This work was presented, in part, at the meeting of the European Symposium on Calcified Tissue in Harrogate, U.K., April 1997, and the ASBMR in Cincinnati, OH, U.S.A., September 1997.
Biochemical markers of bone turnover can be used to study the pathophysiology of osteoporosis. So far there have been few such studies in men. The aims of this study were to determine the effect of aging on bone turnover and to identify which hormones might regulate bone turnover in men. We studied 178 healthy Caucasian men, ages 20–79 years (30 per decade). The data for the effect of age on bone turnover was best fit by a quadratic function (nadirs at age 56, 57, 53, 39, and 58 years for intact propeptide of type I procollagen, osteocalcin, bone alkaline phosphatase, free deoxypyridinoline, and cross-linked N-telopeptides of type I collagen, respectively). For most markers, bone turnover tended to be highest in the third decade, lowest in the fifth and sixth decade, with a small increase in some markers in the eighth decade. Insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3, dehydroepiandrosterone sulfate, testosterone, estradiol, and free androgen index all decreased significantly with age (54, 17, 76, 26, 33, and 57%, respectively), while sex hormone binding globulin and parathyroid hormone increased significantly with age (62% and 43%). IGF-I and sex hormones were positively correlated with bone turnover, and this association was stronger in young men than older men. In conclusion, increased IGF-I and sex hormones may be associated with increased bone turnover in young men, with less influence on bone turnover in older men.
In men, as in women, bone mineral density (BMD) in later life depends on the peak bone mass attained in young adulthood and the subsequent rate of bone loss with age.
Peak bone mass in men is related to hormonal factors, dietary calcium, genetic factors, and lifestyle. After reaching peak bone mass at about age 30 years, men maintain a stable bone density, before losing bone with advancing age.(1)
The effect of age on bone resorption and bone formation in men is still controversial. Some studies reported a decrease in bone formation(1,2) and bone resorption(1) with age, while others reported an increase in bone formation(3) and resorption(2) with age.
There are several possible hormonal determinants of bone turnover and these include parathyroid hormone (PTH), growth hormone (GH), insulin-like growth factors and their binding proteins, and sex hormones. Although these hormones have been studied in relation to BMD,(1) their importance in the attainment of peak bone mass and in the development of age-related bone loss in men is unclear.
In men, PTH levels are known to increase with age.(4) PTH is involved in calcium regulation: in bone it increases the rate of resorption,(5) and in the kidney it increases 1,25-dihydroxyvitamin D (1,25(OH)2D) production.(6) Elevated PTH levels may lead to bone loss in the event of vitamin D insufficiency, vitamin D resistance, or renal impairment.
GH acts directly on bone by causing precursor cells to commit to the osteoblast lineage,(7) and indirectly via insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein (IGFBP3), and 1,25(OH)2D. In men, IGF-I decreases with age,(8–10) possibly due to decreased physical activity(11) and decreased GH secretion.(8) Although IGF-I and IGFBP3 are decreased in osteoporotic men,(12) their relationship to bone density in healthy men is less certain. Johansson et al.(13) found a strong correlation between femoral neck BMD and IGF-I, IGFBP3, and the GH response to GH releasing hormone. Serum IGF-I levels are lower in osteoporotic(14) and elderly(15) men compared with healthy young men.
In men there is a decrease in testicular function and circulating male sex hormones with age,(16,17) which is exacerbated by underlying illness or obesity.(17) Male hypogonadism has been associated with osteoporosis(18) and testosterone replacement therapy has been associated with increased bone formation defined by histomorphometry(19) and increased bone density.(20) However, it is unclear whether there is a threshold below which sex hormone status has a deleterious effect. Free testosterone has been related to bone density at the spine,(16) hip,(21) and forearm,(18) in some studies, but others have been unable to confirm a relationship between free testosterone and BMD at multiple sites after adjusting for age.(1) Testosterone may influence bone metabolism through peripheral conversion to estrogens: there is a rapid, sustained rise in estrogen during treatment with testosterone,(22) and histomorphometry suggests that the effects of testosterone and estradiol are different and complimentary.(23)
A study that evaluates bone turnover and its related factors in men across a broad age range is required to determine the relative importance of different factors at different stages of life.
The aims of this study were to study men across a broad age range to determine the effect of aging on bone formation and bone resorption in men, and identify which factors play a direct role in the regulation of bone turnover in men.
