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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3785-3793
Copyright © 2003 by The Endocrine Society

Effects of Suppression of Estrogen Action by the P450 Aromatase Inhibitor Letrozole on Bone Mineral Density and Bone Turnover in Pubertal Boys

Sanna Wickman, Eero Kajantie and Leo Dunkel

Hospital for Children and Adolescents, University of Helsinki, Helsinki, FIN-00029 HUS, Finland

Address all correspondence and requests for reprints to: Sanna Wickman, Hospital for Children and Adolescents, University of Helsinki, PL 281, FIN-00029 HUS, Finland. E-mail: sanna.wickman{at}helsinki.fi.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The essential role of estrogen (E) in regulation of developing peak bone mass in males was confirmed when young adult men were described who cannot respond to or produce E because of defective E receptor {alpha} or P-450 aromatase enzyme, respectively. These men had significantly reduced bone mineral density (BMD) despite normal or supranormal androgen concentrations, and E administration improved BMD in the men with aromatase deficiency, whereas testosterone (T) was ineffective. Because new P450 aromatase inhibitors may prove to be potential drugs in various growth disorders, the effect of suppression of E action on developing peak bone mass has to be closely evaluated.

In this study, we explored the effects of suppression of E synthesis on bone metabolism in pubertal boys. A total of 23 boys with constitutional delay of puberty were randomized to receive T and placebo or T and a specific and potent P450 aromatase inhibitor, letrozole. We determined BMD in the lumbar spine and the femoral neck. Bone resorption was studied by measuring the serum concentration of cross-linked carboxyterminal telopeptide of type I collagen by two different methods (CTx and ICTP), and bone formation by determining the serum concentrations of carboxyterminal propeptide of type I procollagen (PICP), osteocalcin, and alkaline phosphatase.

We demonstrated previously that, during treatment with T and placebo, the concentrations of androgens and E increased. During treatment with T and letrozole, the E concentrations remained at the pretreatment level, but the androgen concentrations increased; the increase in the T concentration was more than 5-fold higher than during treatment with T and placebo. We did not observe any significant differences in the changes in bone mineral content, BMD, or bone mineral apparent density, an estimate of true volumetric BMD, between the treated groups. Lumbar spine bone mineral apparent density increased in both treated groups; but in the T- plus letrozole-treated group, the increase was statistically significant only 6 months after discontinuation of letrozole treatment. All bone resorption and formation markers increased during treatment with T and placebo. During treatment with T plus letrozole, CTx, PICP, and osteocalcin remained unchanged, whereas ICTP and alkaline phosphatase increased. Thus, 1-yr treatment with this new P450 aromatase inhibitor in pubertal boys is unlikely to be associated with any major harmful effect on developing peak bone mass. However, to convincingly exclude such effects, particularly rare or minor ones, will require a study with a larger sample size; and thus, close follow-up of bone metabolism during treatment with P450 aromatase inhibitors is still warranted.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
BONE MASS AND bone mineral density (BMD) increase throughout childhood, with a maximum increment rate during puberty (1, 2, 3), and peak in males around the age of 20 yr (2, 3, 4). Impaired peak bone mass may predispose to later osteoporosis (5).

The role of estrogen (E) in male bone has been highlighted by reports on males who have suppressed E action attributable to mutations in the genes encoding E receptor (ER){alpha} (6) or P-450 aromatase enzyme, responsible for the conversion of androgens to E (7, 8). These men had markedly reduced BMD, along with unfused epiphyses of the long bones, and elevated indices of bone turnover, despite normal or elevated serum concentrations of androgens. E administration to the men with mutations in the P-450 aromatase gene resulted in increases in BMD and closure of the epiphyses (8, 9), thus confirming the essential role of E in bone metabolism in males.

