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ABSTRACT We investigated whether levels of serum collagen markers and serum osteocalcin are related to pubertal growth and development in a cross-sectional study of 57 healthy boys at 14 y

of age. The level of the soft tissue marker, serum amino-terminal propeptide of type III procollagen(PIIINP) was higher in boys at Tanner stages G3 _versus_ G2(_p_ < 0.01). The levels of

the markers of bone collagen matrix differed only at a more advanced pubertal stage: the formation markers, carboxy-terminal and amino-terminal propeptides of type I procollagen, and the

degradation marker, carboxy-terminal telopeptide of type I collagen were higher only at stage G4 _versus_ G3 (_p_ < 0.01). The marker of bone mineralization, serum osteocalcin was also

higher only at stage G4 _versus_ G3 (_p_ < 0.01). Stage G4 was associated with the pubertal growth spurt. The results demonstrate that pubertal development should be taken into account

when serum levels of collagen markers and osteocalcin are evaluated, and suggest that an increase in serum PIIINP in boys at G3 might predict a normal pubertal growth spurt, but the finding

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CHILDREN AND BONE DEVELOPMENT DURING PUBERTY: A 4-YEAR LONGITUDINAL STUDY Article Open access 04 August 2022 MAIN Body growth and development are accompanied by corresponding changes in

connective tissue growth. Serum markers of the latter (collagen markers) have recently been developed(1), _e.g._ PICP and PINP to reflect synthesis of type I collagen (predominantly in bone)

and ICTP to indicate its degradation. Serum OC levels reflect the rate of mineralization of mature type I collagen(2). PIIINP reflects the synthesis of type III collagen (soft tissues). In

normal boys, height velocity is closely related to the velocity of connective tissue growth, and the effects of estrogens and androgens on bone would be anticipated to strengthen the

correlation between height velocity and the collagen markers. The data on the relationship between bone collagen metabolism and growth during puberty are still scanty and controversial(3–5).

In most previous studies, serum PIIINP(5–7) and serum PICP(3, 4) have been evaluated for age and height velocity, but without reference to pubertal stage. Serum PIIINP, the soft tissue

marker, has been observed to increase at pubertal ages in several studies(5, 6, 8). There are no published data no PINP or ICTP, whereas serum PICP has been observed to increase(3) and, on

the other hand, not to change systematically at pubertal ages(4, 8). We undertook the present study to investigate the relationships of height velocity and pubertal development to serum

collagen markers and serum osteocalcin in 57 healthy boys at 14 y of age. Individual pubertal development was estimated from clinical staging, testicular volume, and serum testosterone

concentration. METHODS This was a cross-sectional study of 57 healthy boys at the same secondary school level in three schools near the Children's Hospital, Helsinki(9). The study was

approved by the parents, the school authorities, and those responsible for school health care, and by the Ethics Committee of the Children's Hospital. Information about the study was

sent to the families. Of the 84 boys, 62 agreed to participate. All the parents and children gave their informed consent. Medical history and physical examination indicated that 60 boys were

healthy; two with bronchial asthma were excluded. There were three dropouts, leaving 57 boys for the study. This cross-sectional study included a physical examination and measurement of

height and weight. Height was measured on a Harpenden scale. Height, similarly measured 6 mo previously(9), was used to calculate height velocity (cm/y). Pubertal development was assessed

from testicular volume. The length and width of the testicles were measured with a ruler to the nearest millimeter. Testicular volume was calculated from the formula: 0.52 × longitudinal

axis in cm × (transverse axis in cm)2(10). The mean testicular volume was used. A cut-off point of 2 mL was used to draw a line between prepuberty and early puberty. Genital stage (G) was

determined according to Tanner(11). The median (range) age of the boys was 13.8(13.0-15.1) y, being similar in boys with different Gs: 13.9 y at G2(_n_ = 9), 13.8 y at G3 (_n_ = 23), 13.7 y

at G4 (_n_= 20), and 13.8 y at G5 (_n_ = 5). A morning blood sample was drawn, and serum was stored at -20 °C until analyzed. Serum testosterone concentration was determined by a RIA(12),

and serum sex hormone-binding globulin concentration with a fluoroimmunoassay (Delfia, Wallac, Turku, Finland). The free androgen index was calculated as total testosterone/sex

hormone-binding globulin × 100. Serum OC levels were determined by a RIA technique (Nichols Institute, San Juan, Capistrano, CA). The sensitivity of the assay is 0.3 μg/L, the intra- and

interassay coefficients being 4.5-6.2% and 4.1-8.9%. Serum PICP, PINP(1, 13). PIIINP(14), and ICTP(15) were measured with RIAs (Orion Diagnostica, Oulunsalo, Finland). The sensitivity of the

PICP assay is 1.2 μ/L, the intra- and interassay coefficients being 2.1-3.7% and 3.5-6.6%. The sensitivity of the PINP assay is 2.3 μg/L, the intra- and interassay coefficients being

