J Pediatr Endocr Met 2015; 28(5-6): 563–569
Joanna Gajewska*, Witold Klemarczyk, Jadwiga Ambroszkiewicz, Katarzyna Szamotulska, Magdalena Chełchowska and Halina Weker
Associations between IGF-I, IGF-binding proteins and bone turnover markers in prepubertal obese children Abstract Objective: To assess the relationships between components of the growth hormone axis, body composition, and bone markers in obese children. Methods: We determined the levels of bone alkaline phosphatase (BALP), C-terminal telopeptide of type I collagen (CTX-I), insulin-like growth factor-I (IGF-I), and IGF-binding proteins (IGFBPs) by immunoenzymatic methods, and body composition by dual-energy X-ray absorptiometry in 45 obese and 20 non-obese children. Results: IGF-I and functional IGFBP-3 levels, IGF-I/total IGFBP-3, and functional IGFBP-3/total IGFBP-3 molar ratios were significantly higher in obese patients than in controls. Multivariate regression analysis in obese patients showed significant associations of BALP with IGF-I (p = 0.047) and percent of body fat mass (p = 0.002). Conclusion: The relationship of IGF-I and functional IGFBP-3 to BALP may support the concept of IGF-I influence on accelerated bone formation process in obesity. Moreover, IGF-I and percentage of body fat mass may be significant predictors of BALP in obese during the prepubertal period. Keywords: bone alkaline phosphatase; collagen type I cross-linked C-telopeptide; functional IGF-binding protein-3; IGF-binding protein-1; total IGF-binding protein-3.
*Corresponding author: Joanna Gajewska, Screening Department, Institute of Mother and Child, Warsaw, Poland, Phone: +48-22-3277260, Fax: +48-22-3277161, E-mail: [email protected] Witold Klemarczyk and Halina Weker: Department of Nutrition, Institute of Mother and Child, Warsaw, Poland Jadwiga Ambroszkiewicz and Magdalena Chełchowska: Screening Department, Institute of Mother and Child, Warsaw, Poland Katarzyna Szamotulska: Department of Epidemiology, Institute of Mother and Child, Warsaw, Poland
DOI 10.1515/jpem-2014-0326 Received July 31, 2014; accepted December 17, 2014; previously published online February 18, 2015
Introduction The insulin-like growth factor (IGF) system appears to be one of the most important factors affecting growth and bone remodeling. IGF-I stimulates the growth plate by promoting chondrogenesis and mediates bone mass accrual by regulating osteoblast differentiation and function (1). Serum IGF-I binds to IGF-binding proteins (IGFBPs), which controls and modulates its tissue bioavailability. Proteolysis of IGFBP3 has been postulated as one of the mechanisms regulating IGF-I action (2). An indicator of IGFBP-3 fragmentation is functional IGFBP-3 (f-IGFBP-3), which may reflect IGF-I binding better than total IGFBP-3 (t-IGFBP-3) (3). Many pathological conditions where the fragmentation level of IGFBP-3 is changed are known. Abnormal IGFBP-3 fragmentation is seen in traumatic and postoperative states, chronic kidney disease, and noninsulin-dependent diabetes (3, 4). The role of the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in the acquisition of peak bone mineral density (BMD) is evident from a number of clinical and animal studies (5). Several cross-sectional studies demonstrated a strong linear relationship between serum IGF-I or IGFBP-3 and BMD in postmenopausal women or in men (6, 7). Changes in concentrations of IGFs and their binding proteins before and during puberty, correlating with skeletal changes and concentrations of bone turnover markers were also observed (8–11). Among bone markers, bone alkaline phosphatase (BALP) and collagen type I cross-linked C-telopeptide (CTX-I), were demonstrated to reflect changes in children’s growth. Physiologically, serum BALP and CTX-I levels increased slightly and were similar in both sexes during the prepubertal period (12). There are conflicting reports on the effects of obesity on IGFs, related binding proteins and IGFBP-3 proteolysis.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 5/20/15 1:55 PM
564 Gajewska et al.: IGF system and bone markers in obesity IGF-I and IGFBP-3 levels in obese children were reported to be high (8, 13–15) or normal (16–19) despite the low rate of growth hormone (GH) secretion (16, 18). However, accelerated growth and bone maturation were observed in these children and were associated with greater height (20). Therefore, in obese children, a positive effect of fat on bone may occur, but this phenomenon in the prepubertal period is not completely understood. The aims of this study were: (1) to analyze the levels of IGF-I, IGF-binding proteins and bone turnover markers in obese and nonobese children, (2) to evaluate the relationships between components of the growth hormone axis, body composition and bone markers in growing obese individuals.
