Clinical Chemistry 57: (2011)

Transcription

1 Clinical Chemistry 57: (2011) Endocrinology and Metabolism Serum Concentrations of Insulin-like Growth Factor (IGF)-1 and IGF Binding Protein-3 (IGFBP-3), IGF-1/IGFBP-3 Ratio, and Markers of Bone Turnover: Reference Values for French Children and Adolescents and z-score Comparability with Other References Corinne Alberti, 1,2,3 Didier Chevenne, 4 Isabelle Mercat, 5 Emilie Josserand, 2 Priscilla Armoogum-Boizeau, 2 Jean Tichet, 5 and Juliane Léger 3,6,7,8* BACKGROUND: A reference model for converting serum growth factor and bone metabolism markers into an SD score (SDS) is required for clinical practice. We aimed to establish reference values of serum insulinlike growth factor-1 (IGF-1) and IGF binding protein 3 (IGFBP-3) concentrations and bone metabolism markers in French children, to generate a model for converting values into SDS for age, sex, and pubertal stage. METHODS: We carried out a cross-sectional study of 1119 healthy white children ages 6 20 years. We assessed concentrations of serum IGF-1, IGFBP-3, carboxyterminal telopeptide 1 chain of type I collagen (CrossLaps), and bone alkaline phosphatase concentrations and height, weight, and pubertal stage, and used semiparametric regression to develop a model. RESULTS: A single regression model to calculate the SDSs with an online calculator was provided. A positive relationship was found between SDS for serum IGF-1 and IGFBP-3, IGF/IGFBP-3 mol/l ratio, and anthropometric parameters (P ), with slightly greater effects observed for height than for body mass index (BMI). There was a negative relationship between serum CrossLaps concentration and BMI, and a positive relationship between serum CrossLaps concentration and height. A comparison of serum IGF-1 reference databases for children showed marked variation as a function of age and pubertal group; smooth changes with age and puberty were observed only in our model. CONCLUSIONS: This new model for the assessment of SDS reference values specific for age, sex, and pubertal stage may help to increase the diagnostic power of these parameters for the assessment of growth and bone metabolism disorders. This study also provides information about the physiological role of height and BMI for the interpretation of these parameters American Association for Clinical Chemistry Serum concentrations of insulin-like growth factor-i (IGF-1) 9 and IGF binding protein-3 (IGFBP-3) are commonly used to assess the growth hormone (GH)- IGF-1 axis in patients with growth disorders (1).It has been suggested that the molar ratio of IGF-1 to IGFBP-3 could be used as a crude indicator of IGF-1 bioavailability (2), and several recent epidemiological studies in adults have shown an association between highly bioavailable IGF-1 concentrations and cancer risk, raising concerns about the long-term safety of high-dose GH treatment (3). The IGF-1/IGFBP-3 ratio is also associated with metabolic syndrome (4). These biological parameters have become essential tools for monitoring GH replacement therapy in various conditions (3). Good clinical practice dictates that patients should be monitored regularly during GH replacement and that serum IGF-1 concentrations should be maintained below the upper limit of the reference interval for the age and sex of the child (3). Serum concentrations of bone alkaline phosphatase (BALP) and the car- 1 INSERM, CIC-EC CIE5, Paris, France; 2 Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Unité d Epidémiologie Clinique, Paris, France; 3 Université Paris Diderot, Paris, France; 4 Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Service de Biochimie-Hormonologie, Paris, France; 5 Regional Institute for Health, Tours, France; 6 Assistance Publique-Hôpitaux de Paris, Hôpital Robert Debré, Service d Endocrinologie Pédiatrique, Paris, France; 7 Assistance Publique-Hôpitaux de Paris, Centre de Référence des Maladies Endocriniennes Rares de la Croissance, Paris, France; 8 INSERM, UMR 676, Paris, France. * Address correspondence to this author at: Pediatric Endocrinology Department, Centre de Référence Maladies Endocriniennes de la Croissance, INSERM U676, Hôpital Robert Debré, 48 Boulevard Sérurier, Paris, France. Fax ; juliane.leger@rdb.aphp.fr. Received May 27, 2011; accepted July 29, Previously published online at DOI: /clinchem Nonstandard abbreviations: IGF-1, insulin-like growth factor I; IGFBP-3, insulinlike growth factor binding protein 3; GH, growth hormone; BALP, bone alkaline phosphatase; CrossLaps, carboxyterminal telopeptide 1 chain of type I collagen; BMI, body mass index. 1424