The effect of E on bone turnover in children has to be critically evaluated, because new aromatase inhibitors may prove to be an efficient treatment in various growth disorders. In treating delayed male puberty with testosterone (T), we have shown that simultaneous treatment with a P450 aromatase inhibitor, letrozole, delays bone maturation and thus potentially increases adult height in these patients (10). We therefore designed this study: 1) to assess, in particular, whether the P450 aromatase inhibitor treatment is associated with changes in BMD accretion, bone collagen, and mineral turnover; and 2) to study, in general, the effects of sex steroids on bone metabolism during male puberty.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

A total of 33 boys were recruited for the study (Table 1Go). The boys were referred to the Hospital for Children and Adolescents, University of Helsinki, for evaluation of delayed puberty and/or short stature. Diagnosis of constitutional delay of puberty was defined as a Tanner genital or pubic hair stage observed at an age older than the mean + 2 SD for healthy Finnish boys (12) or a testis volume of less than 4 ml after 13.5 yr of age. At entry, none of the boys had had any pubertal increase in growth velocity. Neither medical history, nor clinical examination, nor routine laboratory tests revealed any signs of chronic illnesses to account for the delayed puberty in any of the boys. None of the boys had received any previous sex hormone treatment. Two boys were receiving inhaled corticosteroid treatment for asthma: one in the untreated group (from age 11 yr; budesonide, 200 µg twice a day, at the start of the follow-up), and the other in the T- plus placebo-treated group (from age 2.5 yr; fluticasone, 100 µg twice a day, at the start of the treatment).


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  		TABLE 1. Clinical parameters of the three groups at the start (mean ± SD)1

 
Protocol

Informed written consent was obtained from the patient and from his guardian. The protocol was approved by the Ethical Committee of the Hospital for Children and Adolescents and the National Agency for Medicines.

Ten boys, with a mean age of 15.0 ± 0.2 yr (range, 14.4–16.8 yr), decided to wait for spontaneous progression of puberty without medical intervention and thus composed the untreated group. Twenty-three boys, with a mean age of 15.1 ± 0.2 yr (range, 13.5–16.1 yr) desired medical intervention and were randomly assigned to receive one or other of the two treatments. The boys in the T- plus placebo-treated group (12 boys) received T enanthate (Testoviron-Depot-250; Schering AG, Berlin, Germany), six times with a dose of 1 mg/kg im every 4 wk, and placebo orally once a day for 12 months. The T- plus letrozole-treated group (11 boys) received T enanthate (as above) and, in addition, a specific and potent, fourth-generation aromatase inhibitor, letrozole (Femar; Novartis AG, Stein, Switzerland; purchased from the hospital pharmacy), 2.5 mg orally once a day for 12 months. The project was conducted as a randomized, double-blind, placebo-controlled study between the treated groups. The rationale for giving the boys this new P450 aromatase inhibitor was our hypothesis that this treatment, which inhibits E action, would help the boys to achieve their genetic height potential. At the start, the mean testis volume was 5.9 ± 0.9 ml in the untreated group, 6.9 ± 1.2 ml in the T- plus placebo-treated group, and 5.5 ± 0.6 ml in the T- plus letrozole-treated group. The results of growth velocity, bone maturation, progression of puberty, and the serum concentrations of 17ß-estradiol (17ß-E2), T, 5{alpha}-dihydrotestosterone (5{alpha}-DHT), IGF-I, IGF-binding protein (IGFBP)-3, gonadotropins, and trends of changes in BMD of the lumbar spine and femoral neck, from the start to 12 months and to 18 months, have been published previously (10, 13).

Nine boys in the untreated group, 10 boys in the T- plus placebo-treated group, and 11 boys in the T- plus letrozole-treated group completed the 2-month follow-up; 8, 11, and 11 boys, respectively, completed the 5-month follow-up; 8, 11, and 10, respectively, completed the 12-month follow-up; and 7, 10, and 10, respectively, completed the 18-month follow-up. One boy in the T- plus letrozole-treated group was considered noncompliant, and therefore his results were excluded from the analyses.

The subjects were examined at the start of treatment and at 2 months (~7 d after the third T injection), 5 months (~7 d after the sixth T injection), 12 months, and 18 months of treatment. Height was measured on a Harpenden stadiometer to the nearest 0.1 cm. Weight was measured with underwear on. The body mass index (BMI) was calculated from the formula: weight/height2 (kg/m2). Testis volumes were calculated from the formula length x width2 x 0.52 (14) and are presented as means of the two testes. Pubertal stages were assessed according to Tanner (15).