Materials and methods
Electric Healthcare, Madison, WI, USA). All subjects were measured on the same machine using standard positioning techniques.
Dietary intake Dietary intake data were collected using randomly selected 3-day records. Subjects and their parents were asked to record the type and amount of food and beverages consumed for two consecutive weekdays and one weekend day. We assessed the average daily energy intake and the percentage of energy intake from protein, fat, and carbohydrates in the diets of obese and non-obese children. Average daily food rations and their nutritional value were calculated using nutritional analysis software (Dietetyk2®, National Food and Nutrition Institute, Warsaw, Poland) (23). The daily energy intake was similar in both groups (1725±445 in obese vs. 1593±443 kcal/day in controls; p = 0.227). Patients had three to five meals every day. The diet in both groups was similarly composed of about 20% protein, 30% fat, and 50% carbohydrates. Participants in our study did not receive vitamins or mineral supplements.
Subjects We examined 45 obese and 20 non-obese prepubertal children aged 4–10 years, which were recruited between 2012 and 2013 among consecutive patients presenting for dietary counseling to the Department of Nutrition at the Institute of Mother and Child. The study groups were representative of the entire study population. Children were classified as obese [body mass index (BMI) z-score > 2] and nonobese (BMI z-score ). Exclusion criteria were: (1) the presence of endocrine disorders or genetic syndromes, including syndromic obesity; (2) chronic medical conditions; and (3) taking medications that could affect growth, pubertal development, nutritional or dietary status. Pubertal stage was determined according to the Tanner scale, and subjects who showed pubertal development were excluded. The physical activity in both studied groups was estimated using a questionnaire (21). Sports activities at school and out of school of obese and non-obese children were similar (3.9±2.1 h/week vs. 4.3±2.0 h/week, respectively). However, the prevalence of sedentary behaviors related with screen time was higher in the obese group than in normalweight controls. About 80% (38/45 children) of obese and 60% (12/20 children) of controls spent more than 2 h/day watching television. Written informed consent was obtained from the parents of all the examined children. The Ethics Committee of the Institute of Mother and Child approved this study.
Anthropometric parameters Physical examination, including body height and weight, was performed. Body height was measured using a standing stadiometer and recorded with a precision of 1 mm. Body weight was assessed unclothed to the nearest 0.1 kg with a calibrated balance scale. BMI was calculated as body weight divided by height squared (kg/m2). The BMI of each individual was converted to a standard BMI z-score for the child’s age and gender using Polish reference tables (22). Body composition [fat mass, lean mass, total bone mineral content (BMC)], and total BMD, BMD L2-L4 were measured by dual-energy X-ray absorptiometry (DXA) using Lunar Prodigy with pediatric software 9.30.044 (General
Biochemical measurements Venous blood samples were collected between 08:00 and 10:00 am in the morning after an overnight fast and centrifuged (1000 g for 10 min at 4°C). BALP and CTX-I were determined by immunoenzymatic methods. BALP activity was estimated using kits from Quidel Corporation (San Diego, CA, USA) with intra- and inter-assay coefficients (CVs) of 6% and 8%, respectively. CTX-I concentration was measured with serum CrossLaps ELISA kit (IDS, Boldon, UK) specific for the beta-aspartate form of the EKHD-b-GGR epitope derived from the cross-linked degradation products of C-terminal telopeptides of type I collagen. Intra- and inter-assay CVs for CTX were 3% and 10.9%, respectively. IGF-I, IGFBP-1, and t-IGFBP-3 values were determined by immunoassay (ELISA) (Mediagnost, Reutlingen, Germany). The intra- and inter-assay coefficients of variation were less than 4.8% and 7.4% for IGF-I, 6.8% and 7.4% for IGFBP-1, 4.5% and 6.3% for t-IGFBP-3, repectively. We calculated the IGF-I/IGFBP-3 molar ratio, an estimation of free IGF-I concentration, as (IGF-I [ng/mL] × 0.130)/(IGFBP-3 [ng/mL] × 0.036). The IGF-I/IGFBP-3 molar ratio has been used as a surrogate parameter associated with IGF-I bioactivity (23). The f-IGFBP-3 concentration was determined by ligand-binding immunoassay (LIA) (Mediagnost, Reutlingen, Germany) with intraand inter-assay variability less than 5.6% and 6.8%, respectively. This assay for f-IGFBP-3 exclusively detects IGFBP-3 capable of IGF binding. The f-IGFBP-3/t-IGFBP-3 molar ratio was calculated to serve as an index of IGFBP-3 fragmentation. A ratio close to 0 indicates almost complete fragmentation and loss of IGFBP-3 function, whereas a ratio close to 1 indicates the presence of mostly intact protein with an unchanged biological function.