2 GH/IGF-1 Axis and Bone Turnover Metabolism in Children boxyterminal telopeptide 1 chain of type I collagen (CrossLaps) are recognized markers of bone formation and resorption, respectively. These markers are used to assess bone-remodeling status and the response to treatment in patients with bone metabolism disorders (5, 6). Known determinants of these biological parameters include age, sex, and puberty, together with genetic and ethnic factors (7 19). There is some concern that auxological parameters may modify serum concentrations. Illness and nutritional status are also known to affect concentrations of all these serum growth factors and bone metabolism markers. Thus, determination of pediatric reference intervals for healthy children is essential for the assessment of growth and bone metabolism disorders and for the monitoring of response to treatment (20). The establishment of pediatric reference intervals for serum growth factors and bone metabolism markers and the assessment of the relationships between these parameters and sex, age, and puberty require a large population of healthy children and adolescents, preferably specific to a particular ethnic group. Moreover, the concentrations of these markers depend partly on nutritional state and display circadian variation; their determination from morning samples collected from fasting patients greatly decreases individual variability and should therefore be preferred (21). The distribution of these biological parameters is often skewed, so appropriate curve-fitting procedures are also essential. Very few normative studies have fulfilled these conditions, and many have suffered from the inclusion of only small numbers of study participants. There is also considerable variation in the assay methods used (20, 22). On the basis of results of our previous study, in which we investigated the relationship between absolute serum concentrations for components of the GH/ IGF-1 axis and serum markers of bone turnover and metabolism in healthy children (23), we aimed to establish references values for sex, age, and pubertal stage for these parameters in healthy French children and to present a model for converting serum concentrations of these factors into standard deviation scores (SDS) that can be adapted for easy practical use in other ethnic populations. We investigated the relationship between each parameter and body mass index (BMI) and height expressed as SDS. We then compared our model for serum IGF-1 concentrations with previously reported models. Methods The study population (n 1119) has been described elsewhere (23). Briefly, morning serum samples were obtained from healthy fasting white children and adolescents (n 579 boys and n 540 girls) 6 to 20 years old. This cross-sectional study also included pubertalstage assessment by clinical examination, according to Tanner stage, height and weight measurements, and BMI calculations. These anthropometric data are reported in Supplemental Table 1 and Supplemental Fig. 1 in the Data Supplement that accompanies the online version of this article at content/vol57/issue10. The study protocol was reviewed and approved by the faculty ethics committee. It was explained to all study participants and their parents, who signed a written consent form for participation. SERUM ANALYSIS Serum IGF-1 concentrations were originally determined by fully automated 2-site chemiluminescence immunoassays (Nichols Advantage, Nichols Institute Diagnostics), with interassay CVs of 5.3% at a serum concentration of 45 g/l, 4.8% at 413 g/l, and 7.4% at 810 g/l, and a detection limit of 6 g/l. Because the Nichols Advantage system has since been discontinued and is no longer available, we used 93 blood samples from children of both sexes and different ages to carry out a comparison of this method with the IRMA IGF- 1-RIACT assay (Cisbio Bioassays). The correlation between the 2 methods was r 0.986, which yielded similar results for IGF-1 concentration, as shown by the weighted Deming regression equation: IGF-1 Cisbio (1.016 IGF-1 Nichols Advantage) 9.5 (see online Supplemental Fig. 2). The serum IGF-1 concentrations obtained with the Nichols Advantage assay were therefore converted into Cisbio IGF-1 values with this equation, making it possible to establish reference curves that could be used with the Cisbio assay. Serum IGFBP-3 concentrations were measured with an IRMA kit (IGFBP-3 Active IRMA, DSL, BeckmanCoulter), with interassay CVs of 6.5% at a serum concentration of 400 g/l, 5.8% at 4200 g/l and 3.9% at 7200 g/l, and a detection limit of 50 g/l. We calculated the IGF-1/IGFBP-3 mol/l ratio on the basis of the molecular weights of IGF-1 and IGFBP-3. Serum BALP concentrations were determined with an IRMA kit (Ostase, BeckmanCoulter), with interassay CVs of 9.2% at a serum concentration of 17 g/l, 8.5% at 40 g/l, and 7.5% at 82 g/l, and a detection limit of 2 g/l. The serum CrossLaps assay is an ELISA (serum CrossLaps ELISA, IDS) specific for a -aspartate form of the EKAHD- -GGR epitope derived from the cross-linked degradation products of C-terminal telopeptides of type I collagen. For this assay, the detection limit is 20 ng/l and the interassay CVs were 9.7% at a serum concentration of 444 ng/l, 3.9% at 1066 ng/l, and 2.5% at 1967 ng/l. Clinical Chemistry 57:10 (2011) 1425

