabl. 24).
Fig. 21. Trend of total estradiol levels with age (for simplicity 95%CI not shown).

1. Rancho-Bernardo I, USA; CHD-free men (Barret-Conner and Khaw 1987, Mean levels). Mt: RIA
SD%=23-33%, r=-0,17
Mt: (Longcope et al. 1986). SD%=57%,
3. Paris, France; occ-based (Simon et al. 1992, Mean levels). Mt: RIA SD%=21-37%, r=-0,10
4. Quebec, Canada; pop-based (Belanger et al. 1994, Benchmark median concentrations), Mt: RIA.
5. Ghent, Belgium. healthy BMI=20-26kg/m (Vermeulen et al. 1996, Mean levels). Mt: RIA. SD%=26-42%
Mt: RIA SD%=26-52%,
7. Sheffield, UK, healthy (Fatayerji and Eastell 1999, Mean levels). Mt: RIA, r=-0,40
8. Rancho Bernardo II, USA; pop-based (Barrett-Connor et al. 1999, Mean levels). Mt: RIA, SD%=29-38%
9. Hannover, Germany, donors (Our data, Mean levels). Mt: RIA; SD%=19-23%, r=-0,35

Table 24. Calculation of age-trends for total estradiol.
-2
) %/decadese
age-trend/year
age-trend/year
NS
NS
NS
NS
Paris, France; occ-based regression of the means -0,320,11 -3,11,1%
Quebec, Canada; pop-based %/decade reported by authors NS NS
Ghent, Belgium, healthy BMI 20-26 regression of the means 0,00,1 01%
NS non-significant
0
50
100
150
200
250
20 30 40 50 60 70 80 90 100
age (years)
t
o
t
a
l

e
s
t
r
a
d
i
o
l

(
p
m
o
l
/
l
)
1
1
2a
2a
2b
2b
3
3
4 4
5
5
6
6
7
7
9
9
8
8
__________________________________________________________________________________________
_______________________________________________________________________________________ 55
Bioavailable Estradiol
Results were inconsistent. The mean bioavailable estradiol levels differed widely between the
studies. The calculated mean age-trends varied between 5-10%/decade in population-based
studies; in studies on healthy men between 5-12%/decade (Fig. 22, Tabl. 25).
Fig. 22. Trend of bioavailable estradiol levels with age (for simplicity 95%CI not shown).

Calculated from mean levels of SHBG, total testosterone and total estradiol.
Calculated from benchmark median levels of SHBG, total testosterone and total estradiol.
3. Ghent, Belgium. healthy BMI=20-26kg/m (Vermeulen et al. 1996, Mean levels).
Mt: ammonium sulphate precipitation. SD%=33-75%, r=-0,43
Mt: ammonium sulphate precipitation (Tremblay and Dube 1974). SD%=28-41%, r=-0,28
7. Hannover, Germany, donors (Our data, Mean levels).
Calculated from SHBG, total testosterone and total estradiol. SD%=24-33%, r=-0,45

Table 25. Calculation of age-trends for bioavailable estradiol.
-2
) %/decadese
-0,52
0,48
-5%
-5%
Ghent, Belgium, healthy BMI 20-26 regression of the means -0,540,08 -5,20,8%
0
20
40
60
80
100
120
140
20 30 40 50 60 70 80 90 100
age (years)
b
i
o
a
v
a
i
l
a
b
l
e

e
s
t
r
a
d
i
o
l

(
p
m
o
l
/
l
)
1
1
2a
2b
2a
2b
3
3
4
4
5
5 7
7
6
6
__________________________________________________________________________________________
_______________________________________________________________________________________ 56
Estrone
Results were inconsistent. The mean estrone serum levels differed widely between the studies.
The calculated mean age-trends varied between +6% and -31%/decade in population-based
studies; in studies on healthy men between 0 and -4%/decade (Fig. 23, Tabl. 26).
Fig. 23. Trend of estrone levels with age (for simplicity 95%CI not shown).

Mt: RIA, SD%=36-54%, r=0,16
Mt: RIA (Longcope et al. 1986). SD%=59%
3. Paris, France; occ-based (Simon et al. 1992, Mean levels). Mt: RIA SD%=15-39%
4. Quebec, Canada; pop-based (Belanger et al. 1994, Benchmark median concentrations, not shown). Mt: RIA
Mt: RIA. SD%=32-85%
6. Hannover, Germany, donors (Our data, Mean levels). Mt: RIA SD%=37-42%, r=-0,11

Table 26. Calculation of age-trends for estrone serum levels.
-2
) %/decadese
Rancho Bernardo I, USA; CHD-free regression of the means 0,560,16 5,81,7%
age-trend/year
age-trend/year
NS
NS
NS
NS
Paris, France; occ-based regression of the means 0,050,14 0,41,4%
Quebec, Canada; pop-based %/decade reported by authors -1,20 -11%
Rochester, USA; pop-based/healthy -11,5% decrease between 25-85 yrs -0,20 -2,0%
NS non-significant
0
50
100
150
200
250
20 30 40 50 60 70 80 90
age (years)
e
s
t
r
o
n
e

(
p
m
o
l
/
l
)
1
1
2a
2a
2b
2b
3 3
5 5
6
6
__________________________________________________________________________________________
_______________________________________________________________________________________ 57
Insulin-like growth factor-I
The mean IGF-I serum levels were relatively similar in the studies. However, the calculated
age-trends were inconsistent. The mean exponential age-trends varied between -10-
16%/decade in population-based studies; in studies on healthy men between 10-15%/decade
(Fig. 24, Tabl. 27). The mean linear age-trends varied between 2,6 and 4,3 nmol/l/decade in
population-based studies; in studies on healthy men between 2,6 and 3,4 nmol/l/decade.
Fig. 24. Trend of IGF-I levels with age (for simplicity 95%CI not shown).

1. Izumo, Japan, pop-based, (Jamomoto et al. 1991, mean levels). Mt: RIA. SD%=18-41%, r=-0,48
2. Copenhagen, Denmark, occ-based (Juul et al. 1994, regression curve). Mt: RIA, r=-0,71
3. Gteborg, Sweden, pop-based (Landin-Wilhelmsen et al. 1994, mean levels) Mt: RIA SD%=20-27%,
r=-0,46
4. Linkping, Sweden, pop-based (Nystrm et al. 1997, mean levels), Mt: RIA. SD%=24-37%, r=-0,61
5. Baltimore, USA, healthy (OConnor et al. 1998, regression curve), Mt: RIA. r=-0,51
6. Stockholm, Sweden, healthy (Hilding et al. 1999, regression curve), Mt: RIA. SD%=29%, r=-0,77
7. Sheffield, UK, healthy (Fatayerji and Eastell 1999, mean levels), Mt: not reported (probably RIA). r=-0,64
8. Hannover, Germany, donors (Our data, mean levels). Mt: RIA. SD%=23-27%, r=-0,46.

Table 27. Calculation of age-trends for IGF-I serum levels.
-2
) %/decadese
Izumo, Japan, pop-based regression of the means -1,110,14 -10,51,4%
Copenhagen, Denmark, occ-based regression equation (quadratic) -1,720,05 -15,80,5%
Gteborg, Sweden, pop-based regression of the means -1,030,20 -9,82,0%
Linkping, Sweden, pop-based regression of the means -1,430,09 -13,40,9%
Baltimore, USA, healthy regression equation (linear) -1,150,05 -10,80,5%
Stockholm, Sweden, healthy regression equation (exponential) -1,62 -15%
Sheffield, UK, healthy regression of the means -1,220,17 -11,51,7%
Hannover, Germany, healthy regression from original data -1,090,09 -10,31,0%

0
5
10
15
20
25
30
35
40
45
20 30 40 50 60 70 80 90 100
age (years)
i
n
s
u
l
i
n
-
l
i
k
e
-
g
r
o
w
t
h
-
f
a
c
t
o
r
-
I

(
n
m
o
l
/
l
)
4
3
8
7
2
6
5
1
6
3
1
8
5
7
2
4
__________________________________________________________________________________________
_______________________________________________________________________________________ 58
DISCUSSION
The main aim of our study was to estimate the mean trends of age-related changes in serum
concentrations of SHBG, testosterone and estradiol fractions as well as estrone and IGF-I in
adult men. We performed our own cross-sectional investigation on blood donors, identified
relevant studies on this issue, calculated mean age-trends pro decade of investigated
parameters for these studies and analysed these results concerning their validity to reflect age-
related hormone changes. Some methodological problems arising from study designs, subject
selections, hormone measurements and our calculations should be pointed out.

Methodological issues
A potential confound, common to both cross-sectional and longitudinal studies on changes of
hormone parameters with age, is a effect of age-related variables, such as chronic illness or
exposure to alcohol, tobacco etc. Thus, studies may reflect hormone changes with age in
general population rather than the proper effect of age.
Because of a higher rate of diseases in old age, it may be expected that studies with and
without exclusions for chronic illnesses may observe different age-trends in changes of some
parameters. However, although many studies investigated hormone changes in population-
based samples; in all of them some exclusions for health status were performed. Moreover, it
should be noted that even studies with only few exclusions for chronic illnesses more likely
represent subjects healthier than the baseline population (s. below). Our own investigation,
performed on blood donors, as well as some other studies exclusively involved persons in
general good health, i.e. without chronic diseases.
However, it should be noted that the interrelation between ageing, diseases and endocrine
changes is not clear. It remains to be demonstrated whether hormone levels are dependent on
the effect of diseases. An alternative model might view some diseases as the endpoint, and a
change in endocrine function as one of several causing factors.
Exclusions for other age-related variables, such as alcohol or tobacco, also thought to be a
confounder or effect modifier, were not performed in the studies and adjustments for some of
these factors were done only in few analyses.
One of the potential confounds of cross-sectional and longitudinal studies on ageing is a
secular effect. It is one that results when there is progressive alteration of a critical condition
in the environment, producing the appearance of a change with age. Such an effect could have
occurred because of a change in environmental exposure over time, such as an increase in
estrogenic substances in food and /or water, which has been suspected to cause a decrease in
sperm counts and a higher incidence of male infertility (Harman et al. 2001). The impact of
secular effect on observed changes in serum hormone concentrations is unclear.
__________________________________________________________________________________________
_______________________________________________________________________________________ 59
The data presented in most studies are cross-sectional and it cannot be ruled out that observed
age-related changes in serum hormone levels represents a cohort rather than a true effect of
age. A cohort effect is one that occurs when individuals of similar age vary significantly from
older and/or younger groups in measured parameters because of a historical factor common to
their generation (Harman et al. 2001). However, it seems to be unlikely that birth cohort can
be a substantial driving factor behind the age-trends observed in the studies.