MATERIALS AND METHODS
We studied 178 men, ages 20–79 years. Letters were sent to men on a local general practitioner's register, inviting then to participate in the study. Letters were sent until ∼30 men per decade were recruited (uptake rate was 20%, of which 70% were eligible). The study was approved by the North Sheffield Local Research Ethics Committee, and all subjects gave written informed consent.
The selection process ensured that all volunteers were healthy Caucasian men, taking no medication known to affect bone or calcium metabolism. Potential volunteers completed a questionnaire on health status and medication: individuals with a history of steroid use, diuretics, thyroid medication, chemotherapy, epilepsy, diabetes, or stroke were excluded from the study. Spine radiographs of men over 50 years enabled those with vertebral fractures to be excluded.
Recruitment was carried out from June 1994 to April 1995. Total body bone mineral content (BMC) was determined by dual-energy X-ray absorptiometry with a Lunar DPX (Lunar Corp., Madison, WI, U.S.A.) to enable bone marker data to be corrected for bone size. Fasting blood and 24 h urine samples were obtained from all volunteers and checked for completeness by interview and by measuring urinary creatinine (Cr). The blood samples were collected between 9:00 a.m. and 10:00 a.m. after an overnight fast. All samples were stored at −80°C.
Urinary free deoxypyridinoline (iFDpd) was determined by enzyme-linked immunosorbent assay (Pyrilinks D; Metra Biosystems, Mountain View, CA, U.S.A.), with an intra-assay variation 7.8%, and an interassay variation 9.1% at 15.8 nM), as was urinary N-telopeptides of type I collagen (NTx) (Osteomark; Ostex International, Seattle, WA, U.S.A.) with intra-assay variation 7.7% and interassay variation 8.4%.
Serum osteocalcin was determined by immunoradiometric assay (ELSA-OSTEO; CIS BioInternational, Cedex, Saclay, France) with intra-assay variation 2.7% and interassay variation 3.2%, as was bone alkaline phosphatase (bALP) (Tandem-R Ostase; Hybritech Europe, S.A., Belgium) with intra-assay variation 6.5% and interassay variation 9.6%. Intact propeptide of type 1 procollagen (PINP) was determined by radioimmunoassay (RIA) (Intact PINP; Orion Diagnostica, Oulu, Finland) with intra-assay variation 8.6%, interassay variation 8.6%.
Urine Cr was determined by the Jaffe method, and urine calcium was determined by colorimetric assay. Measurements were performed by the Clinical Chemistry Department, Royal Hallamshire Hospital, Sheffield.
Serum Cr, calcium, and phosphate were measured by standard colorimetric dry chemistry assay at the Clinical Chemistry Department, Northern General Hospital, Sheffield.
PTH was determined by immunoradiometric assay (Allegro; Nichols, San Juan Capistrano, CA, U.S.A.) with intra-assay variation 9.7%, interassay variation 2.4% at 240 pg/ml.
Total testosterone, dehydroepiandrosterone sulfate (DHEAS) and estradiol were determined by RIA (Coat-A-Count, DPL Division; EURO/DPC Limited, Gwynedd, U.K.) with intra-assay variation 4.6, 4.9, and 10.7% and interassay variation 5.6, 2.8, and 5.6%, respectively). Sex hormone binding globulin (SHBG) (Coat-A-Count, DPL Division, EURO/DPC Limited) with intra-assay variation 3.3%, and interassay variation 4.1% at 29 nmol/l. Free androgen index (FAI) was calculated as the ratio of testosterone to SHBG. The free estradiol index (FEI) was calculated as the ratio of estradiol to SHBG.
IGFBP3 was determined by RIA (Nichols Institute) with intra-assay variation 8.7%, and interassay variation 12.3%. IGF-I was measured after removal of IGFBPs by the department of Clinical Chemistry, Royal Hallamshire Hospital, Sheffield (Medginex Diagnostics, Fleurus, Belgium), with intra-assay variation 7% and interassay variation 5.6% at 170 μg/l.
The relationship between variables and age was evaluated by linear and multiple linear regression analysis (age, height, and weight). Regression with polynomial models (quadratic and cubic) were performed to detect a possible nonlinear relationship between the markers and age. The percentage change in BMC between age 20 and 79 years was calculated by dividing the regression coefficient for age from the regression analysis by the mean BMD and multiplied by 6000. The level of significance was taken as p < 0.05. The relationship between variables and age were examined by one-way analysis of variance and the Sheffe test applied to compare changes between decades.