BMD measurements

The bone mineral content (BMC; grams) of the first through fourth lumbar spines and the femoral neck were determined by dual-energy x-ray absorptiometry (Hologic QDR-1000; Hologic Inc., Waltham, MA) at the start of treatment and at 5, 12, and 18 months of treatment. The BMD (grams per square centimeter) was calculated by dividing the quantity of bone mineral within the scan area (BMC) by the projected area within the region of interest (area). The coefficient of variation of BMD in the lumbar spine is 0.6%, and in the femoral neck, it is 1.5%, in a normal population, according to the manufacturer. The areal BMD obtained by this method can, however, be confounded by changes in bone thickness. To minimize the contributions of bone dimensions, we determined, in addition, bone mineral apparent density (BMAD; grams per cubic centimeter), an estimate of volumetric bone density. Lumbar spine BMAD was calculated using the formula: BMC ÷ (area)1.5, and femoral neck BMAD was calculated using the formula: BMC ÷ [(area)2 ÷ 1.6], corresponding to the length of the scanned area (1.6 cm) (16).

Biochemical measurements

All the venous blood samples were drawn between 0730 and 1015 h. Bone resorption was estimated by measuring the serum concentrations of the carboxyterminal cross-linking telopeptide of type I collagen by two different methods (CTx and ICTP); and bone formation, by measuring the serum concentrations of carboxyterminal propeptide of type I procollagen (PICP), osteocalcin (OC), and alkaline phosphatase (ALP). Serum CTx concentrations were measured by Serum CrossLaps ELISA (Nordic Bioscience Diagnostics, Herlev, Denmark). Serum ICTP and PICP concentrations were determined by RIA (Orion Diagnostica, Espoo, Finland). The serum OC concentrations were measured by an immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). Serum ALP concentrations were determined by a Hitachi 917 Modular autoanalyzator. Serum 17ß-E2 concentrations were determined by a modified RIA using the coated tube technology (Spectria E2; Orion Diagnostica) after diethyl ether extraction (700 µl serum plus 5 ml diethyl ether) (17). The detection limit of the assay was 6 pM. Serum T and 5{alpha}-DHT concentrations were measured by RIA after separation of the steroid fractions on a Lipidx-5000 microcolumn (Packard-Becker, B.V. Chemical Operations, Groningen, The Netherlands) (18). Serum concentrations of IGF-I and IGFBP-3 were determined by RIA [DiaSorin (Stillwater, MN) and Nichols Institute Diagnostics, respectively]. Markers of bone turnover were measured at the start of treatment and at 5, 12, and 18 months; and concentrations of sex steroids, IGF-I, and IGFBP-3, at the start of treatment and at 2, 5, 12, and 18 months.

Statistical analysis

Values are expressed as means ± SEM unless otherwise reported. Analyses were conducted with the SPSS statistical software for Windows, release 10.0.7 (SPSS, Inc., Chicago, IL). One-way ANOVA or Kruskal-Wallis nonparametric ANOVA was used to compare the three groups at the start. For analysis of serial measurements, the summary measures, the differences from the start, were calculated for each subject, and these values were treated as raw data for the appropriate statistical analysis. The differences in the summary measures were only compared between the two treated groups, and the unpaired t test or the Mann-Whitney U test was used. The paired t test or the Wilcoxon matched pairs signed rank test was used for analyses of the changes from the start within groups. Parametric tests were used if the data were normally distributed; and nonparametric tests, if the distribution of the data was nonnormal or if the number of boys in a group was less than nine. To assess the relationship between BMD and hormonal factors and BMI, we compared the changes in lumbar spine and femoral neck BMAD during the treatment period (between 0 and 12 months) with the means of the serum concentrations of sex steroids, IGF-I, and IGFBP-3, and BMI at 2, 5, and 12 months by Spearman’s correlation. To investigate the relationship of bone turnover markers with growth velocity and change in BMD, we compared the means of serum bone turnover markers of 5 and 12 months with growth velocity and changes in lumbar spine and femoral neck BMAD between 0 and 12 months by linear regression analysis. All statistical tests were two-sided. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
At the start of the follow-up, no differences among three groups or between the two treated groups were observed in the BMCs, BMDs, and BMADs nor in the concentrations of serum CTx, ICTP, PICP, OC, ALP, 17ß-E2, T, 5{alpha}-DHT, IGF-I, and IGFBP-3. BMI at the start was lower in the untreated boys than in the treated boys, but it was similar in the two treated groups. In the two treatment groups, the clinical stages of puberty progressed in a similar way during the follow-up (10).