On the other hand, problems arising from the longitudinal studies, may overestimate the
influence of the cohort-effect, inherent for the cross-sectional studies:
Blood samples in the longitudinal studies should be measured immediately after the collection
(Massachusetts Male Aging Study) or storaged for a long time (Baltimore Longitudinal Study
on Aging). In the first case, the differences in the employed assays (or assays drift over time
by the same assay) could overestimate or underestimate real age-trends in the hormone serum
levels. In the second case, a long storage time suggests a progressive alteration in the frozen
samples, leading in the longitudinal studies to systematic variations in measured values (for
example, SHBG can be progressively depleted). However, a long storage time of blood
specimens is also possible in the cross-sectional studies. The alterations in blood samples
during storage were explicitly reported in the Rancho Bernardo Study (Ferrini and Barrett-
Connor 1998) and in the Baltimore Longitudinal Study on Aging (Harman et al. 2001).
The next potential problems in the longitudinal studies are missing data and mathematical
models, used for the analysis. Selection by losses to follow-up exclusively healthier or sickier
subjects, as well as subjects with more or less healthier behaviour may affect the results of
these studies (selective attrition biases). Likewise, statistical models (for example mixed-
effects model or mixed procedure), which have been used in the longitudinal studies to
combine cross-sectional and longitudinal observations as well as to adjust for the above
mentioned biases, had also some mathematical assumptions.

As far as persons in many studies were not randomly assigned from the eligible for inclusion
community, recruited subjects may not be representative for the investigated in these studies
populations (selection biases). Moreover, because of low response rates in the population-
based random-sampled studies (commonly less than 80%), even these samples probably
represent subjects somewhat different than the baseline population, more likely somewhat
healthier, as far as the participants had to be, for example, capable to visit a clinic. The
problem of losses to follow-up, inherent only for longitudinal studies, was discussed
previously.
Volunteers would probably have a healthier risk behaviour. Some behavioural factors, when
differ in young or in old age from a baseline population, can bias results of the studies. For
__________________________________________________________________________________________
_______________________________________________________________________________________ 60
example, as far as smokers were found to have higher levels of total testosterone (s.
testosterone), the higher prevalence of smokers in old age would probably underestimate the
real mean hormone changes, and vice versa.

In most studies blood was drawn, if reported, consistently in the morning, between 8.00-11.00
h. Although this time is more suitable for clinical routine, specimens collected in the morning
may poorly reflect the average daily hormone concentration, which is known to be higher at
this time for most measured hormone parameters (Bremner et al. 1983, Plymate et al. 1989,
Volero-Politi and Fuentes-Arderiu et al. 1996, Ahokoski et al. 1998). Because of diurnal
variation, which is more pronounced in younger than in older men (Bremner et al. 1983), age-
trends, calculated for these studies, would overestimate the real mean hormone changes.
In most of the studies blood measurements were performed from the single sample for each
individual, i.e. not corrected for episodic secretion. This can diminish precision of the
measurements but does not appear to constitute a limitation to the reliability of the data. The
possible seasonal variation in the hormone levels could only theoretically affect results of the
studies, as far as all of the classes of age in the studies were more likely represented equally
over the seasons.

Different methods and assays were employed for the hormone measurements in the studies.
These assays may have different accuracy, precision and some other parameters. Moreover,
some assays, which work well in a normal range, may have problems at the low-concentration
end of measurements because of cross-reactivity with other substances (for example, with
some steroids for testosterone assay; Klee and Heser 2000). Thus, in one study a higher (26-
41%) significant between-kit variability of six different commercially available testosterone
assays was reported, which was higher in samples with the lowest SHBG levels (Boots et al.
1998). This is especially important for the studies measured on average lower hormone serum
levels (not SHBG), which might generally somewhat underestimate real changes, because of
lower hormone levels in the elderly.

The estimation of the free and bioavailable serum fractions of testosterone and estradiol is not
a routine procedure and the data in literature concerning these fractions are scarce. Therefore,
these parameters were calculated for each individual of our investigation and for average
hormone values of the reported studies. When multiple measurements are used, each variable
has its own variance. When these are combined, the final estimate may have fairly large
variances, which may limit the utility of these estimations. However, calculated free
testosterone levels were shown to be a reliable parameter of free testosterone fraction
(Vermeulen et al. 1999).
__________________________________________________________________________________________
_______________________________________________________________________________________ 61
One problem may also arise when population in the study is not equally distributed between
the age-groups. The calculated regression lines may only partially represent changes in
hormone levels for the whole age-interval of these studies (for example, from 810 men aged
23-90 yrs in Rancho Bernardo Study only 27 persons were below 50 yrs).

For some studies exponential age-trends were calculated from the mean serum levels for age-
groups. As distribution of the investigated hormones is positively skewed (contributing to a
higher difference between mean and median values in ages with higher hormone
concentrations), these age-trends would probably slightly overestimate the real exponential
age-trends, which may be calculated by the regression analysis of the individual data.
However, by analysing data from the studies, where both mean and median data for age-
groups were available, no meaningful differences in the calculated age-trends were found.
Another problem of calculations of age-trends from the mean values may appear because of
limited precision of these parameters (se), increasing a random error of the calculated age-
trends (ses of the b-regression coefficients). Such errors may also be suggested by the visual
inspection of the plotted mean values (extremely large values in one of the age-groups).

The majority of studies was performed in the USA, subjects were predominantly Caucasian
and generally well educated. Hence, results of these studies cannot be extrapolated to the
general population.
__________________________________________________________________________________________
_______________________________________________________________________________________ 62
Sex-Hormone Binding Globulin
Mean levels and effect of age
SHBG mean levels in our study were similar to the levels obtained in the Ghent and Sheffield
studies on healthy subjects (Vermeulen 1993, Vermeulen et al. 1996, Fatayerji and Eastell
1999), but were higher than mean levels obtained in the population-based Massachusetts and
Rochester studies (Gray et al. 1991, Field et al. 1994, Khosla et al. 1998, Feldman et al. 2002)
and substantially higher than levels obtained in the Rancho Bernardo population-based group
(Barrett-Connor and Khaw 1987; precipitation by ammonium sulfate).
Such differences in mean SHBG levels are probably due to the differences in the assays used
for investigations or due to the alterations in frozen samples in some investigations because of
long storage time, as has been shown in the Baltimore Longitudinal Study on Aging (Harman
et al. 2001). Also low mean SHBG levels in some studies had a trend to a higher SHBG
variability, as indicated by SD%. It is important that the SHBG serum levels obtained in our
study are identical to the real values of SHBG-binding sites, which were recently estimated by
some investigations (Vermeulen et al. 1999). Hence, mean SHBG serum levels of our study
fit closely to the real SHBG-binding capacity.
In our study SHBG serum levels increased exponentially with age in the regression model and
in the ANOVA-model remained relatively stable between the 2
nd
and the 5
th
decade of ages
with an increase thereafter. Our results do not confirm observations of exclusively early
increases of SHBG levels with age (Vermeulen et al. 1996) and are more in line with the
models of a later (Barrett-Connor and Khaw 1987), exponential (Gray et al. 1991, Fatayerji
and Eastell 1999, Feldman et al. 2002) or, generally, curvilinear (Harman et al. 2001) increase
of SHBG levels (therefore, it is not surprising that studies observing older populations show a
higher linear association of SHBG levels with age than studies on younger age-groups).
Variable age was accounted in the studies for up to 25% in total variability of SHBG serum
levels (r-model) .
Because of a higher rate of hyperinsulinism in the old age, known to decrease SHBG levels (s.
below), it may be expected that in the studies with less exclusion criterias for health status
lower mean age-trends in the SHBG changes would be observed. However, it was an opposite
trend in the studies, 13-19%/decade vs. 8-14%/decade.
One explanation of this observation is that assays used in population-based studies, generally
measuring a lower SHBG-binding capacity, also overestimated SHBG age-trends. Another
possible explanation is that in a subtle age-interval between 40 and 80 years, which was
mostly investigated in these studies, serum SHBG changes are somewhat higher than on
average in a larger age-intervals. The high mean age-trend, calculated for the Rancho-
Bernardo study, was accompanied by the large standard error (se) indicating the large
confidence interval of these estimations.
__________________________________________________________________________________________
_______________________________________________________________________________________ 63
No study was ideal to describe the proper effect of age on SHBG serum levels. After
summarising data from the studies it may be concluded that increase of SHBG with age may
be approximated by an exponential function and that in healthy adult men SHBG increases on
average between 8-14% pro decade. Using these age-trends it may be estimated that at 65, 75
or 85 years serum SHBG levels in healthy men exhibit on average between 136-169%, 147-
193% or 159-219% of serum levels of 25-year-old persons. These estimations confirms the
Baltimore Longitudinal Study on Aging, showing nearly two-fold higher SHBG levels at 80-
90 years compared to 20-30-year-old men (Harman et al. 2001, data from the figure).