2.3-3.5% and 2.5-5.2%. The sensitivity of the ICTP assay is 0.4 μg/L, the intra- and interassay coefficients of variation being 3.8-7.2% and 4.1-7.3%, respectively. Correlations were

calculated using Spearman's rank correlation test. Between-group comparisons were made by Kruskall-Wallis analysis for nonparametric data. After logarithmic transformation of the data

(to obtain Gaussian distribution), multiple _R-_square (explanation rate) was calculated using stepwise regression analysis to evaluate the separate effects of serum testosterone and height

velocity on serum collagen markers. RESULTS _GROWTH AND PUBERTAL STAGE_. Table 1 lists the median values for serum androgen indices and for body growth in the boys at different genital

stages. The differences in testicular volume were significant between the boys at different genital stages, as determined by Tanner staging. Height velocity and serum testosterone level were

higher at more advanced genital stages, the differences being significant between the boys at stages G3 and G4 (_p_ < 0.01). _SERUM LEVELS OF THE MARKERS_. Table 1 lists the median

values for the serum markers of collagen metabolism and serum osteocalcin in boys at different genital stages. The serum levels of all the peptides were higher in the boys in advanced

puberty (Fig. 1). The difference in PIIINP was already significant between boys at stages G2 and G3 (_p_ < 0.01), whereas the difference in PICP, PINP, ICTP, and OC levels were

significant only between G3 and G4 (_p_ < 0.01). The PINP levels were about 4-fold as compared with the PICP levels. The serum PINP/PICP molar ratio did not change significantly during

puberty: the medians (range) were 4.7 (3.3-6.4), 4.7 (2.5-7.1), 4.3 (2.7-6.6), and 4.0(3.6-4.3) at G2-G5, respectively. _CORRELATIONS BETWEEN MARKERS_. The serum PICP and PINP levels were

directly related (_r_ = 0.78, _p_ < 0.001). Both PICP and PINP were directly related to PIIINP (_r_ = 0.80 both, _p_< 0.001) and to OC (_r_ = 0.48, _r_ = 0.64, respectively,_p_ <

0.001 both). The PIIINP and OC levels were directly related as well (_r_ = 0.53, _p_ < 0.001). The degradation marker of type I collagen, ICTP, was also directly related to the formation

markers(_p_ < 0.001, all); to PICP and PINP (_r_ = 0.72, both), to PIIINP (_r_ = 0.84), and to OC (_r_ = 0.52). _CORRELATIONS BETWEEN MARKERS, GROWTH, AND PUBERTAL DEVELOPMENT_. Height

velocity, testicular volume, serum testosterone level, and free androgen index were all directly correlated with the serum levels of all the collagen markers and of osteocalcin (Table 2). In

stepwise regression analysis, the variances in PICP, PINP, PIIINP, and OC levels were best explained by the variances in serum testosterone levels, by 44, 47, 60, and 23% (multiple

_R-_square, respectively). Addition of height velocity to the analysis improved the explanation rates of PINP and PIIINP only by 4 and 6%, respectively, and did not improve the explanation

rate of PICP or OC. The variance in ICTP was best explained, by 51%, by the variance in height velocity, and addition of serum testosterone levels to the analysis improved it only by a

further 5%. DISCUSSION Hypogonadism with long-standing testosterone deficiency is known to be associated with osteoporosis(16). However, the data on the relationship of androgens and the

bones are scanty(17). The present study evaluated the relationship between bone and soft tissue collagen metabolism and pubertal development in boys. Bone matrix is formed by type I collagen

fibers and noncollagenous proteins, which are later mineralized(18). During type I collagen synthesis, the amino- and carboxy-terminal propeptides PICP and PINP are cleaved off from the

larger procollagen proteins(19) and during its degradation the cross-links between the collagen molecules, _e.g._ ICTP, are separated(1). The serum levels of PICP and ICTP, respectively,

have been observed to correlate with bone formation and resorption(20). Levels of serum PICP(3, 4, 8) and the soft tissue collagen formation marker, serum PIIINP, have been observed(5–8) to

correlate with height velocity. In the present study, type I collagen synthesis was evaluated with two markers, PICP and the novel marker PINP, and degradation with ICTP, in boys of the same

age but at different pubertal stages. The results of this cross-sectional study demonstrate that the serum markers of collagen metabolism and of mineralization are associated not only with

body growth but also with pubertal development. All the markers correlated positively with height velocity, with the traditional Tanner staging, with serum testosterone concentration, and

with testicular volume. In the present study, the ratio of the serum molar levels of the amino-terminal to the carboxy-terminal propeptides of type I collagen was about 4-fold. This may be

due to differences in the catabolism of these peptides; PICP being cleared mainly via mannose(21) and PINP via scavenger receptors in the liver(22). The receptors may be differently

sensitive to hormones, for in some studies glucocorticoids seem to up-regulate the mannose receptors(23), whereas they do not seem to affect the scavenger receptors(24). The unchanged

PINP/PICP ratio at different pubertal stages argues against an effect of testosterone in their clearance In the present study, higher serum PIIINP levels were observed in boys at stages G3