Serum 17ß-E2, T, 5{alpha}-DHT, IGF-I, and IGFBP-3 concentrations

In the two treated groups, the concentrations of sex steroids (Table 2Go), IGF-I, and IGFBP-3 changed differently. During treatment with T and placebo, compared with the start, an increase in all concentrations was observed (Table 2Go). In contrast, during treatment with T and letrozole, the 17ß-E2, IGF-I, and IGFBP-3 concentrations remained at the pretreatment level, but the concentrations of T and 5{alpha}-DHT increased; the increase in the T concentration was more than 5-fold higher than during the T- plus placebo treatment (Table 2Go). In the untreated group, the concentrations of 17ß-E2, T, 5{alpha}-DHT, and IGFBP-3 increased during the follow-up, and the IGF-I concentration remained unchanged (Table 2Go).


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  		TABLE 2. Serum 17ß-E2, T, and 5{alpha}-DHT concentrations during treatment and follow-up [mean ± SD (n)]1

 
Lumbar spine BMD

In both treated groups, the mean BMC increased during the treatment, and the increase from the start was of similar magnitude at all time points (except for a borderline higher increase within the first 5 months in the T- plus placebo-treated group, P = 0.06; Fig. 1Go). An increase was also observed in the BMD and the BMAD in both groups; although, in the T- plus letrozole-treated group, the increase in BMAD was statistically significant only at 18 months, i.e. 6 months after discontinuation of letrozole treatment (Figs. 1Go and 2Go). When the changes from the start in the BMD and the BMAD were compared between the treated groups, no difference was observed at any time-point. In the untreated group, the BMC increased, but the BMD and the BMAD did not change during the follow-up (except for a borderline significant increase in the BMD from the start to 18 months, P = 0.08; Figs. 1Go and 2Go).



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  		FIG. 1. Medians and interquartile ranges (50% of values) of the lumbar spine and the femoral neck BMC and BMD. Whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. Boys in the no group did not receive any treatment, boys in the T plus placebo (t + pl) group received T for 5 months and placebo for 12 months, and boys in the T plus letrozole (t + lz) group received T for 5 months and letrozole for 12 months. Asterisks denote significant changes within the group, compared with values at the start: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 


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  		FIG. 2. Medians and interquartile ranges (50% of values) of the lumbar spine and the femoral neck BMAD. Whiskers are lines that extend from the box to the highest and lowest values, excluding outliers. Boys in the no group did not receive any treatment, boys in the t + pl group received T for 5 months and placebo for 12 months, and boys in the t + lz group received T for 5 months and letrozole for 12 months. Asterisks denote significant changes within the group, compared with values at the start: *, P < 0.05; **, P < 0.01.

 
Femoral neck BMD

In both treated groups, an increase from the start in the mean BMC was observed during the treatment, and the magnitude of the increases was similar in both groups at all time points (Fig. 1Go). The mean BMD increased in the T- plus placebo-treated group but showed no change in the T- plus letrozole-treated group (Fig. 1Go). In contrast, changes in BMAD showed a different pattern. In the T- plus placebo-treated group, the mean BMAD was lower at 5 months (P = 0.03), similar at 12 months, and borderline lower at 18 months than at the start (P = 0.07; Fig. 2Go). In the T- plus letrozole-treated group, the mean BMAD did not change except for a borderline significant decrease from the start to 12 months (P = 0.08; Fig. 2Go). However, when the changes from the start in the BMD and in the BMAD were compared between the treated groups, no differences were seen. In the untreated group, neither the mean BMC, the mean BMD, nor the mean BMAD changed during the follow-up (except for a borderline significant increase at 18 months in the mean BMC and the mean BMD; P = 0.08 for both; Figs. 1Go and 2Go).