The cause of changes and biological regulation of SHBG serum levels
The association of variables, investigated in our study, with SHBG serum levels was small;
however, their role becomes more clear from a hypothesis of SHBG biological regulation,
which arises from the comparison of SHBG changes with age in men with those changes in
women (Fig. 23).
SHBG levels progressively decrease during puberty, more pronounced in boys than in girls
(Pugeat et al. 1996). In adult women, SHBG substantially (almost 10-fold) increases during
pregnancy; in adult men curvelinearly (nearly twofold) increases during ageing, reaching at
the age of 80-90 years practically female values (summarised from the data reported in
Goodman-Gruen and Barrett-Connor 1996, Khosla et al.1998, Lecomte et al. 1998).
Fig. 23. Schematic representation of SHBG changes with age in men and women.

Such pattern of changes supports a hypothesis that SHBG probably plays a substantial role
only during puberty and pregnancy and is suppressed in adult women and to a higher extent in
men; but mechanisms, which suppress SHBG binding capacity, are impaired with advancing
age as well as in male person as in old females.
women
Pregnancy
Puberty
Ageing
SHBG
men
years
10 20 30 40 50 60 70 80 90
__________________________________________________________________________________________
_______________________________________________________________________________________ 64
Indeed, although biological function of SHBG remains unclear and a number of theories have
been suggested (Pardridge 1998, Joseph 1994 for review), the presence of SHBG (and also
albumin) in body fluids is not essential for steroid homeostasis. This can be inferred from
analbuminemic rats which possess neither SHBG nor albumin. In these rats, which are fertile,
the total (and free) concentration of testosterone is within the range of the affinity constant of
the androgen receptor (Rommerts 1998). Nevertheless, some factors may affect SHBG-
binding capacity and regulate SHBG serum levels.

The only substantial increase in SHBG levels is during pregnancy; in young adults SHBG
serum levels are almost twofold higher in women than in men. Therefore, sex-hormone levels
appear to play an important role in the regulation of serum SHBG. In subjects presenting with
a complete form of androgen insensitivity syndrome SHBG decreases during puberty only to
female values; estrogen treatment is known to increase SHBG levels and the administration of
testosterone generally decreases SHBG-levels as in hypogonadal male person as well as in
women presenting hyperandrogenism; effects of testosterone and estradiol on SHBG were
also shown in vitro studies on human hepatocarcinoma (Hep G2) cells (Pugeat et al. 1996 for
review).
Although our study as well as one further study (Longcope et al. 1990) could not find any
significant effect of the ratio of estradiol to testosterone fractions on SHBG concentrations, in
physiological concentrations these steroids are poor predictors of SHBG levels. The effect of
sex-hormones on SHBG is only apparent, when the concentration of these steroids, especially
of androgens, is substantially high (Pugeat et al. 1996). But in general, changes in sex-
hormone concentrations with age may partially contribute to the increase of sex-hormone-
binding-capacity.
Structurally and functionally similar substances, viz. insulin and insulin-like growth factors
also appear to be determinant of SHBG levels; and body mass index via the accompanying
hyperinsulinism can partially reflect such effects (Vermeulen et al. 1996). SHBG levels
decrease at puberty, which parallels the pubertal rise in GH and IGF-I. This decrease occurs
even in boys with isolated gonadotropin deficiency or with incomplete androgen sensitivity,
suggesting that, at least in part, it is independent from circulating androgen levels
(Pfeilschifter et al. 1996). Subjects with diabetes mellitus or with insulin resistance syndrome
have lower SHBG levels (Barrett-Connor et al. 1990, Stellato et al. 2000); as well as patients
with acromegaly (Pugeat et al. 1996). In isolated GH deficiency SHBG levels are increased
and decrease during treatment with substitutive doses of GH (Vermeulen et al. 1996).
Although GH therapy induces insulin resistance, the effect of insulin and insulin-like growth
factors on SHBG level appear to be independent (Vermeulen et al. 1996). The inhibitory
effect of insulin and several growth factors on SHBG levels was also shown in the studies on
human hepatocarcinoma (Hep G2) cells (Pugeat et al. 1996 for review).
__________________________________________________________________________________________
_______________________________________________________________________________________ 65
In most clinical studies an inverse unadjusted association between insulin and SHBG
(Vermeulen et al. 1996, Haffner 1996), IGF-1 and SHBG (Pfeilschifter et al. 1996,
Vermeulen et al. 1996) and BMI and SHBG (Barrett-Connor and Khaw 1987, Longcope et al.
1990, Field et al. 1994, Vermeulen et al. 1996, Haffner 1996, Denti et al. 2000) has been
reported. In our study, similar to other researches (Field et al. 1994, Longcope et al. 2000), we
found an age-independent association between SHBG and BMI. We also found an age- and
BMI-independent association between SHBG and IGF-1 serum levels; such association was
previously reported only for population-based study (Pfeilschifter et al. 1996). Including BMI
in multiple linear regression increased from 6% to 14% (r-model) the prediction value of
SHBG serum levels compared to the model when only age was included as an independent
variable. Thus, in general, the age-associated decrease in GH and IGF-1 levels may also be
partially responsible for the increase of SHBG levels with advancing age.

Some other factors are also responsible for the large interindividual variability of SHBG
serum levels. Heredity appears to play one of the most important roles in the determination of
SHBG and up to 30% of the variability of SHBG levels, after normalisation for body surface
area, may be attributable to genetic factors (Kaufman and Vermeulen 1997). Smoking (Field
et al. 1994), thyroid status (Pugeat et al. 1996, Kaufman and Vermeulen 1998) and total daily
circadian variation (Plymate et al. 1988, Valero-Politi and Fuentes-Arderiu 1996) are also
accounted for the wide-range of SHBG serum concentrations.

In conclusion, SHBG in blood serum having a very high affinity for sex-steroids produces a
very low biodisposal to target cells SHBG-bound hormone fractions. Therefore, the
concentrations of non-SHBG-bound hormones, thought to be bioavailable to the peripheral
tissues (s. below), may be underestimated by measurements of only total testosterone and
estradiol levels, especially in elderly men. As the measurement of bioavailable hormone
fractions is not a routine procedure, SHBG measurements concomitant to measurements of
total testosterone or estradiol for assessing their bioavailable activity are of great clinical
importance.
__________________________________________________________________________________________
_______________________________________________________________________________________ 66
Testosterone
The mean levels of total testosterone and calculated levels of free and bioavailable
testosterone in our study were grossly similar to the age-related mean levels of these
testosterone fractions, obtained in or calculated for healthy subjects in Ghent (Vermeulen
1991, 1993, Vermeulen et al. 1996), Sheffield (Fatayerji and Eastell 1999) and for
Massachusetts II population (Feldman et al. 2002). The mean levels of total testosterone in
our study were also similar to the age-related mean levels of total testosterone obtained in
Paris Telecom employers (Simon et al. 1992) as well as in Rochester (Khosla et al 1998)
and in Rancho Bernardo I (Barret-Connor and Khaw 1987) populations.
Some populations such as Quebec (Belanger et al. 1994), Massachusetts I (Gray et al. 1991)
Rancho Bernardo II (Ferrini and Barrett-Connor 1998, Barrett-Connor et al. 1999, 2000)
showed very low mean concentrations of total testosterone, even low for the studied ages (40-
90 years). This occurred because of the assays used in these studies or, more likely, because
of alterations in serum by storage. The Baltimore Longitudinal Aging Study (Harman et al.
2001) revealed a significant increase in total testosterone levels with length of storage and
adjusted their measurements for data effect (which after adjustment became grossly similar to
the age-related mean levels obtained in our study). The later report of the research group from
Quebec also showed higher mean total testosterone levels (Labrie et al. 1997).
Although average levels of total testosterone obtained in Rancho Bernardo I population were
within normal range, calculated mean free and bioavailable testosterone levels for this study
were high. This was due to the very low in this study measured SHBG-binding capacity
(precipitation by ammonium sulfate, s. SHBG).
Observed levels of free or bioavailable testosterone in our study were on average higher than
age-related mean serum levels of these fractions obtained in Massachusetts I (Gray et al.
1991), Rochester (Khosla et al 1998) and Rancho Bernardo II (Ferrini and Barrett-Connor
1998, Barrett-Connor et al. 1999) population-based groups.
Levels of free and albumin-bound testosterone fractions in Massachusetts I study (Longcope
et al. 1990, Gray et al. 1991, Field et al. 1994), measured in this study by ultrafiltration assay
as a percentage of total testosterone, were very low because of low total testosterone levels.
These levels were on average even lower than reference values for this Hospital (Case
records... 1992). In the Massachusetts II population levels of free and albumin-bound
testosterone fractions were within normal range, and by longitudinal analyse Massachusetts I
blood samples were subsequently reassayed by newer method (Feldman et al. 2002). The
reassayed mean levels of total, free and albumin-bound testosterone were grossly similar to
the age-related mean levels obtained in our study.
__________________________________________________________________________________________
_______________________________________________________________________________________ 67
These examples suggest the importance of the effect of storage-time on the observed hormone
levels, which may also lead to systematic errors in calculated age-trends (s. methodological
issues). These examples also suggest the reliability of our total testosterone measurements.
However, in the case of bioavailable testosterone it remains to be investigated, if our
calculations somewhat overestimate serum levels, or if measured in the Rochester (Khosla et
al. 1998) and Rancho Bernardo II (Ferrini and Barrett-Connor 1998, Barrett-Connor et al.
1999) studies somewhat lower concentrations are also the result of the alterations of these
fractions by storage or the property of the used assays.