Correlations were performed between the markers of bone turnover and the other variables measured, and the level of significance was taken as p < 0.05. This was done for different age groups: men of all ages, young men (younger than 40 years), and older men (older than 40 years). The earliest time point when a marker of bone turnover reached a nadir was in the fifth decade, therefore we selected 40 years as the cut-off age.
All statistical analyses were performed using Statgraphics® for Windows version 1.4 (Manugistics, Rockville, MD, U.S.A.) and GraphPad Prism™ (Intuitive Software for Science, San Diego, CA, U.S.A.).
The men were between 20 and 79 years (mean 49.9 years). Height decreased across the age range, weight increased until the fifth decade and decreased thereafter, and body mass index increased until the seventh decade (Table 1).
Table Table 1.. Mean (SD) Values of Characteristics of Study Population by Age
Bone mineral content
There was a linear decrease in BMC of 14% in men between age 20 and 79 years (Fig. 1 and Table 2).
Effect of age
The data for the effect of age on bone turnover was best fit by a quadratic model, with nadirs at age 56, 57, 53, 39, and 58 years for PINP, osteocalcin, bALP, iFDpd/Cr, and NTx/Cr, respectively (Fig. 2). The effect of age on iFDpd and NTx expressed as either an output (nmol/day) or in relation to total body BMC (nmol/g) was also best fit by a quadratic (graphs not shown).
Compared with men in the third decade, two of the bone formation markers (osteocalcin and PINP) decreased with age. This decrease progressed until the fifth decade, with little change thereafter. There was no significant change in bALP across the age range since the decrease in the third decade was masked by an increase in the eighth decade (Table 2).
NTx decreased across the age range when expressed in relation to Cr, total body BMC, or as an output. iFDpd also decreased across the age range when expressed as an output, but remained unchanged when expressed in relation to total body BMC, or increased (despite an initial decrease until the fifth decade) when expressed in relation to Cr. In general, there was a decrease in bone turnover between the second and fourth decade, with little change thereafter (Table 2).
There was a log-linear increase in PTH with age (43% between ages 20 and 79 years) (Fig. 3). IGF-I decreased with age (54%) and the data was best fit by a quadratic function after log transformation, with the nadir being reached at age 76 years. There was a log-linear decrease in IGFBP3 with age (17%) (Fig. 4).
Table Table 2.. Relationship Between Biochemical Variables and Age (Ten Year Age Groups)
There was a log-linear decrease in DHEAS (76%), total testosterone (26%), and estradiol (33%) with age (Fig. 5). The log-linear increase in SHBG (62%) with age resulted in a large decrease in FAI with age (57%) and FEI (42%).
There was a weak negative correlation between PTH with osteocalcin and NTx/Cr, but the relationship was not present when young or old men were considered separately.
IGF-I was positively correlated with bone turnover and the relationship was stronger for young men. IGFBP3 was not related to bone turnover (Table 3).
Table Table 3.. Correlation Between Markers of Bone Turnover and Hormonal Determinants
There were significant intercorrelations between the sex hormones, except for estradiol and SHBG, and the strongest correlation was between estradiol and DHEAS (r = 0.54, p = 0.0001). There were also significant intercorrelations between IGFI and the sex hormones (r < 0.49, p < 0.0001). Testosterone, estradiol, FAI, and FEI were positively correlated with bone turnover, while SHBG was negatively correlated with bone turnover. The relationship between DHEAS and bone turnover was variable. All relationships were stronger in young men (Table 3).
The main findings of the study were that bone turnover was highest in young men with little change thereafter, and in men increased bone turnover may be associated with high levels of sex hormones and IGF-I, and this relationship may be stronger in young men than in older men.
Effect of age
The average height reported in this male population (174.5 cm) is identical to the male normative data for England,(24) although the decrease with age in this population was slightly greater (10.6 cm compared with 8.6 cm). The average weight reported in this male population was similar to the male normative data for England(24) (80.1 kg compared with 79.5 kg), and followed a similar increase until the fifth decade with a decrease thereafter (3 kg compared with 9 kg).