Serum bone turnover markers

During treatment with T and placebo, there was a simultaneous increase in both bone resorption and formation markers (Table 3Go; Fig. 3Go). During treatment with T and letrozole, the concentrations of CTx, PICP, and OC remained unchanged, whereas there was an increase in the concentrations of ICTP, and ALP (Table 3Go and Fig. 3Go). The untreated group showed a similar pattern of changes: no change in the concentrations of CTx, PICP, and OC, but an increase in the ICTP and ALP concentrations (Table 3Go).


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  		TABLE 3. Serum CTx, ICTP, PICP, OC, and ALP concentrations during treatment and follow-up [mean ± SD (n)]

 


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  		FIG. 3. Mean ± SEM serum (S-)17ß-E2, T, CTx, ICTP, PICP, OC, and ALP concentrations at the start and at 5 months of treatment. Boys in the t + pl group received T and placebo, and boys in the t + lz group received T and letrozole. Asterisks denote significant changes within the group: *, P < 0.05; **, P < 0.01.

 
Power of the study to detect differences between groups

With 80% power and 5% significance level, the sample size of the present study is able to detect a difference of 1.25 SD in each variable between the two treated groups (19). With regard to the difference of BMD and BMAD gain by 12 months, for example, this limit is equivalent to a between-groups difference of 0.044 g/cm2 in lumbar spine BMD increase, 0.0052 g/cm3 in lumbar spine BMAD increase, and 0.053 g/cm2 in femoral neck BMD increase.

Relationship between BMAD and 17ß-E2, T, 5{alpha}-DHT, IGF-I, and IGFBP-3 concentrations and BMI

To assess the association of BMAD with hormonal factors and BMI, we compared the changes in the lumbar spine and the femoral neck BMAD between 0 and 12 months with the mean concentrations of 17ß-E2, T, 5{alpha}-DHT, IGF-I, IGFBP-3, and the mean BMI of 2, 5, and 12 months. The changes in the lumbar spine BMAD correlated with the 17ß-E2 (r = 0.5, P < 0.01) and the IGF-I (r = 0.5, P < 0.02) concentrations, and the changes in the femoral neck BMAD with the IGFBP-3 concentrations (r = 0.4, P < 0.05). No significant associations with other variables were observed. Figure 4Go illustrates the similarity of the lumbar spine BMAD changes between the two treated groups, despite sizable differences in the 17ß-E2 concentrations.



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  		FIG. 4. Relationships between the changes in lumbar spine BMAD from the start to 12 months and the mean concentrations of 17ß-E2 of 2, 5, and 12 months. •, Boys who received T for 5 months and placebo for 12 months; {circ}, boys who received T for 5 months and letrozole for 12 months.

 
Relationships between serum bone turnover markers and growth velocity and BMAD

To study the association of growth velocity and BMAD with bone turnover, we compared growth velocities and changes in the lumbar spine and the femoral neck BMAD, from the start to 12 months, with the means of serum bone turnover markers of 5 and 12 months. Growth velocity correlated with all of the serum bone turnover markers (Table 4Go). In contrast, the change in BMAD associated less well with the bone turnover markers; a significant correlation was observed only between the change in lumbar spine BMAD and the ICTP concentration (r = 0.5, P < 0.02).


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  		TABLE 4. Relationship between serum concentrations of bone turnover markers and growth velocity

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This randomized, prospective study was designed to describe the effects of suppression of E synthesis on bone metabolism and, in particular, on the developing peak bone mass in pubertal boys. We were unable to show any significant differences in the changes in BMC, BMD, or BMAD of lumbar spine or proximal femur between the groups treated with T and placebo or with T and new, specific, and potent P450-aromatase inhibitor, letrozole. These findings suggest that 1-yr treatment with a P450 aromatase inhibitor in pubertal boys is unlikely to have any major harmful effect on BMD. However, one must recognize the limited power of a study of this size, in particular in detecting minor or rare side effects. It is of note that, during 1-yr treatment, a decrease in the lumbar spine BMAD was observed in four letrozole-treated boys but only in one of the boys treated with T and placebo. Thus, it is possible that, in some subjects, the probable increase in adult height induced by letrozole treatment may take place at the expense of decreased skeletal strength. Definite assessment of the safety of this treatment requires thus a larger study sample, and it remains essential to follow up BMD regularly during suppression of E action.