Although our data as well as Massachusetts (Feldman et al. 2002), Quebec (Belanger et al.
1994) and Sheffield (Fatayerji and Eastell 1999) studies suggest an exponential decrease of
total testosterone as well as of free and bioavailable testosterone fractions, Ghent researchers
confirm only an exponential decrease of free testosterone, but suggest more stable levels of
total testosterone until the age of 55 years (Vermeulen et al. 1996). Nonetheless, this study
was relatively small and the proportion of smokers in this study was probably higher in the
old age. In earlier reports of these researches (Vermeulen 1991, Vermeulen 1993) and by
separate analyse of smokers and non-smokers in the latter publication (Vermeulen et al. 1996)
a decrease of total testosterone levels was observed at earlier ages. In our subset of subjects
with BMIs comparable to this study (between 20-26 kg/m) total testosterone started to
decrease also in the young adults.
In some reports changes of total testosterone (Ferrini and Barrett-Connor 1998, Feldman et al.
2002) and bioavailable testosterone (Ferrini and Barrett-Connor 1998) concentrations with
age were approximated by linear regression model. Linear model was also used in two
longitudinal studies on small age-intervals (Zmuda et al. 1997, Morley et al. 1997). This
model seems to be less appropriate because of the skewed distribution of the hormone levels.
None of the other investigated regression models (quadratic, cubic and inverse) showed a
meaningfully higher prediction value of serum levels of testosterone fractions in our study.
The calculated mean age-trends for the studies varied between 3-16%; -11-25% and 10-
22% pro decade for total, free and bioavailable testosterone, respectively. The highest age-
trends in serum hormone concentrations were shown in the Quebec study (Belanger et al.
1994) and by the analysis of longitudinal changes in the Massachusetts subjects (Feldman et
al. 2002). These studies were population-based and may represent mean serum hormone age-
trends in population, but not changes caused by ageing itself (what was also suggested by the
authors, Feldman et al. 2002). Nevertheless, such age-trends seems to overestimate average
age-trends even for population and are probably caused by inaccuracies in measurements,
such as drift in assays measurements or long storage time of the samples (s. methodological
issues). In confirmation, calculated from the linear age-trend (-0,124nmol/l/yr) and mean
levels (from the figure) the mean age-trend for exponential regression of total testosterone for
__________________________________________________________________________________________
_______________________________________________________________________________________ 68
Baltimore Longitudinal Aging Study was between 5-8%/decade. Studies performed on
healthy subjects showed the mean age-trends for total, free and bioavailable testosterone
between -4-10%; -11-17% and 10-17% pro decade, respectively.
No study was ideal to describe the proper effect of age on testosterone serum levels. After
summarising data from the reported studies it may be concluded that the decrease of serum
testosterone fractions with age may be approximated by an exponential function and that in
healthy adult men total testosterone decreases on average between 4-10% pro decade,
whereas free and bioavailable testosterone fractions between 10-17% pro decade. In the
general population, most likely due to a higher rate of diseases in old age, the mean decrease
of testosterone fractions is accentuated.
Using these age-trends it may be estimated that at the age of 65, 75 and 85 years in healthy
men remain on average between 66-85%, 59-82% and 53-78% of the total testosterone serum
levels of 25 year-old persons, respectively. However, at the same age the concentrations of
serum bioavailable testosterone remain on average between 47-66%, 39-59% and 33-53%
(Fig. 24). Remaining percentage of free testosterone is largely similar to that of bioavailable
testosterone fraction.
Fig. 24. Conclusions: average changes of total and bioavailable testosterone serum levels
and remaining hormone concentrations in healthy adult men with age.

However, variable age was accounted in the studies for up to 20%, 40% and 40% in total
variability of total, free and bioavailable testosterone, respectively, (r-model) and at any age
these fractions exhibited a large interindividual variability (SD%>50% in some studies).
0
5
10
15
20
25
20 30 40 50 60 70 80 90 100
age (years)
t
o
t
a
l

a
n
d

b
i
o
a
v
a
i
l
a
b
l
e

t
e
s
t
o
s
t
e
r
o
n
e

(
n
m
o
l
/
l
)
100%
100%
33%
39%
47%
53%
59%
66%
total testosterone
decrease 4-10%/decade
bioavailable testosterone
remaining concentrations:
-4%/decade
-17%/decade
-10%/decade
-10%/decade
55%
60%
67%
78%
82%
85%
__________________________________________________________________________________________
_______________________________________________________________________________________ 69
Effect of SHBG, BMI and other factors
The differences in age-related decreases in total and free or bioavailable testosterone fractions
are caused by the age-related increase in sex-hormone binding globulin concentrations. But
the common used expression that the increase in SHBG with age leads to the more profound
decrease in free and bioavailable testosterone concentrations is probably not correct.
The bioavailable testosterone fraction but not total testosterone is probably the index of
steady-state of negative feedback regulation of testosterone secretion. In this case total
testosterone should only be a subject of SHBG variability. In experiment the chronic infusion
of SHBG into male rats produced marked increase in total plasma testosterone, but had little
effect on the concentration of free testosterone and the maintenance of the sexual accessory
glands (Joseph 1994). In hyperthyroidism levels of SHBG and total testosterone are elevated,
whereas levels of free or bioavailable testosterone are normal (Kaufman and Vermeulen 1997,
Vermeulen et al. 1999). The mean age-trends of FSH and LH are also more similar to the
mean age-trend of bioavailable than total testosterone (Khosla et al. 1998, Feldman et al.
2002).
This steady-state of feedback regulation probably alters with advancing age and changes in
the bioavailable testosterone fraction reflect this; i.e. the age-related increase in SHBG serum
levels masks the age-related decrease of bioavailable testosterone fraction, leading to only
mild decrease in total serum testosterone concentrations; however, the failure of
neuroendocrine regulation in old age to normalise bioavailable testosterone levels by a
substantial increase in SHBG concentrations (Kaufman and Vermeulen 1997) must also be
taken into account. In confirmation, there was a positive age-adjusted association of total
testosterone and SHBG in our study and the decrease of all testosterone fractions was seen in
our study earlier than age-related increase in SHBG levels.

We found an inverse correlation of BMI with total testosterone level in our study and this
association remained significant when age was taken into account. The negative association of
BMI with total testosterone was also previously reported by some authors (Barret-Connor and
Khaw 1987, Field et al. 1994, Vermeulen et al. 1996, Ferrini and Barret-Connor 1998) and in
one study this association also remained significant after adjustment for age and smoking
status (Field et al. 1994). The negative correlation of BMI with free or with bioavailable
testosterone concentrations in our study was marginally significant and the absence or very
poor association of BMI with these testosterone fractions has also been reported by some
authors previously (Field et al. 1994, Vermeulen et al. 1996, Ferrini and Barret-Connor 1998).
In our study BMI had a significant but not meaningful effect on total, free and bioavailable
testosterone levels in a multiple linear regression analysis, including age and BMI as
independent variables. However, in this context it is important to note that BMI only partially
reflects adiposity and body fat distribution (see methods).
__________________________________________________________________________________________
_______________________________________________________________________________________ 70
Adiposity affect total testosterone levels, probably through an effect of hyperinsulinism on
SHBG levels (Vermeulen et al. 1996). Furthermore, although free testosterone levels in
moderate obesity remains comparable with levels in non-obese subjects, they are also
decreased in morbid obesity (BMI>40) as a result of alterations in the neuroendocrine
regulation of testicular function (Giagulli et al. 1994).
However, the relationship between testosterone and body composition may also be reciprocal.
Lean body mass is decreased in hypogonadal person and testosterone treatment increase fat-
free mass in hypogonadal and eugonadal men (Bhasin et al. 1998, Vermeulen et al. 1999),
suggesting the role of testosterone on body fat distribution.

Some other factors are also responsible for the large interindividual variability of testosterone
serum levels. One of the most important factors in the determination of testosterone is
heredity and up to 60% of the variability of serum testosterone levels, after normalisation for
body surface area, may be attributable to genetic factors. Stress may be the cause of a
profound, but generally transient decrease of testosterone. Ultradian, circadian and circannual
variations play an important role in the wide range of normal testosterone levels (Kaufman
and Vermeulen 1997, 1998 for review). Smoking has also been found to be associated with
higher (5-15%) testosterone levels (Vermeulen et al. 1996), but the significance of this
association remains controversial (Barret-Connor and Khaw 1987, Simon et al. 1992, Field et
al. 1994). Several metabolic and hormonal factors such as insulin, IGF-1 and thyroxin
influence total testosterone levels over the SHBG binding capacity (s. SHBG).