3 STATISTICAL ANALYSIS Quantitative variables are reported as medians (range or percentiles) or mean (SD). We established age-specific reference intervals for all biological parameters by using the parametric method described by Royston and Wright, in line with the recommendations of the WHO (24, 25). This method can be used (a) to estimate outer percentiles precisely, (b) to estimate percentiles simultaneously such that they are constrained not to cross, (c)to estimate z scores and percentiles from direct formulas, (d) to account for both skewness and kurtosis when necessary, and (e) to apply continuous age smoothing. This last aspect is particularly important when choosing the statistical method to be used. Indeed, reference intervals are sometimes calculated as the mean (2 SD) for each age group, these groups being arbitrarily defined, often by using the middle of the class. This procedure results in the absence of a smooth change in the mean with age, as would be expected on biological grounds, and the same is true for the SD curve (24, 25). A summary of the method is given below. For more details see the online Supplemental Methods file. Assuming a gaussian distribution of the variable at each age, we calculated a percentile according to a widely used formula: percentile mean (U SD), where the mean and SD are defined for a given age and U is the corresponding percentile constant of the standard gaussian distribution (for example, for the 2.5th and 97.5th percentiles curves, U 1.96). The aim was to identify functions that adequately represented the changes in mean and SD with age. Initial mathematical transformation was applied, if required, to reduce the positive skewness and heteroscedasticity of the measurements of interest. Suitable functions for the mean and SD based on age were obtained from regression splines. The fitted values based on the regression gave the estimated mean curve and the SD. We assessed the fit of the model by calculating the SDSs (z score) as: z (measurement mean)/sd. The ordered z scores were plotted to provide a graphical check of normality. If the hypothesis of normality was accepted, then no further modeling was required. If not, then an exponential transformation was applied to z, and the skewness curve was fitted by the maximum likelihood method, using fractional polynomials as powers of the model of regression against age. We calculated the percentile estimates and reference intervals by introducing the fitted curves of the mean, SD, and skewness into the percentile equation, which becomes a little more complex when adding skewness (see the online Supplemental Methods file for more details). For variables that have previously been transformed, we obtained percentile curves on the original scale by applying a back transformation to the calculated curves. At the end of the process, we checked several internal validity measures (26). Sex-specific curves were generated and the effect of puberty was explored by adding the variable to the reference interval model. We used linear regression analysis to investigate the relationships between individual biological parameters (expressed in SDS) and BMI and height (also expressed in SDS), as a function of age and sex. We explored the differences between our model and reference curves for IGF-1 as a function of age and puberty stage generated from published data, by plotting the sex-specific median curves of 6 databases (7, 8, 10, 11, 14, 15, 19) together with our data. All statistical analyses were carried out with SAS 9.1 (SAS Institute), STATA (version 9) and Method Validator (for Deming regression) software. Results Reference interval curves (medians and 2.5th 97.5th percentiles) for IGF-1, IGFBP-3, IGF-1/IGFBP-3 ratio, CrossLaps, and BALP for age and sex are displayed in Fig. 1 and reference values per midclass age integer are given in Table 1. All mean and scale curves were modeled with natural cubic splines. An online calculator is available ( IRSA_web/index.html). As expected, despite considerable interindividual variability, marked variation with age and sex was observed for all parameters. Median serum concentrations of IGF-1 and IGFBP-3, and the IGF-1/IGFBP-3 ratio increased between the ages of 6 and years in boys and 6 and years in girls. Consistent with the timing of the pubertal period, peak concentrations occurred at the ages of years in girls and at years in boys, with slightly higher values in girls than in boys. Beyond the ages of 16 years in boys and 14 years in girls, serum concentrations of IGF-1 decreased, whereas those of IGFBP-3 reached a plateau. In both sexes, median serum concentrations of CrossLaps and BALP remained fairly constant during the prepubertal period; increased during puberty, with slightly higher values obtained for boys than for girls; and decreased rapidly thereafter. At midpuberty (approximately age years in girls and years in boys), all reference curves other than that for IGFBP-3 displayed a larger spread of values than before or after puberty. Fig. 2 shows the effect of puberty stage, adjusted for age and sex. For serum IGF-1 concentration and IGF-1/ IGFBP-3 ratio in girls, distinct curves were obtained for stages I, II, and III, whereas the curves for stages IV and V were confounded. Puberty stages could be assigned to 2 groups (I-II vs III-IV-V) for serum IGF-1 and IGFBP-3 concentrations and IGF-1/IGFBP-3 ratios in 1426 Clinical Chemistry 57:10 (2011)