Statistical analysis Statistical analysis was performed using SPSS version 18.0 software (SPSS Inc, Chicago, IL, USA). The results are presented as means±standard deviation (SD) for normally distributed data or medians and interquartile range (25th–75th percentiles) for
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 5/20/15 1:55 PM
Gajewska et al.: IGF system and bone markers in obesity 565
non-normally distributed variables. The Kolmogorov-Smirnov test and graphical inspections of data were used for evaluating distribution for normality. Differences in anthropometric characteristics and biochemical parameters of obese and non-obese children were assessed using Student’s t-test for normally distributed data and non-parametric Mann-Whitney test for non-normally distributed variables. Spearman correlations between parameters were calculated. To evaluate relationships between bone markers and growth factors in obese children, multivariate linear regression models with BALP as a dependent variable and two independent variables were estimated. As one independent variable we selected one of the components of the IGF system (IGF-I, IGFBP-1, IGFBP-3, f-IGFBP-3, IGF-I/tIGFBP-3 molar ratio or f-IGFBP-3/t-IGFBP-3 molar ratio – models 1–6) and BMI z –score or fat mass (%) as the other independent variable. Statistical significance was set at p
Joanna Gajewska*, Witold Klemarczyk, Jadwiga Ambroszkiewicz, Katarzyna Szamotulska, Magdalena Chełchowska and Halina Weker
Associations between IGF-I, IGF-binding proteins and bone turnover markers in prepubertal obese children Abstract Objective: To assess the relationships between components of the growth hormone axis, body composition, and bone markers in obese children. Methods: We determined the levels of bone alkaline phosphatase (BALP), C-terminal telopeptide of type I collagen (CTX-I), insulin-like growth factor-I (IGF-I), and IGF-binding proteins (IGFBPs) by immunoenzymatic methods, and body composition by dual-energy X-ray absorptiometry in 45 obese and 20 non-obese children. Results: IGF-I and functional IGFBP-3 levels, IGF-I/total IGFBP-3, and functional IGFBP-3/total IGFBP-3 molar ratios were significantly higher in obese patients than in controls. Multivariate regression analysis in obese patients showed significant associations of BALP with IGF-I (p = 0.047) and percent of body fat mass (p = 0.002). Conclusion: The relationship of IGF-I and functional IGFBP-3 to BALP may support the concept of IGF-I influence on accelerated bone formation process in obesity. Moreover, IGF-I and percentage of body fat mass may be significant predictors of BALP in obese during the prepubertal period. Keywords: bone alkaline phosphatase; collagen type I cross-linked C-telopeptide; functional IGF-binding protein-3; IGF-binding protein-1; total IGF-binding protein-3.