The cause and the clinical importance of testosterone changes with age
The origin of the age-related changes in testosterone secretion may be generally explained by
Four Models of Medicine: ecological, genetic, accumulative and ontogenetic (Dilman
1987). Pathogenically, this decrease is the consequence of both primary testicular changes and
an altered neuroendocrine regulation of Leydig cell function with an inappropriate LH
secretory response to the prevailing hypoandrogenism. This results most probably from a
decreased size of the bolus of GnRH, intermittently released by the hypothalamus into the
pituitary portal circulation, notwithstanding the increased sensitivity to the negative feedback
effects of sex-steroids (Kaufman and Vermeulen 1997).
Ageing in men is generally accompanied by symptoms seen in hypogonadal young men
(Blackman 1995, Bhasin et al. 1998, Finkelstein 1998, Kaufman and Vermeulen 1998). It
certainly seems a reasonable hypothesis that at least some of the clinical changes observed in
ageing men are causally related to a progressive decline of testosterone levels. However, the
studies revealed mostly only weak correlations between total testosterone and clinical
parameters in the elderly (Kaufman and Vermeulen 1998 for review, also: Boonen et al. 1997,
__________________________________________________________________________________________
_______________________________________________________________________________________ 71
Booth et al. 1998, Khosla et al. 1998, Rapado et al. 1999, Vermeulen et al. 1999, Barrett-
Connor et al. 1999, 2000, Stellato et al. 2000, Khosla et al. 2001). Nevertheless, bioavailable
testosterone was found to be strongly positively related with muscle strength (Van den Beld et
al. 2000), bone mineral density (Greendale et al. 1997, Khosla et al. 1998, Van den Beld et al.
2000, Khosla et al. 2001), with better scores on the Blessed Information-Memory-
Concentration Test and the Selective Reminding Test (Barrett-Connor et al. 1999) and
negatively related with fat mass (Van den Beld et al. 2000) and with Beck Depression
Inventory score (Barrett-Connor et al. 1999) in old male persons.
Testosterone administration improves symptoms in young hypogonadal men (Bhasin et al.
1998, Finkelstein 1998, Leifke et al. 1998), increases bone mineral density in eugonadal men
with idiopathic primary osteoporosis (Anderson et al. 1996). However, there is limited
information about the effects of testosterone therapy in old age. Most studies showed an
increase in fat-free mass and decrease in body fat (Vermeulen et al. 1999 for review, also
Snyder et al. 1999). Testosterone supplementation significantly lowered cholesterol serum
level (Zgliczynski et al. 1996, Sih et al. 1997). It also significantly improved muscle strength
in older men in one RCT, randomised controlled trial (Sih et al. 1997), but did not increase
the strength and did not increase lumbal spine bone density in another (Snyder et al. 1999).
One of the explanations for these data is the possible existence of threshold concentrations for
the action of the androgens on tissues (Kaufman and Vermeulen 1998). Persons with very low
hormone levels appear to have more profound clinical symptoms (Kaufman and Vermeulen
1998, Barrett-Connor et al. 1999, Center et al. 1999) and more pronounced effects from
testosterone therapy (Kaufman and Vermeulen 1998). The first above mentioned RCT (Sih et
al. 1997) included old men with bioavailable testosterone levels below 60ng/dl (2nmol/l). In
the second RCT (Snyder et al. 1999) testosterone therapy increased lumbar spine bone density
only in men with low pre-treatment serum testosterone concentrations.
The proportion of men with low testosterone levels increases substantially with age. In the
group of healthy men, aged 20-100 years (Kaufman and Vermeulen 1997) more than 20% of
those between 60-80 years and about 40% of those over 80 years had total or free testosterone
levels below 11nmol/l or 0,18nmol/l respectively. In the Baltimore Longitudinal Study on
Aging (Harman et al. 2001) 12%, 19%, 28% and 49% of the persons in their 50s, 60s, 70s
and 80s, respectively, exhibit total testosterone levels below 11,3nmol/l. In our study, 9%,
13% and 26% of the persons between 50-72 years had total, free or bioavailable testosterone
levels below the lowest levels of the 20-29-year-old persons (11nmol/l, 0,16nmol/l and 4
nmol/l, respectively). Such persons, especially with low bioavailable testosterone levels, may
be in future the primary target for the testosterone substitution therapy in the elderly.
__________________________________________________________________________________________
_______________________________________________________________________________________ 72
Estrogens
The mean levels of total estradiol in our study were substantially lower than age-related mean
levels of this hormone obtained in Gent and Sheffield healthy subjects (Vermeulen et al.
1996, Fatayerji and Eastell 1999), by Quebec researches (Belanger et al. 1994) and in
Rancho-Bernardo I populations (Barrett-Connor and Khaw 1987), somewhat lower than in
Telecom employers (Simon et al. 1992) and in Rochester (Khosla et al. 1998) subjects, but
were relative similar to the levels reported for Massachusetts I (Gray et al. 1991) and for
Rancho-Bernardo II (Ferrini and Barrett-Connor 1998, Barrett-Connor et al. 1999, 2000)
populations.
The mean levels of bioavailable estradiol in our study were slightly lower than age-related
mean levels of this hormone fraction calculated for Gent and Sheffield healthy subjects
(Vermeulen et al. 1996, Fatayerji and Eastell 1999), for Massachusetts I (Gray et al. 1991)
and for Rancho-Bernardo I populations (Barrett-Connor and Khaw 1987). The cause is a
higher levels of total estradiol in the first two, lower levels of total testosterone in the third
and lower levels of SHBG in the fourth of the above mentioned studies.
In the new study on Rancho-Bernardo II population (Ferrini and Barrett-Connor 1998,
Barrett-Connor et al. 1999, 2000), which measured both total and bioavailable estradiol
levels, albeit only in 50-80 years old subjects, observed mean levels were also grossly similar
to the age-related levels of our investigation.
There is only one study, the Rochester study (Khosla et al. 1998), which measured
bioavailable estrogens in men in the wide range of ages. Although the mean levels of total
estradiol and estrone in our study were slightly lower than in this report, mean levels of
bioavailable estradiol were comparable.
The mean levels of serum estrone in our study were a bit lower than age-related mean levels
obtained in Rancho-Bernardo I subjects (Barrett-Connor and Khaw 1987) and in Telecom
employers (Simon et al. 1992), but similar to the mean estrone levels observed in
Massachusetts I (Gray et al. 1991), Rochester (Khosla et al. 1998) as well as in Massachusetts
II (Feldman et al. 2002) populations. Surprisingly high mean estrone levels (400-700 pmol/l)
were reported by Quebec researches in their population-based study (Belanger et al. 1994).
Nevertheless, it is not clear, measurements of which study reflect mean concentrations of total
estradiol and estrone in blood serum better, and whether calculations of bioavailable estradiol
reflect serum levels of this hormone fraction accurately. However, bioavailable estradiol,
calculated with the same method in one other study (Van den Beld et al. 2000), has been
shown to be the best parameter of serum bioactive estradiol in describing its positive relation
with bone mineral density in old male persons.
__________________________________________________________________________________________
_______________________________________________________________________________________ 73
Our study as well as Massachusetts I (Gray et al. 1991), Quebec (Belanger et al. 1994) and
Sheffield (Fatayerji and Eastell 1999) studies suggest an exponential decrease of total
estradiol, and our study also of bioavailable estradiol, with age. One study, Rancho Bernardo
II (Ferrini and Barrett-Connor 1998), approximate changes of total and bioavailable estradiol
concentrations with age by linear regression. This model seems to be less appropriate because
of the skewed distribution of the hormone levels. With regard to estrone, only exponential
model was used in the studies to describe its age-trends. None of the other investigated
models (quadratic, cubic and inverse) showed a meaningfully higher prediction value of
serum levels of estrogen fractions in our study.
Population-based studies showed no or weak association of total estradiol levels with age and
calculated for these studies mean age-trends were between 3-0%/decade. However, the
calculated mean age-trend of total estradiol for Sheffield healthy subjects were similar to our
observation (-7%/decade). These differences between the studies are probably because of a
higher proportion in the population-based studies of adipose old men or men with
cardiovascular disorders, factors known to increase total estradiol levels (Barrett-Connor and
Khaw 1987). Although the Ghent study on healthy subjects also showed stabile total estradiol
levels with advancing age, this may be because of higher proportion of smokers in old age-
groups of this study, subjects known to have also increased estradiol values (Barrett-Connor
and Khaw 1987). However, calculated mean age-trends for serum bioavailable estradiol
fraction showed more similarities in the studies on population-based and on healthy subjects
and varied between 5%-10% and 5%-12% pro decade, respectively.
Most of the studies showed none or very low association of estrone serum levels with age and
the calculated mean age-trends for these studies varied between negative and positive values
(8% and +6% pro decade). However, Quebec (Belanger et al. 1994) and especially
longitudinal analyse of Massachusetts population (Feldman et al. 2002) reported substantially
higher decreases of estrone levels, -11% and 31% pro decade, respectively.
Studies with higher age-trends for estrone serum levels were population-based and may
represent mean serum hormone age-trends in population, but not changes caused by ageing
itself (what was also suggested by the authors, Feldman et al. 2002). Nevertheless, age-trend
of 31%/decade (95%CI: -24%; -27%) seems to overestimate the mean age-trend even for
population and is probably caused by inaccuracies, such as drift in assays measurements or a
long storage time of the samples (s. methodological issues). Care should also be taken
concerning data from Quebec researches because of extremely high estrone levels and
concerning results of calculations from the mean levels for Rancho Bernardo I population. In
studies on healthy men age-trends varied between 0% and 4% pro decade.
Rochester researchers also presented levels of bioavailable estrone fraction, which showed
age-trends in the same degree as bioavailable estradiol fraction. This is very surprising as far
as estrone practically has no SHBG-bound-fraction (Sdergard et al. 1982).
__________________________________________________________________________________________
_______________________________________________________________________________________ 74
No study was ideal to describe the proper effect of age on estrogens serum levels. After
summarising data from the reported studies it may be concluded that the decrease of serum
estrogen fractions with age may be approximated by an exponential function and that in
healthy adult men total estradiol decreases on average up to 7% pro decade, whereas
bioavailable estradiol fraction between 5%12% pro decade. In the general population,
however, the mean decrease of estradiol fractions is diminished, probably because of the
increase with age of adipose tissue. Serum estrone concentration decreases in healthy adult
men most likely similar to that of total estradiol; however, its changes, especially in the
general population, remain to be further investigated.
Using these age-trends it may be estimated that at the age of 65, 75 and 85 years in healthy
men remain on average over 75%, 70% and 65% of the total estradiol serum levels of 25-
year-old persons, respectively. However, at the same age the concentrations of bioavailable
estradiol remain on average between 60-81%, 53-77% and 46-74% (Fig. 25). Remaining
percentage of estrone is largely similar to that of total estradiol.
Fig. 25. Conclusions: average changes of total and bioavailable estradiol
serum levels and remaining hormone concentrations in healthy adult men with age.