The key role of E for developing peak bone mass was confirmed by reports of young adult men who cannot respond to or produce E (6, 7, 8). These men were osteopenic, despite normal or supranormal androgen concentrations (6, 7, 8); and administration of E, but not T, improved BMD in the men with aromatase deficiency (8, 9). Consistently, we found no increase in femoral neck BMD or lumbar spine BMAD during suppression of E action by letrozole, despite high androgen concentrations, whereas these parameters showed an increase in the T- plus placebo-treated group with intact P-450 aromatase activity. Because of the small size of the present study, these findings must be interpreted with caution. However, they are consistent with findings in pre- and early-pubertal boys, showing a correlation of bioavailable E2 levels with BMD in the arms (20), as well as with the relationship between an ER gene polymorphism and BMD in late-pubertal boys (21). Together with our observation of the positive correlation of 17ß-E2 concentrations with the changes in lumbar spine BMAD, these findings further support the concept of the important role of E in accretion of bone mass during growth and maturation in males.

We found that combining T treatment with aromatase inhibition by letrozole resulted in remarkably high T concentrations and low 17ß-E2 concentrations. That there was no significant difference in the changes of BMC, BMD, or BMAD between the T- plus placebo-treated and the T- plus letrozole-treated groups suggests an important compensatory role for androgens in conditions of suppressed E action, at least in the case of supraphysiological androgen concentrations. This is consistent with previous observations showing T to be positively, and independently of E, related to BMD in adult men (22, 23, 24). The importance of androgens in bone mass accretion is further supported by the finding that periosteal bone formation is stimulated by T and 5{alpha}-DHT in male rats (25), which is consistent with the fact that, in humans, males have larger bones with thicker cortical widths than females. Importantly, patients with androgen insensitivity syndrome have decreased BMD even before attainment of peak bone mass, reflecting the important role of androgens in developing peak bone mass (26, 27).

GH has a significant role in bone growth and in the development of bone mass. The reduced BMD in children with GH deficiency increases during GH treatment (28, 29, 30, 31). Our finding of an increase in the lumbar spine BMAD and the femoral neck BMD during the T- plus placebo treatment, but not during the T- plus letrozole treatment, may also be attributable to a difference in the activity of the GH-IGF-I axis, because the IGF-I and IGFBP-3 concentrations only increased during treatment with T plus placebo (10). Furthermore, we found that the IGF-I concentrations correlated positively with the changes in lumbar spine BMAD, and the IGFBP-3 concentrations correlated positively with the changes in femoral neck BMAD. Because E augments GH secretion (32), the effects of GH on BMD may also, at least in part, reflect the action of E.

Measuring serum concentrations of bone turnover markers offers an indirect way to study various aspects of bone metabolism. Bone resorption is reflected by type I collagen degradation products, CTx and ICTP (33, 34). Bone formation is mirrored by the serum concentration of PICP, which is a by-product of type I collagen synthesis (33, 34). Additional markers of bone formation include OC, which is secreted during the mineralization process (35), and ALP, the bone isoform of which constitutes 75–90% of total serum ALP activity in growing children over the age of 4 yr (36). The effects of sex steroids on bone resorption and formation have been explored in elderly men in a direct interventional study in which gonadotropin secretion and the activity of the P-450 aromatase enzyme were suppressed; and, concomitantly, E alone, T alone, both, or neither were administered (37). The results of this study indicate that bone resorption is regulated predominantly by E, T having a smaller effect, whereas both E and T have an important role in maintaining bone formation (37). Furthermore, the suppressed E synthesis by administration of the P-450 aromatase inhibitor, anastrozole, to elderly men was accompanied by an increase in the bone resorption marker and decreases in the bone formation markers (38). In our study, during T- plus placebo treatment, concomitant increases were observed in all bone resorption and formation markers. During treatment with T and letrozole, with low 17ß-E2 concentrations and high androgen concentrations, one resorption marker, CTx, and two of the formation markers, OC and PICP, remained unchanged, whereas an increase in another resorption marker, ICTP, and in one formation marker, ALP, was observed. These findings do not suggest significant imbalance between bone resorption and formation during either of the two treatments.