4 GH/IGF-1 Axis and Bone Turnover Metabolism in Children Fig. 1. Reference curves for IGF-1 concentration, IGFBP-3 concentration, IGF-1/IGFBP-3 mol/l ratio, and BALP and CTX concentrations in boys (left column) and girls (right column), as a function of chronological age. Curves represent the 2.5th percentile (- - -), 50th percentile (...), and 97.5th percentile ( ). Clinical Chemistry 57:10 (2011) 1427

5 Table 1. Age- and sex-specific reference values for IGF-1 concentration, IGFBP-3 concentration, IGF-1/IGFBP-3 molar ratio, and BALP and CrossLaps concentrations. a Age, years IGF-1 males, g/l 2.5th Percentile Median th Percentile IGF-1 females, g/l 2.5th Percentile Median th Percentile IGFBP-3 males, g/l 2.5th Percentile Median th Percentile IGFBP-3 females, g/l 2.5th Percentile Median th Percentile IGF-1/IGFBP-3 males 2.5th Percentile Median th Percentile IGF-1/IGFBP-3 females 2.5th Percentile Median th Percentile BALP males, g/l 2.5th Percentile Median th Percentile BALP females, g/l 2.5th Percentile Median th Percentile CrossLaps males, ng/l 2.5th Percentile Median th Percentile CrossLaps females, ng/l 2.5th Percentile Median th Percentile a Reference values were calculated from reference equations, according to sex and the age in the middle of the age class. For example, for the age class of 6to 7 years, we chose an age of 6.5 years to estimate the expected median IGF-1 concentration for a boy. This equation gives a value of 160 g/l Clinical Chemistry 57:10 (2011)

6 GH/IGF-1 Axis and Bone Turnover Metabolism in Children Fig. 2. Reference curves for IGF-1 concentration, IGFBP-3 concentration, IGF-1/IGFBP-3 mol/l ratio, and BALP and CTX concentrations in boys (left column) and girls (right column), as a function of Tanner stage and chronological age. Curves represent stage I ( ), stage II (---), stage III ( ),stage IV ( ), stage V(- - -). Clinical Chemistry 57:10 (2011) 1429

7 Table 2. Relationship of biological markers to BMI and height (all expressed in SDS as a function of age and sex), as studied by multivariate linear regression. a Intercept BMI Height IGF ( to 0.126) (0.074 to 0.159) (0.131 to 0.233) IGFBP ( to 0.045) (0.064 to 0.151) (0.043 to 0.146) IGF-1/IGFBP ( to 0.085) (0.025 to 0.112) (0.107 to 0.210) CrossLaps ( to 0.106) (0.073 to 0.160) BALP ( to 0.046) ( to 0.018) ( to 0.090) a Results are expressed as regression coefficients with 95% CIs, and are illustrated in Fig. 3. b All P values are less than 0.001, except for BALP concentration, for which P 0.47 for the intercept; P 0.25 for BMI; and P 0.17 for height. boys and IGFBP-3 concentrations in girls, and were less contrasted for serum CrossLaps and BALP concentrations in boys. Thus, for certain markers, puberty provides additional information that can be taken into account when estimating SDS. Table 2 shows the relationship of all biological parameters to height and BMI, all expressed in SDS for age and sex. There were significant positive relationships between serum IGF-1 and IGFBP-3 concentrations, IGF-1/IGFBP-3 mol/l ratio, and anthropometric parameters. The effect of height was slightly greater than that of BMI, as shown by the magnitude of the coefficients. For bone markers, there was a negative relationship between serum CrossLaps concentration and BMI, and a positive relationship between serum CrossLaps concentration and height. Fig. 3 illustrates the predicted 2.5th, 50th, and 97.5th percentile curves, based on the equations given in Table 2, for serum IGF-1 SDS values as a function of height SDS for a study participant with a BMI fixed at 2 SDS (Fig. 3A) and as a function of BMI SDS for a study participant with a height fixed at 2 SDS (Fig. 3B). These curves demonstrate the positive association of serum IGF-1 SDS values with BMI SDS and height SDS. We plotted graphical comparisons of reference curves of serum IGF-1 concentrations as a function of age, sex, and puberty from different reference populations and our models (Figs. 4 and 5). Despite known differences in absolute circulating total IGF-1 concentrations between different commercially available IGF-1 immunoassays, in analyses of data with respect to age continuity, the median values obtained during the pubertal peak were generally less pronounced for both boys and girls in studies other than this study (Fig. 4). Furthermore, differences between databases varied considerably between pubertal groups, with smooth changes with age and puberty observed only in our model (Fig. 5). Fig. 3. Relationship of IGF-1 SDS to BMI (A) and height (B), both expressed in SDS and based on the equations given in Table 2. A linear model shows predicted curves for the relationship between serum IGF-1 SDS and height SDS for a child with a BMI arbitrarily fixed at 2 SDS and for the relationship between serum IGF-1 SDS and BMI SDS for a child with a height arbitrarily fixed at 2 SDS. Curves represent the 2.5th percentile ( ), 50th percentile ( ) and 97.5th percentile ( ). For example, 95% of serum IGF-1 concentration lies between 0.27 and 0.58 SDS, with the 50th percentile at 0.43 SDS for a child with a BMI of 2 SDS and height of 0 SDS, and 95% of serum IGF-1 concentration lies between 0.39 and 0.73 SDS, with the 50th percentile at 0.56 SDS for a child with a height of 2 SDS and a BMI of 0 SDS Clinical Chemistry 57:10 (2011)