However, variable age was accounted in the studies for up to 15%, 20% and 2% in total
variability of total, bioavailable estradiol and estrone serum levels, respectively, (r-model)
and at any age these fractions exhibited a large interindividual variability (SD%>50% in some
studies).
0
30
60
90
120
150
20 30 40 50 60 70 80 90 100
age (years)
t
o
t
a
l

a
n
d

b
i
o
a
v
a
i
l
a
b
l
e

e
s
t
r
a
d
i
o
l

(
p
m
o
l
/
l
)
46%
53%
60%
100% 100% 100%
100%
100%
bioavailable estradiol
total estradiol
decrease up to 7%/decade
74%
77%
81%
65%
70%
75%
-12%/decade
-5%/decade
0%/decade
-7%/decade
__________________________________________________________________________________________
_______________________________________________________________________________________ 75
Effect of SHBG, BMI and other factors
As in the case for testosterone, the differences in age-related decreases in total and
bioavailable estradiol fractions are caused by the age-related increase in sex-hormone binding
globulin concentrations; however, the commonly used expression that increase in SHBG with
age leads to a more profound decrease in bioavailable estradiol concentrations is probably
also misleading.
Bioavailable estradiol is likely to reflect estrogen activity and the steady-state of feedback
hormone regulation better as total estradiol serum levels. In this case total estradiol should
only be a subject of SHBG variability. This suggests the association of bioavailable
testosterone and estradiol levels, the highest of all associations between testosterone and
estradiol fractions in our study. Additionally, the age-trends of FSH and LH in some studies
were also more similar with the age-trend of bioavailable than total estradiol fraction (Khosla
et al. 1998, Feldman et al. 2002). Although there was only a weak age-adjusted association of
total estradiol with SHBG levels in our study, it is probably a sign of different effects of
adipose tissue on bioavailable estradiol (positive) and SHBG (negative) concentrations.

Similar to the Rancho Bernardo study (Barrett-Connor and Khaw 1987, Ferrini and Barrett-
Connor 1998), we found no association of serum total estradiol and estrone levels with body-
mass-index. Also age-adjusted effect of BMI on total estradiol and estrone serum levels was
very small, as has been shown in our study by multiple regression of age and BMI on serum
levels of these estrogens. In contrast, by a similar model, we found a high age-adjusted
association of BMI with bioavailable estradiol fraction, probably indicating that this
parameter reflect the testosterone-converting activity of the adipose tissue better than total
estradiol concentration.
As testosterone, precursor of estradiol, decreases but BMI increases with advancing age, it
may be postulated that an age-related increase of adipose tissue (and BMI) compensates the
decrease of the bioavailable estradiol fraction with age. According to the b-coefficient of
multiple regression model, the age-related decrease of bioavailable estradiol without age-
related increase of body-mass-index could be substantially higher (-16%/decade), practically
as high as the decrease of bioavailable testosterone fraction.

Some other factors are responsible for the large interindividual variability of estrogens serum
levels. All factors affecting testosterone levels are also responsible for the wide range of
estrogen concentrations (see testosterone). In confirmation, there were positive age-adjusted
associations of all testosterone with all estrogen fractions in our study. Moreover, all estrogen
fractions can be converted to each other and estrone considered to be practically non-active in
men may act as a precursor of bioactive estradiol. In our study all estrogen fractions were
__________________________________________________________________________________________
_______________________________________________________________________________________ 76
associated with each other, also after controlling for age. Smoking increases total estradiol
and estrone levels (Barrett-Connor and Khaw 1987, Simon et al. 1992) and alcohol probably
increases estrone (Simon et al. 1992). As in the case of testosterone, estradiol was also shown
to have a significant daytime variation (Ahokoski et al. 1998).

The cause and the clinical importance of changes in estrogens with age
Most circulating estradiol in the male derives from the adipose tissue and the testis. The
activity of these two tissues plays a central role in the timing of puberty and its associated
growth effects (Sharpe 1998). The age-related decrease in testosterone is probably the most
important factor in the decrease in serum estradiol levels. However, the increase in adipose
tissue and its aromatase activity with age appear to compensate partially these changes,
increasing conversion of testosterone in estradiol. Additionally, in the peripheral tissues
estrogens are also derived by the enzyme aromatase by conversion of both testicular and
adrenal androgens, such as androstenedione and DHEA (De Kretser et al. 1995) and over 50%
decline in serum concentrations of adrenal sex steroid precursors and total androgens during
ageing, previously described by some authors (Belanger et al. 1994, Labrie et al. 1997),
appear to contribute substantially to the decline in estrogens in peripheral tissues.

Decreased serum total and especially bioavailable estradiol levels in men are associated with
decreased bone density (Greendale et al. 1997, Slemenda et al. 1997, Khosla et al. 1998,
Center et al. 1999, Van den Beld et al. 2000, Khosla et al. 2001) and a graded association
between higher concentrations of serum total as well as bioavailable estradiol and lower
fracture prevalence in men was reported (Barrett-Connor et al. 2000). A recent interventional
study on elderly men revealed that estrogens play the major role in regulating bone resorption,
and that both estrogens and testosterone are important in maintaining bone formation (Khosla
et al. 2001). It was hypothesised, that estrogens deficiency plays a major role in involutional
bone loss not only in women, but also in men (Riggs et al. 1998, Finkelstein 1998). It is also
interesting that men and women have relative similar serum levels of bioavailable estradiol
fraction, that may be also in agreement with similar function of this fraction. The crucial role
of estrogens for both sexes would not be surprising, if we consider the general principle of
parsimony, operating in evolutionary genetic (Tarlov 1969).
It may be also suggested that some effects seen by testosterone supplementation, especially on
bone mineral density, are mediated by conversion of testosterone in estradiol. Indeed, non-
aromatisable androgens cannot develop the full spectrum of testosterone activities (Bhasin et
al. 1998). Estrogens replacement effects on both epiphyseal closure and bone mineral density
in patients with aromatase deficiency and causes a complete suppression of serum
gonadotropins, whereas androgen treatment has no such effects (Sharpe 1998, Faustini-Fustini
__________________________________________________________________________________________
_______________________________________________________________________________________ 77
et al. 1998 for review, Bilezikian et al. 1998). Some experts advocate the use of estrogens in
male senescence, in particular because of anticipated positive cardiovascular effects.
However, in the new study of a 9-week therapy with micronized estradiol on men with
osteopenia of the femoral neck was concluded that it is feasible to give low dose estrogen to
healthy older men, but that the effects on bone turnover are not consistent (Taxel et al. 2000).
On the other hand, estrogens have been suggested to cover only some of the metabolic facets
of testosterone action (Bhasin et al. 1998).
It is not clear if some threshold concentration exists for estrogens action, as in the case for
testosterone. However, elderly men with bioavailable estradiol levels below 40pmol/L were
found to have significantly higher rates of bone loss and levels of bone resorption markers
than men with levels above 40pmol/L (Khosla et al. 2001). The proportion of men with very
low bioavailable estradiol levels, as seen in our investigation, increases substantially with age
and 16% person between the ages of 50-70 years had bioavailable estradiol levels below the
lowest values of the 20-29-year-old persons (28pmol/l).
__________________________________________________________________________________________
_______________________________________________________________________________________ 78
Insulin-like-growth-factor-I
IGF-I mean levels obtained in our study were a little lower than age-related mean IGF-I levels
obtained by other researchers in young men but grossly similar to the levels obtained in older
subjects (Jamomoto et al. 1991, Juul et al. 1994, Landin-Wilhelmsen et al. 1994, Nystrm et
al. 1997, OConnor et al. 1998, Hilding et al. 1999, Fatayerji and Eastell 1999), Small
differences between the studies in measured IGF-I levels are due to the different assays used
in the investigations. It is important that IGFBP-blocked assay employed in our study
eliminate the interference with IGFBP's better than conventional used methods such as acid-
ethanol extraction and others (Blum and Breier 1994).
All reported studies showed relative strong negative associations of IGF-I levels with age,
with correlation coefficients ranging between -0,46 and -0,77. Most studies employed a linear
regression of untransformed (Jamomoto et al. 1991, Landin-Wilhelmsen et al. 1994, Nystrm
et al. 1997, O'Connor et al. 1998), of log-transformed (Hilding et al. 1999, Yu et al. 1999), or
of square-root-transformed (Juul et al.1994) IGF-I serum levels on age; however, in one study
this association was best-fitted by a quadratic function after log-transformation of the data,
with the nadir being reached at the age of 76 years (Fatayerji and Eastell 1999).
Although most authors approximated IGF-I changes in their studies with linear regression
model, these studies were relative small to detect the skewed distribution of the hormone
levels. In our investigation (with the largest sample-size) logarithmical transformation of the
data increased the normality of distribution, although the fittings of these two models differed
only marginally. However, we thought that the exponential model would be more appropriate
to describe age-related serum IGF-I changes. Thus, after estimation of mean levels for age-
groups from regression equations reported in the studies, we calculated mean age-trends for
exponential regression.
The calculated mean age-trends of serum IGF-I levels in the population-based studies varied
between 10% and -16% pro decade; in the studies on healthy subjects these age-trends
varied between 10% and -15% pro decade. No associations of the calculated age-trends with
age-intervals, investigated in these studies, were found. The mean age-trend of about -16%
pro decade, calculated from the quadratic equation for the Copenhagen occupation-based
study (Juul et al. 1994), appear to overestimate the proper effect of age on IGF-I levels.
Estimated from the studies mean age-trends for linear regression varied in population-based
studies between 2,6 and 4,3 nmol/l/decade; in studies on healthy subjects between 2,6 and
3,4 nmol/l/decade. Extrapolating these trends for the whole range of investigated ages (20-90
yrs), mean age-trends about 4,3 nmol/l/decade appear to overestimate the mean age-related
changes of serum IGF-I levels even in the general population (Fig. 26).
__________________________________________________________________________________________
_______________________________________________________________________________________ 79
No study was ideal to describe the proper effect of age on IGF-I serum levels. After
summarising data from the studies it may be concluded that the mean decrease of serum IGF-I
with age may be approximated by an exponential function and that in healthy adult men IGF-I
decreases on average between 10-15% pro decade. Using these age-trends it may be estimated
that at the age of 65, 75 or 85 years serum IGF-I levels in healthy men exhibit on average
between 52-66%, 44-59% or 38-53% of serum levels of 25-year-old persons (Fig. 26). The
mean age-related decrease of IGF-I serum levels in healthy adult men can also be (although
badly) approximated by linear function and this increase is on average between 2,63,4
nmol/l/decade. In the general population, however, most likely due to a higher rate of diseases
in old age, the mean decrease of IGF-I is, probably, weakly accentuated.
Fig. 26. Conclusions: average changes of Insulin-like factor I serum levels and remaining
hormone concentrations in healthy adult men with age.