*Corresponding author: Joanna Gajewska, Screening Department, Institute of Mother and Child, Warsaw, Poland, Phone: +48-22-3277260, Fax: +48-22-3277161, E-mail: [email protected] Witold Klemarczyk and Halina Weker: Department of Nutrition, Institute of Mother and Child, Warsaw, Poland Jadwiga Ambroszkiewicz and Magdalena Chełchowska: Screening Department, Institute of Mother and Child, Warsaw, Poland Katarzyna Szamotulska: Department of Epidemiology, Institute of Mother and Child, Warsaw, Poland
DOI 10.1515/jpem-2014-0326 Received July 31, 2014; accepted December 17, 2014; previously published online February 18, 2015
Introduction The insulin-like growth factor (IGF) system appears to be one of the most important factors affecting growth and bone remodeling. IGF-I stimulates the growth plate by promoting chondrogenesis and mediates bone mass accrual by regulating osteoblast differentiation and function (1). Serum IGF-I binds to IGF-binding proteins (IGFBPs), which controls and modulates its tissue bioavailability. Proteolysis of IGFBP3 has been postulated as one of the mechanisms regulating IGF-I action (2). An indicator of IGFBP-3 fragmentation is functional IGFBP-3 (f-IGFBP-3), which may reflect IGF-I binding better than total IGFBP-3 (t-IGFBP-3) (3). Many pathological conditions where the fragmentation level of IGFBP-3 is changed are known. Abnormal IGFBP-3 fragmentation is seen in traumatic and postoperative states, chronic kidney disease, and noninsulin-dependent diabetes (3, 4). The role of the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in the acquisition of peak bone mineral density (BMD) is evident from a number of clinical and animal studies (5). Several cross-sectional studies demonstrated a strong linear relationship between serum IGF-I or IGFBP-3 and BMD in postmenopausal women or in men (6, 7). Changes in concentrations of IGFs and their binding proteins before and during puberty, correlating with skeletal changes and concentrations of bone turnover markers were also observed (8–11). Among bone markers, bone alkaline phosphatase (BALP) and collagen type I cross-linked C-telopeptide (CTX-I), were demonstrated to reflect changes in children’s growth. Physiologically, serum BALP and CTX-I levels increased slightly and were similar in both sexes during the prepubertal period (12). There are conflicting reports on the effects of obesity on IGFs, related binding proteins and IGFBP-3 proteolysis.
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 5/20/15 1:55 PM
564 Gajewska et al.: IGF system and bone markers in obesity IGF-I and IGFBP-3 levels in obese children were reported to be high (8, 13–15) or normal (16–19) despite the low rate of growth hormone (GH) secretion (16, 18). However, accelerated growth and bone maturation were observed in these children and were associated with greater height (20). Therefore, in obese children, a positive effect of fat on bone may occur, but this phenomenon in the prepubertal period is not completely understood. The aims of this study were: (1) to analyze the levels of IGF-I, IGF-binding proteins and bone turnover markers in obese and nonobese children, (2) to evaluate the relationships between components of the growth hormone axis, body composition and bone markers in growing obese individuals.
Materials and methods
Electric Healthcare, Madison, WI, USA). All subjects were measured on the same machine using standard positioning techniques.
Dietary intake Dietary intake data were collected using randomly selected 3-day records. Subjects and their parents were asked to record the type and amount of food and beverages consumed for two consecutive weekdays and one weekend day. We assessed the average daily energy intake and the percentage of energy intake from protein, fat, and carbohydrates in the diets of obese and non-obese children. Average daily food rations and their nutritional value were calculated using nutritional analysis software (Dietetyk2®, National Food and Nutrition Institute, Warsaw, Poland) (23). The daily energy intake was similar in both groups (1725±445 in obese vs. 1593±443 kcal/day in controls; p = 0.227). Patients had three to five meals every day. The diet in both groups was similarly composed of about 20% protein, 30% fat, and 50% carbohydrates. Participants in our study did not receive vitamins or mineral supplements.
Subjects We examined 45 obese and 20 non-obese prepubertal children aged 4–10 years, which were recruited between 2012 and 2013 among consecutive patients presenting for dietary counseling to the Department of Nutrition at the Institute of Mother and Child. The study groups were representative of the entire study population. Children were classified as obese [body mass index (BMI) z-score > 2] and nonobese (BMI z-score ). Exclusion criteria were: (1) the presence of endocrine disorders or genetic syndromes, including syndromic obesity; (2) chronic medical conditions; and (3) taking medications that could affect growth, pubertal development, nutritional or dietary status. Pubertal stage was determined according to the Tanner scale, and subjects who showed pubertal development were excluded. The physical activity in both studied groups was estimated using a questionnaire (21). Sports activities at school and out of school of obese and non-obese children were similar (3.9±2.1 h/week vs. 4.3±2.0 h/week, respectively). However, the prevalence of sedentary behaviors related with screen time was higher in the obese group than in normalweight controls. About 80% (38/45 children) of obese and 60% (12/20 children) of controls spent more than 2 h/day watching television. Written informed consent was obtained from the parents of all the examined children. The Ethics Committee of the Institute of Mother and Child approved this study.