The fact that, in growing children and adolescents, different biological processes (bone growth, modeling, and remodeling) occur simultaneously in bone complicates the use of bone turnover markers, which are not specific to these different processes. Serum concentrations of ICTP, PICP, OC, and ALP correlated strongly with height velocity in previous studies (39, 40, 41, 42), as well as in our study. In growing boys and girls, changes in BMC also associate positively with concentrations of bone formation and with those of bone resorption (42). In contrast, the changes in BMD have not been shown to correlate with bone turnover markers in growing children (28, 43, 44). We found that, of the bone turnover markers, only the ICTP concentration was significantly associated with the changes in lumbar spine BMAD. Thus, in healthy, growing children, circulating concentrations of bone turnover markers seem to reflect bone growth, rather than changes in true BMD.

The DEXA measures BMC within the scan area and the projected area within the region of interest, and thus does not take into account changes in bone thickness. Mathematical models have been developed to calculate apparent BMD (BMAD; grams per cubic centimeter) from DEXA-derived data, which better estimate the true BMD (grams per cubic centimeter) (16, 45). We found that, in the lumbar spine, the BMAD increased during the follow-up, which is in accord with a previous finding of an increase of lumbar spine BMAD, with age, in boys (46). However, in the femoral neck, we observed a decreasing trend in BMAD. This may have resulted from inaccuracy of estimated BMAD to represent the true BMD, because the model for calculating BMAD had been validated by a direct measurement of the volume of the lumbar spines but not of the femoral neck (16). However, the decreasing trend in the femoral neck BMAD did not reach statistical significance at 12 or 18 months in any of the groups, which is in accord with previous findings of unchanged values of the femoral neck BMAD in boys with increasing age (46, 47).

It is unclear whether the timing of puberty is a significant determinant of peak BMD in men. Men with a history of constitutionally delayed puberty have been observed to have osteopenia in adult life (48, 49, 50), but volumetric BMD has also been demonstrated to be normal in these men (51). Reasons for this discrepancy remain uncertain, although they may be related to the fact that, in the latter study, most of the men had received androgen therapy for induction of puberty (51). In our study, the BMAD values were similar to those in another study of healthy growing boys (47). However, the present study was not designed to detect whether boys with delayed puberty had a lower BMD than normally maturing boys. Further studies are needed to explore this important issue.

In conclusion, we treated boys with delayed puberty with T and placebo or with T and a P450 aromatase inhibitor, letrozole, which inhibits the conversion of androgens to E. We did not observe any significant differences in the changes in BMC, BMD, or BMAD between the T- plus placebo-treated and the T- plus letrozole-treated groups. Neither did we observe any significant imbalance between bone resorption and formation in either of the treated groups. Thus, our observations suggest that 1-yr treatment with new P450 aromatase inhibitors in pubertal boys is unlikely to have any major harmful effects on developing BMD. However, to convincingly exclude such effects, particularly rare or minor ones, will require a study with a larger sample size. Thus, before the ultimate cost vs. benefit of the use of aromatase inhibitors in various growth disorders has been clarified, these, by themselves promising treatments, should still only be used in research trials.


   Acknowledgments
 
We gratefully acknowledge the help of Carina Ankarberg-Lindgren and Ensio Norjavaara (of Göteborg Pediatric Growth Research Center, Göteborg University, Sweden) for performing the 17ß-E2 assays and of Ilkka Sipilä (of the Hospital for Children and Adolescents) for helping in determination of bone ages.


   Footnotes
 
This work was supported by the Foundation for Pediatric Research, Helsinki, Finland; the Helsinki University Central Hospital Research Fund; and Finska Läkaresällskapet.

Abbreviations: ALP, Alkaline phosphatase; BMAD, bone mineral apparent density; BMC, bone mineral content; BMD, bone mineral density; BMI, body mass index; DHT, dihydrotestosterone; E, estrogen; E2, estradiol; ER, estrogen receptor; IGFBP, IGF-binding protein; OC, osteocalcin; PICP, procollagen; T, testosterone.

Received October 23, 2002.

Accepted April 21, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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