8 GH/IGF-1 Axis and Bone Turnover Metabolism in Children Fig. 4. Comparison of our reference curve models for serum IGF-1 concentration (fitted mean, solid line) with 3 pediatric models (fitted mean, dotted line) as a function of age and sex. Data were analyzed with age continuity preserved. (A), Nichols Advantage. (B), Immulite; Siemens. (C), Immulite; Siemens. References in which authors reported the serum IGF-1 measurements obtained with these assays are also shown. Discussion In this cross-sectional study, we provide reference values specific for age, sex, and pubertal stage for serum IGF-1 and IGFBP-3 concentrations, the IGF-1/ IGFBP-3 ratio, and CrossLaps and BALP concentrations in healthy French white children between ages 6 20 years. We constructed a model formula to calculate the SD scores for all of these biological parameters as a function of age, sex, and pubertal stage. This single regression model makes possible the simultaneous estimation of all these known explanatory variables and takes into account the interactions between variables, rather than dividing the sample into subgroups corresponding to each pubertal stage and fitting separate regression models for each subgroup. Our model also Clinical Chemistry 57:10 (2011) 1431

9 Fig. 5. Comparison of our reference curve models for serum IGF-1 concentration (fitted mean, solid line) with 4 pediatric models (fitted mean, dotted line) as a function of age, sex and puberty. Data were analyzed pubertal groups preserved. (A), DSL-5600 ACTIVE IGF-1 IRMA; BeckmanCoulter). (B), In-house RIA. (C), Mediagnost. (D), Immulite; Siemens. References in which authors reported the serum IGF-1 measurements obtained with these assays are also shown Clinical Chemistry 57:10 (2011)