The data for the effect of age on bone turnover was best fit by a quadratic, with increased bone turnover rates in young and old men. Previous studies also reported a nonlinear decrease in bone turnover with age(1) while studies which excluded men in the third decade reported an increase in bone turnover with age.(3)
In young men, bone formation was highest, corresponding to attainment of peak bone mass. Thereafter, there was no change in osteocalcin or PINP with aging, but there was a small increase in bALP in the eighth decade. Since PINP is also metabolized by the liver, the increase in bALP in elderly men cannot be attributed to decreased liver function. The increase in bALP may be due to the fact that different markers reflect different processes of bone formation: PINP is a by product of collagen synthesis, bALP is a product of osteoblasts and osteoblast precursors during early stages of mineralization, and osteocalcin is expressed by osteoblasts during later stages of mineralization. Alternatively, the increase in bALP may reflect an increase in liver alkaline phosphatase (ALP), since the measurement techniques for bALP show a cross-reactivity of 14% to 20% with liver ALP.(25)
With the exception of iFDpd expressed as a ratio to Cr, bone resorption was highest in young men, corresponding to attainment of peak bone mass. Thereafter there was little change in bone resorption with age. The small increase in iFDpd expressed in relation to Cr cannot be explained by decreased kidney function, degenerative joint disease, or diseases affecting nonskeletal collagen such as chronic obstructive pulmonary disease,(26) since these factors would have affected NTx also. Since an increase in iFDpd was not seen when it was expressed as an output or in relation to total body BMC, a change in the proportion of free cross-links in urine with age(22) is not a valid explanation.
Histologic studies on trabecular bone found an age-related decrease in bone formation, with unchanged or decreased bone resorption.(2) Markers of bone turnover have the advantage of allowing study of large numbers of subjects across a broad age range since the technique is not invasive. A decrease in bone formation and resorption with age using histomorphometry is compatible with a decrease in bone turnover after attainment of peak bone mass.
Overall we found markers of bone turnover to be elevated in the third decade, coinciding with attainment of peak bone mass, and to remain fairly constant thereafter. In women, there is a significant increase in bone turnover with age which is believed to be due to increased PTH levels.
Influence of hormones on bone turnover
The increase in PTH with age is consistent with previous studies.(4) The decrease in both IGF-I and IGFBP3 with age is consistent with previous studies.(8–10,13) The decrease in testosterone, DHEAS, estradiol, and FAI, and increase in SHBG with age is also consistent with previous studies.(16–17,21)
The relationship between PTH and bone turnover was weak. This suggests that PTH is not an important determinant of bone turnover in men.
In young men, IGF-I was positively correlated with bone turnover. This is consistent with the theory that IGF-I may stimulate bone formation(27) and bone resorption(28) by increasing cell number and activity. The relationship was stronger in young men, suggesting that IGF-I is more important for attainment of peak bone mass than bone loss in the elderly.
IGFBP3 was not related to bone turnover, but other studies have reported a correlation with BMD.(10) It has been proposed that IGFBP3 influences bone indirectly via IGF-I, and it has been reported to potentiate or inhibit the effect of IGF-I on bone, depending on the experimental conditions.(29)
In young men, there were weak correlations between sex hormones and markers of bone turnover. The association was stronger in young than in older men, suggesting that sex hormones are more important for attainment of peak bone mass than bone loss in the elderly. Despite lack of association between DHEAS and bone density,(30–32) these data and others(2) suggest that adrenal androgens may be important determinants of bone turnover in healthy men. Further, in older men DHEAS was the only sex hormone related to bone turnover, being negatively correlated with iFDpd. A role for DHEAS in bone loss has previously been proposed in animal studies.(33)
The theory that sex hormones act on bone via paracrine or autocrine production of IGF-I(33) is supported by the positive correlation between IGF-I and the sex hormones. However, the mechanism of action of sex hormones on bone remains controversial: testosterone may act directly on bone via androgen receptors on osteoblasts,(34) or indirectly by metabolism to estrogen,(19) or by stimulation of calcitonin secretion.(18)
There are several limitations with this study. First, although we recruited a random sample of men from a general practice we were concerned about including men with diseases or drugs that may have major effects on bone turnover. However, by excluding such men these results cannot be extrapolated to the general population. Second, this study looked at the correlation between bone turnover and variables, but correlation does not necessarily imply causation. Furthermore, we examined many relationships therefore type one errors are likely. The data from this study are useful in generating hypotheses, and these need to be tested experimentally.
In summary, PTH does not directly determine bone turnover in men. In men, increased bone turnover may be associated with high levels of sex hormones and IGF-I, and this relationship may be stronger in young men than older men: sex hormones and IGF-I may be important determinants of peak bone mass but have less influence on bone turnover in elderly men.
We would like to thank Hybritech for providing kits and Dr. R. Hannon for her laboratory supervision of assays. This work was funded by an Arthritis program grant.