Variable age was accounted in the studies for up to 60% in total variability of IGF-I serum
levels (r-model) and at any age serum IGF-I exhibited a large interindividual variability
(SD%>40% in some studies).

Effect of BMI, sex-steroids and some other factors
Similar to some other reports (Landin-Wilhelmsen et al. 1994, Nystrm et al. 1997, O'Connor
et al. 1998), serum IGF-I level in our study was inversely related to BMI, but this relation
disappeared when age was taken into account. In contrast, significant age-independent inverse
correlations of various measures of adiposity with basal GH concentrations, spontaneous GH
0
5
10
15
20
25
30
35
40
20 30 40 50 60 70 80 90 100
age (years)
I
G
F
-
I

s
e
r
u
m

l
e
v
e
l
s

(
n
m
o
l
/
l
)
Insulin-like growth factor I
(for comparison shown linear age-trends:
2,6-3,4nmol/l/decade - dashed lines)
100%
38%
44%
52%
53%
59%
66%
-15%/decade
-10%/decade
__________________________________________________________________________________________
_______________________________________________________________________________________ 80
release and integrated daily plasma GH concentrations are well known (Corpas et al. 1993)
and reduced IGF-I levels in obese subjects vs. non-obese age-matched controls were found
previously by some authors (Copeland et al. 1990, Vermeulen et al. 1996). The absence of the
age-adjusted associations between IGF-I and BMI in the reported studies suggests that such
relationship does not truly exist at the BMI levels of the subjects included in these studies.
However, age-related IGF-I hyposecretion in men appear to be more strongly related to the
proportion of abdominal fat, as assessed by waist/hip ratios, than to total body fat, as assessed
by body-mass-index (Corpas et al. 1993).
As both ageing and obesity are associated with elevation of plasma insulin, a hormone
structurally similar to IGF-I, one of the possible mechanisms of the effect of body
composition on IGF-I level is an increase in insulin action on hypothalamic and/or pituitary
IGF-I receptors, resulting in enhanced feedback inhibition of GH and, hence, IGF-I secretion.
Therefore, one can speculate that obesity, hyperglycaemia, insulin, nutrition and physical
activity, known to affect GH and IGF-I levels (Corpas et al. 1993, Nystrm et al. 1997,
Hilding et al. 1999), may have a common causative mechanism of their action. In addition,
GH and IGF-I concentrations might also increase after weight loss (O'Connor et al. 1998,
Vermeulen et al. 1996), although remain lower than in non-obese controls (Vermeulen et al.
1996).
It has been also suggested that there is a reciprocal relationship between adiposity and GH
secretion, with more body fat leading to less GH secretion and reduced GH secretion leading
to increased body fat (Corpas et al. 1993). GH-deficient person have increased body fat mass
and GH administration increases lean body mass and reduces adipose mass in older men and
in men with GH deficiency (Corpas et al. 1993, O'Connor et al. 1998).

Somatotropic axis is associated with gonadal axis. Pubertal increase in plasma estradiol and
testosterone augments pulsatile GH secretion, what may be important for the timing and
synchronisation of human puberty. A strong positive relationship between estradiol levels and
GH secretion in adult women and a correlation between serum testosterone levels and daily
GH secretion in old men were also reported. Although our study revealed no age- and BMI-
adjusted association of serum IGF-I levels with total testosterone and marginally significant
with total estradiol, a comparable to another study (Pfeilschifter et al. 1996) age- and BMI-
adjusted association of IGF-I with bioavailable testosterone, and a significant age- and BMI-
adjusted association of IGF-I with bioavailable estradiol levels were found. These
associations were stronger as age- and BMI-adjusted association of IGF-I with SHBG levels.
These findings deny the suspicion that they resulted from the calculations of the bioavailable
hormone fractions. However, the interrelation of the somatotropic axis with sex-steroids in
adults appear to be of minor physiological importance (Corpas et al. 1993 for review).
__________________________________________________________________________________________
_______________________________________________________________________________________ 81
Androgen administration enhances GH secretion in peripubertal boys, in men with
hypogonadotropic hypogonadism (Corpas et al. 1993 for review) and in normal men (Hobbs
et al. 1993), but the latter was significant only for administration of aromatisable but not for
non-aromatisable testosterone. This fact and the fact, that spontaneous and stimulated GH
secretion are higher in women than in men, suggests the primary importance of estradiol for
this action. However, some facts are in contradiction to this hypothesis: premenopausal
women show a decline in somatotropic activity despite high endogenous estradiol levels,
menopause has no impact on the concentration of IGF-I in peripheral tissues, such as bone,
and estrogen replacement therapy is not capable of changing the decline in somatotropic
activity with age (Pfeilschifter et al. 1996).
The potential mechanisms of the association of gonadal and somatotropic axes remain unclear
and, moreover, these interrelations may be reciprocal. The influence of the somatotropic axis
on gonadal activity, probably at the pituitary level, was evident in some experiments on
animals (Pfeilshifter et al. 1996 for review).

Some other factors are also responsible for the large interindividual variability of IGF-I levels.
Serum concentration of IGF-I in postnatal life is primarily dependent upon GH, and factors
influencing GH serum levels can also affect serum IGF-I concentrations. Smoking status and
serum fibrinogen (Landin-Wilhelmsen et al. 1994) or haemoglobin (Nystrm et al. 1997)
were found to be positively (fibrinogen and haemoglobin) or negatively (smoking status)
associated with IGF-I serum levels in population-based studies when age was taken into
account. Genetics, season, nutritional status, thyroid hormones and other variables also affect
GH and IGF-I concentrations (Blum and Breier 1994).

The cause and the clinical importance of IGF-I changes with age
As in the case of testosterone, the origin of the age-related changes in GH and IGF-I secretion
may be generally explained by Four Models of Medicine: ecological, genetic, accumulative
and ontogenetic (Dilman 1987). Although pathogenically no central unifying basis has been
proven to underlie these changes, some evidence suggests that primarily, the depression of
central cholinergic tone in the ageing brain leads to enhanced somatostatinergic activity,
which in turn depresses the somatotropic axis (Marcus and Hoffman 1998).

The clinical importance of changes in GH/IGF-I axis with age and possible benefits in
supplementation of somatotropic axis have been reviewed by some authors (Corpas et al.
1993, Blackman et al. 1995, Marcus and Hoffman 1998). Briefly, both ageing and GH
deficiency are associated with reduced protein synthesis, decreases in lean body mass and
bone mass, and increases in body fat. Therefore, it may be presumed that reduced GH and
__________________________________________________________________________________________
_______________________________________________________________________________________ 82
IGF-I secretion might account, at least in part, for one or more of the effects of ageing, and
that some older people might benefit from GH/IGF-I axis supplementation. GH treatment of
GH-deficient adults or old men with reduced IGF-I levels increases plasma IGF-I, nitrogen
retention, lean body mass and decreases the percentage of body fat, but does not offer a
clinically useful improvement in bone mass.
It is not clear if some threshold concentration exists for IGF-I action, as in the case for
testosterone. However, the prevalence of person with low IGF-I serum levels increases with
advancing age (Corpas et al. 1993 for review, Blackman et al. 1995 for review, Abbassi et al.
1993, O'Connor et al. 1998). In one study (Abbassi et al. 1993) 85% of healthy older men,
aged 59-98 years, and in one other study (O'Connor et al. 1998) 59% of older men exhibited
reduced, below the 2,5
th
percentile of values of young persons, aged 20-29 years. In our study
19% persons between the age of 50-70 had IGF-I levels below the lowest levels of young
persons, aged 20-29 years (16nmol/l).