10 GH/IGF-1 Axis and Bone Turnover Metabolism in Children shows that all these biological variables are related to age at each stage of puberty. A similar model was previously proposed for serum IGF-1 and IGFBP-3 concentrations and their ratio (7, 15, 16, 19). Our findings confirm those of previous studies showing an agerelated increase in serum IGF-1 and IGFBP-3 concentrations and their ratio during the prepubertal and early pubertal stages, followed by a decrease in late puberty for IGF-1 and the ratio, but not for IGFBP-3, with slightly higher peak values in girls than in boys. Our study is also the first to develop a model suitable for use in children that relates serum BALP and CrossLaps concentrations to age, sex, and puberty simultaneously and converts these biological parameters of bone turnover into SDS with a high degree of accuracy. Consistent with other studies (17, 27 29), serum BALP and CrossLaps concentrations during puberty markers describing only net skeletal formation and resorption, respectively, and unable to distinguish between growth, modeling, and remodeling were found to be higher in boys than in girls, probably reflecting the higher peak height velocity and peak bone mass in boys than in girls, resulting in a greater degree of bone remodeling, but also of bone modeling, during this critical period of growth and bone mass accretion. We also investigated the determinants of these biological parameters over the whole spectrum of height and BMI in this large cohort of carefully selected healthy white children. Previous studies on the relationships between growth factors and anthropometric measurements have yielded conflicting results, with some, but not all, studies indicating positive associations between these parameters and the serum concentrations of IGF-1 and IGFBP-3 and their ratio in children (7, 11 13, 15, 16, 19, 30 33) or adults (34 37).It has been suggested that such associations may be attributable to the effects of IGF-1 on growth velocity rather than adult height, with no persistent effect on adult height. Nevertheless, the molar ratio of IGF-1/ IGFBP-3, which provides an indication of the amount of bioavailable IGF-1, has been shown to be weakly positively associated with the leg-to-trunk ratio, an indicator of relatively long leg length in relation to total stature in adults (38). A positive association was found between serum IGF-1 and IGFBP-3 concentrations, and also between each of these concentrations and height growth over the next 2 years, in prepubertal children of ages from 5 years to 9 10 years (39). We have shown in this study that there are potentially useful positive relationships between serum IGF-1 concentration, IGFBP-3 concentration, and their ratio, and height and BMI expressed in SDS for age and sex. Our findings provide new insight for the interpretation of these biological parameters in clinical practice and, for example, in short children, with respect to normative data appropriate for sex, age, pubertal age, and BMI, which indicated that height SDS was a significant determinant of serum IGF-1 and IGFBP-3 concentrations and of their ratio, expressed as SDS values, over the whole BMI spectrum, including the lowest end of the range. However, although GH secretion and chronic and acute starvation are known to be important determinants of IGF-1 production, the determinants of serum IGF-1 concentrations across the entire weight or height spectrum are poorly understood. We were therefore unable to establish definitively that the relationship between IGF-1 and height, for example, was comparable within groups of children with anorexia nervosa or obesity or children who were very short or tall. Further studies in large groups of patients with growth disorders would be required for this comparison. Our results for bone metabolism markers are consistent with those previously reported for serum CrossLaps concentration, a marker of bone resorption, but not with those for serum BALP concentration, for which a weak positive correlation was found with the SDS of anthropometric markers, suggesting greater bone formation in children that are tall or heavy for their age (17), which was not seen in our study. The use of different assays with different antibodies and ethnic groups, and differences in nutrition and age at onset of puberty between the populations studied may account for the differences in absolute serum IGF-1 concentration observed (the most commonly used parameters in clinical practice and the most frequently studied). We show here that our model, which preserves age continuity as children pass from one pubertal stage to another, increases the precision of the smooth physiological changes observed with age and pubertal stage. This model increases the diagnostic power of SDS values in clinical practice, particularly in children with puberty onsets that deviate from the mean. This study included a substantial number of clinically well-characterized healthy homogeneous white children and adolescents, for whom determinations were carried out after an overnight fast, but it nonetheless had several limitations. First, the withdrawal of the assay initially used for the determination of serum IGF-1 concentrations in this study made it necessary to convert each serum IGF-1 value obtained to an equivalent value for the assay currently in use. The validity of this conversion was higher than the interassay CV of each assay. Second, circannual variations in biological markers of bone turnover have been reported (40) and may contribute to the biological variability of bone turnover. These variations may be at least partly attributed to subclinical vitamin D deficiency during the winter period, which is common in countries of the northern hemisphere. In our study, our blood samples Clinical Chemistry 57:10 (2011) 1433

11 were obtained over an 18-month period and did not take into account the potential circannual variation. Finally, we observed no longitudinal changes in biochemical and auxological markers because the study design was cross-sectional. In conclusion, we present a newly developed model for assessing age-, sex-, and pubertal stage specific SDS reference values for serum IGF-1 and IGFBP-3 concentrations and their ratio and BALP and CrossLaps concentrations. Informative, widely accepted biological norms for the assessment of growth and bone metabolism disorders are required to help clinicians make optimal decisions about patient management. These reference values may also help to increase the diagnostic power of these parameters in France. However, we believe that this model is potentially suitable for use with other assays and in other countries, provided it is correctly validated. Based on this extensive normative data set, our findings highlight the importance of interpreting serum IGF-1 and IGFBP-3 concentrations and their ratio in children as a function of height and BMI SDS values. Further studies are required to determine whether the relationship between serum and auxological data are comparable in children with growth disorders or obesity and to explore the associations between growth and growth factors, with the use of longitudinal growth measurements from childhood to early adulthood. References Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: Grant from Pfizer-France to the Regional Institute for Health (Tours, France). Expert Testimony: None declared. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. Acknowledgments: We thank Professor Paul Czernichow for his help in initiating the study and all doctors, nurses and others from the Regional Institute for Health in Western France who gave up time to help us with the fieldwork. 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