Anthropometric parameters Physical examination, including body height and weight, was performed. Body height was measured using a standing stadiometer and recorded with a precision of 1 mm. Body weight was assessed unclothed to the nearest 0.1 kg with a calibrated balance scale. BMI was calculated as body weight divided by height squared (kg/m2). The BMI of each individual was converted to a standard BMI z-score for the child’s age and gender using Polish reference tables (22). Body composition [fat mass, lean mass, total bone mineral content (BMC)], and total BMD, BMD L2-L4 were measured by dual-energy X-ray absorptiometry (DXA) using Lunar Prodigy with pediatric software 9.30.044 (General
Biochemical measurements Venous blood samples were collected between 08:00 and 10:00 am in the morning after an overnight fast and centrifuged (1000 g for 10 min at 4°C). BALP and CTX-I were determined by immunoenzymatic methods. BALP activity was estimated using kits from Quidel Corporation (San Diego, CA, USA) with intra- and inter-assay coefficients (CVs) of 6% and 8%, respectively. CTX-I concentration was measured with serum CrossLaps ELISA kit (IDS, Boldon, UK) specific for the beta-aspartate form of the EKHD-b-GGR epitope derived from the cross-linked degradation products of C-terminal telopeptides of type I collagen. Intra- and inter-assay CVs for CTX were 3% and 10.9%, respectively. IGF-I, IGFBP-1, and t-IGFBP-3 values were determined by immunoassay (ELISA) (Mediagnost, Reutlingen, Germany). The intra- and inter-assay coefficients of variation were less than 4.8% and 7.4% for IGF-I, 6.8% and 7.4% for IGFBP-1, 4.5% and 6.3% for t-IGFBP-3, repectively. We calculated the IGF-I/IGFBP-3 molar ratio, an estimation of free IGF-I concentration, as (IGF-I [ng/mL] × 0.130)/(IGFBP-3 [ng/mL] × 0.036). The IGF-I/IGFBP-3 molar ratio has been used as a surrogate parameter associated with IGF-I bioactivity (23). The f-IGFBP-3 concentration was determined by ligand-binding immunoassay (LIA) (Mediagnost, Reutlingen, Germany) with intraand inter-assay variability less than 5.6% and 6.8%, respectively. This assay for f-IGFBP-3 exclusively detects IGFBP-3 capable of IGF binding. The f-IGFBP-3/t-IGFBP-3 molar ratio was calculated to serve as an index of IGFBP-3 fragmentation. A ratio close to 0 indicates almost complete fragmentation and loss of IGFBP-3 function, whereas a ratio close to 1 indicates the presence of mostly intact protein with an unchanged biological function.
Statistical analysis Statistical analysis was performed using SPSS version 18.0 software (SPSS Inc, Chicago, IL, USA). The results are presented as means±standard deviation (SD) for normally distributed data or medians and interquartile range (25th–75th percentiles) for
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 5/20/15 1:55 PM
Gajewska et al.: IGF system and bone markers in obesity 565
non-normally distributed variables. The Kolmogorov-Smirnov test and graphical inspections of data were used for evaluating distribution for normality. Differences in anthropometric characteristics and biochemical parameters of obese and non-obese children were assessed using Student’s t-test for normally distributed data and non-parametric Mann-Whitney test for non-normally distributed variables. Spearman correlations between parameters were calculated. To evaluate relationships between bone markers and growth factors in obese children, multivariate linear regression models with BALP as a dependent variable and two independent variables were estimated. As one independent variable we selected one of the components of the IGF system (IGF-I, IGFBP-1, IGFBP-3, f-IGFBP-3, IGF-I/tIGFBP-3 molar ratio or f-IGFBP-3/t-IGFBP-3 molar ratio – models 1–6) and BMI z –score or fat mass (%) as the other independent variable. Statistical significance was set at p