The extent to which age-related changes of GH and IGF-I contribute to alterations in body
composition and function remains to be elucidated and a great deal of research is necessary to
determine whether normalisation of GH and IGF-I levels in older men would lead to
improvements in their physical and psychological functional capacity and quality of life.
Observations about the interrelation of gonadal and somatotropic axis, the increase in
prevalence of person with simultaneously low gonadal steroids and IGF-I levels, as seen in
our study, as well as the 11-fold increase in HIP-fracture prevalence in old people with
simultaneously three subnormal hormonal parameters (Center et al. 1999) highlight the
importance of considering the interrelation of these axes in studies of the effect of ageing.
__________________________________________________________________________________________
_______________________________________________________________________________________ 83
Conclusions
Our investigation on 514 blood donors aged 20-72 years revealed that in healthy men SHBG
serum levels increases, whereas serum concentrations of all hormone fractions decreases with
advancing age. Cross-sectional age-trends of SHBG and hormonal parameters pro decade
(meanse) are 91% for SHBG, -101% for total testosterone, -171% for bioavailable
testosterone, -171% for free testosterone, -71% for total estradiol, -121% for bioavailable
estradiol, -41% for estrone and -101% for IGF-I serum levels. BMI, SHBG, IGF-I and sex-
hormone fractions are interrelated, however, the magnitude of these interrelations is small.
Only BMI exerts a meaningful age-adjusted effects on SHBG levels. Of all sex-steroid
fractions bioavailable estradiol appear to be the best predictor of serum IGF-I concentrations.
The prevalence of men with low serum levels of sex-steroids and IGF-I increases with
advancing age.
No study reporting changes of SHBG, IGF-I and sex-steroids serum levels in adult men with
age 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 it may be concluded that exponential function
appear to be the most appropriate to describe age-related changes and that in healthy adult
men 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.
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 (Tabl. 28).
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.
age-trend % changes % remaining levels
%/decade 65 years 75 years 85 years 65 years 75 years 85 years
SHBG 8-14 36-69 47-93 59-119 136-169 147-193 159-219
Total testosterone -4-10 -15-34 -18-41 -22-47 66-85 59-82 53-78
Bioavailable testosterone -10-17 -34-53 -41-61 -47-66 47-66 39-59 33-53
Free testosterone -10-17 -34-53 -41-61 -47-66 47-66 39-59 33-53
Total estradiol -0-7 -0-25 -0-30 -0-35 75-100 70-100 65-100
Bioavailable estradiol -5-12 -19-40 -23-47 -26-54 60-81 53-77 46-74
Estrone -0-7 -0-25 -0-30 -0-35 75-100 70-100 65-100
Insulin-like growth factor I -10-15 -34-48 -41-56 -47-62 52-66 44-59 38-53

__________________________________________________________________________________________
_______________________________________________________________________________________ 84
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Zmuda-JM; Cauley-JA; Kriska-A; Glynn-NW; Gutai-JP; Kuller-LH. Longitudinal relation
between endogenous testosterone and cardiovascular disease risk factors in middle-aged men. A 13-
year follow-up of former Multiple Risk Factor Intervention Trial participants. Am-J-Epidemiol. 1997
Oct 15; 146(8): 609-617.

__________________________________________________________________________________________
_______________________________________________________________________________________ 93
ANHANG
Danksagungen

Ich bedanke mich ganz herzlich bei Herrn Professor Dr. Georg Brabant fr die berlassung
des Dissertationsthemas und fr die hilfsbereite Betreuung.

Mein offenherzigen Dank gilt auerdem Herrn Dr. Eckhard Leifke fr die sachliche, kritische
und freundliche Untersttzung und fr die stetige Diskussionsbereitschaft.

Ich bin Herrn Professor Dr. Sandeep Khosla (Mayo Clinic, Rochester, Minnesota, USA) sehr
dankbar fr die bersendung der zustzlichen Information zu seinem Artikel.

Auerdem bedanke ich mich ganz herzlich bei Frau Renee Klie fr die sprachliche
berprfung und Korrektur des Manuskriptes.

Ein ganz besonderer Dank gilt meiner Ehefrau, Svetlana, die durch ihre Geduld, ihr
Verstndnis und ihre Liebe eine unschtzbare Untersttzung bei der Erstellung der
Dissertation leistete.

Ich bin besonders dankbar meinem ersten Lehrer, Herrn Professor Dr. Michail Dilman (St.
Petersburg, Ruland; gestorben), der durch seine Ideen und Werke mein Interesse an
Wissenschaft seit den studentischen Jahren geprgt hat.

__________________________________________________________________________________________
_______________________________________________________________________________________ 94
Vitali Gorenoi geboren am 12.12.66 in St.Petersburg (ehem. Leningrad)
Staatsangehrigkeit: Russische Fderation (ehem. UdSSR)

Lebenslauf
Schulausbildung
1974-1984 Grund- und hhere Schule Nr. 105
St. Petersburg, Russische Fderation (ehem. UdSSR).
Abschlu: Staatsexamen, entspr. Abitur.
Studium der Humanmedizin
1984-1993 1. Leningrader (akad. I.P.Pavlov) Medizinische Hochschule
St. Petersburg, Russische Fderation. /Whrend des Studiums:
-Sprachkurs Englisch fr Mediziner
-Famulatur an der Universitt Padova, Italien
-Teilnahme an wissenschaftlichen Ttigkeiten
Abschlu: Diplom; entspr. 3. Staatsexamen
1985-1988 Studiumunterbrechung wegen Wehrdienstpflicht:
Militrdienst bei der Marine, Schwarzmeerflotte.
Deutscher Sprachkurs
1994-1995 Intensivsprachkurs beim DGB-Bildungswerk in Hannover
rztliche Weiterbildung
1995-1996 6-monatiges Fortbildungsseminar der rztekammer Nordrhein
Qualifizierung fr Klinik und Praxis in Kln.
1996-1999 Ttigkeit als Arzt im Praktikum
-1/2 Jahr - Klinik fr medizinische Rehabilitation und Geriatrie
Henriettenstiftung Hannover (im Rahmen des Seminars)
-1/2 Jahr - Praxis fr Allgemeinmedizin in Hannover
-1/2 Jahr - Abteilung fr Allgemeinmedizin, MHH
Wissenschaftliche Mitarbeit im Bereich der Endokrinologie
1998-2000 Untersuchung der altersabhngigen hormonellen Vernderungen bei
Mnnern und mnnlicher Osteoporose
Abteilung fr Endokrinologie, Medizinische Hochschule Hannover.
2000 Untersuchung der Knochendichte bei lteren gesunden Frauen
Abteilung fr Endokrinologie, Medizinische Hochschule Hannover.
Weiterbildung im Bereich Bevlkerungsmedizin und Gesundheitswesen
2000- bis heute Ergnzungsstudiengang Bevlkerungsmedizin und Gesundheitswesen,
(Public Health); Medizinische Hochschule Hannover
2001- bis heute Erstellung eines HTA-Reports: Stenting vs. Ballondilatation bei
koronarer Herzkrankheit Abt. fr Epidemiologie, Sozialmedizin und
Gesundheitssystemforschung, Medizinische Hochschule Hannover

Vitali Gorenoi
__________________________________________________________________________________________
_______________________________________________________________________________________ 95
Liste der wissenschaftlichen Verffentlichungen

Hager-K, Gorenoi-V, Breidung-R.
Schmerzen in einer geriatrischen Klinik (Abstrakt bei dem 3. Kongress der Deutschen
Gesellschaft fr Gerontologie und Geriatrie; Leipzig, Deutschland; 18-21 September, 1996).
In Schtz-RM, Ries-W, Tews-HP. Altern in Gesundheit und Krankheit. 3. Kongress der
Deutschen Gesellschaft fr Gerontologie und Geriatrie. Melsungen: Bibliomed.

Hager-K, Gorenoi-V, Breidung-R.
Prvalenz von Schmerzen in einer geriatrischen Klinik.
Geriatrie Forschung, 1999: Vol. 9(1): 35-39.

Leifke-E, Wichers-C, Gorenoi-V, Lucke-C, von-zur-Mhlen-A, von-Bren-E, Brabant-G.
Serum testosterone, not estrogens are lowered in men with HIP Fractures (Poster by the 81
st

Annual Meeting of the Endocrine Society /ENDO 1999/; San Diego, USA; 12-15 June, 1999).

Leifke-E, Gorenoi-V, von-zur-Mhlen-A, von-Bren-E, Brabant-G.
Age-related changes of serum levels of total, bio-available and free sex hormones, insulin-
like growth factor-1 and sex-hormone binding globulin in a cohort of 572 healthy, physically
active, non-obese and German men from 20 to 80 years (Abstract by the 44
th
Symposion of
the German Society of Endocrinology; Munich, Germany; May 31th -June 3
rd
, 2000).
Experimental and Clinical Endocrinology and Diabetes, 2000: Vol. 108(Suppl. 1): S2.

Leifke-E, Gorenoi-V, Wichers-C, von-zur-Mhlen-A, von-Bren-E, Brabant-G.
Age-related changes of serum sex hormones, insulin-like growth factor-1 and sex-hormone
binding globulin levels in men: cross-sectional data from a health male cohort.
Clinical Endocrinology, 2000: Vol. 53(6): 689-695.

Gorenoi-V, Leifke-E, Brabant-G.
Age-related changes of testosterone and estradiol fractions in men (Abstract by the 17
th

Congress of the International Association of Gerontology; Vancouver, Canada; Juli 1-6,
2001).
Gerontology, 2001: Vol. 47(Suppl. 1): 540.

Gorenoi-V, Dintsios-ChM, Perleth-M.
Stenting versus Ballondilatation bei koronarer Herzkrankheit. Systematische bersicht zur
medizinischen Effektivitt. (Im externen Review beim Deutschen Institut fr Medizinische
Dokumentation und Information).

Vitali Gorenoi
__________________________________________________________________________________________
_______________________________________________________________________________________ 96
Erklrung nach 2 Abs. 2. Nrn. 5 und 6

Ich erklre, dass ich die der Medizinischen Hochschule Hannover zur Promotion eingereichte
Dissertation mit dem Titel:

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

an der Abteilung fr Gastroenterologie, Hepatologie und Endokrinologie der Medizinischen
Hochschule Hannover unter Betreuung von Professor Dr. Brabant ohne sonstige Hilfe
durchgefhrt und bei der Abfassung der Dissertation keine anderen als die dort ausgefhrten
Hilfsmittel benutzt habe.

Ich habe diese Dissertation bisher an keiner in- oder auslndischen Hochschule zur Promotion
eingereicht. Weiterhin versichere ich, dass ich den beantragten Titel bisher noch nicht
erworben habe.

Ergebnisse der Dissertation wurden teilweise in der Zeitschrift Clinical Endocrinology
(Oxford) im Dezember 2000 (Vol. 53: 689-695) und im Kongreband des 17
th
Congress of
the International Association of Gerontology (Gerontology. 2001, Vol. 47, Suppl. 1: 540)
verffentlicht.

Hannover, den ______________________________

___________________________________________
(Vitali Gorenoi)

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Aus der Abteilung fr Gastroenterologie, Hepatologie und Endokrinologie

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- total concentration of steroid Si,

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