_versus_ G2. Significant differences in PICP, PINP, ICTP, and OC were observed between boys only at stages G3 and G4. This was accompanied with a significant difference in serum testosterone

levels and height velocity. These results suggest that, in early puberty, adrenal androgens may play a role in the growth of soft tissues, before testosterone production increases, and may

then stimulate the later pubertal bone growth spurt. An increase in serum PIIINP level has been suggested to predict a good response in height velocity in children during growth hormone

treatment(6). Our results suggest that an increase in serum PIIINP in boys at stage G3 may predict a normal pubertal growth spurt, but the finding remains to be confirmed in longitudinal

studies. In multiple stepwise regression analysis, the variance in ICTP was best explained by the variance in height velocity. In contrast, the variance in the other markers was best

explained by the variation in serum testosterone levels. The results suggest independent roles for serum testosterone in the formation of bone and soft tissues, and in addition, emphasize

the close association between increased degradation and remodeling of bone (ICTP) and the growth of the skeleton (height velocity). The present study did not allow for the evaluation of the

effect of growth hormone secretion in pubertal growth. The direct relationship between serum testosterone and the type I collagen peptide levels in the boys of the present study contrasts

with the inverse relationship between serum estradiol and bone turnover markers observed in girls(25). This may be attributed to the differences in the timing of the peak peptide levels: in

boys the peak is found in advanced puberty, when serum testosterone levels are higher, whereas in girls it occurs in early puberty, when serum estradiol levels are still continuing to

rise(25). The causes are unknown, as the regulation of bone metabolism in puberty is still unclear, but the inverse relationship between serum estradiol and the peptides may reflect an

appropriate decrease in tissue sensitivity to sex steroids by the end of body growth. OC is a noncollagenous protein of bone synthesized by osteoblasts and incorporated into the bone

matrix(26, 27). Serum OC levels possibly reflect the bone mineralization rate(2). Serum OC levels have mostly been evaluated in relation to age and height velocity(28–31), but ignoring

pubertal stage. Serum OC levels are higher in children than in adults(32) and the levels correlate with skeletal growth in children(28, 29). An association between pubertal development and

bone mineralization has been demonstrated in other ways: bone mineral density was lower in boys with constitutional delay of puberty(33) and in adult males with such a history(33). The wide

variation in timing of puberty may be critical for the development of peak bone mineral density(17, 33–35). The present study, showing peak OC levels at Tanner stage G4, suggests a parallel

timing of maximal bone matrix formation and mineralization of bone in boys at puberty. The regulation of bone mineralization is still unclear: in the present study only 23% of the variance

in serum OC was explained by the variation in serum testosterone levels, and addition of height velocity in the analysis did not improve the explanation rate at all. The results of this

cross-sectional study suggest that pubertal development in boys should be taken into account when serum levels of collagen markers and osteocalcin are evaluated. The serum level of PIIINP,

the soft tissue collagen marker, was already higher in boys at stage G3 _versus_ G2, whereas the markers of the bone collagen matrix, serum PICP, PINP, and ICTP, and the marker of bone

mineralization, OC, were not higher until stage G4_versus_ G3. The latter differences were associated with the pubertal growth spurt. The results suggest that an increase in serum PIIINP in

boys at stage G3 may predict a normal pubertal growth spurt, but the finding remains to be confirmed in longitudinal studies. ABBREVIATIONS * ICTP: carboxy-terminal telopeptide of type I

collagen * G: Tanner's genital stage * OC: osteocalcin * PICP: carboxy-terminal propeptide of type I procollagen * PIIINP: amino-terminal propeptide of type III procollagen * PINP:

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framework for the future. _Arch Dis Child_ 72: 177–179. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of

Allergic Diseases, Helsinki, Finland Ritva Sorva & Markku Turpeinen * Department of Allergic Diseases the Children's Hospital, Helsinki, Finland Raija Anttila, Martti A Siimes &

 Antti Sorva * University of Helsinki, Antti Sorva * Malmi Hospital, Helsinki, Finland Antti Sorva * Department of Clinical Chemistry, University of Helsinki, and United Laboratories,

Helsinki, Finland Riitta Tähtelä Authors * Ritva Sorva View author publications You can also search for this author inPubMed Google Scholar * Raija Anttila View author publications You can

also search for this author inPubMed Google Scholar * Martti A Siimes View author publications You can also search for this author inPubMed Google Scholar * Antti Sorva View author

publications You can also search for this author inPubMed Google Scholar * Riitta Tähtelä View author publications You can also search for this author inPubMed Google Scholar * Markku

Turpeinen View author publications You can also search for this author inPubMed Google Scholar ADDITIONAL INFORMATION Supported by the Emil Aaltonen Foundation, Tampere, Finland. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Sorva, R., Anttila, R., Siimes, M. _et al._ Serum Markers of Collagen Metabolism and Serum Osteocalcin in Relation

to Pubertal Development in 57 Boys at 14 Years of Age. _Pediatr Res_ 42, 528–532 (1997). https://doi.org/10.1203/00006450-199710000-00018 Download citation * Received: 11 June 1996 *

Accepted: 11 February 1997 * Issue Date: 01 October 1997 * DOI: https://doi.org/10.1203/00006450-199710000-00018 SHARE THIS ARTICLE Anyone you share the following link with will be able to

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