Clinical Biochemistry 47 (2014) 1341–1343
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Urinary glycosaminoglycan (uGAG) excretion in healthy pediatric and adolescent population Katarzyna Komosinska-Vassev a,⁎, Dorota Blat a, Paweł Olczyk b, Anna Szeremeta a, Agnieszka Jura-Półtorak a, Katarzyna Winsz-Szczotka a, Katarzyna Klimek c, Krystyna Olczyk a a b c
Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Silesia, Poland Department of Community Pharmacy, Medical University of Silesia, Poland Department of Statistics, Medical University of Silesia, Poland
a r t i c l e
i n f o
Article history: Received 1 December 2013 Received in revised form 9 June 2014 Accepted 10 June 2014 Available online 20 June 2014 Keywords: Urinary glycosaminoglycans Chondroitin/dermatan sulfates Heparan sulfates Hyaluronan Healthy children Extracellular matrix remodeling
a b s t r a c t Objectives: The influence of age and gender factor on the urinary excretion of total glycosaminoglycans (uGAGs) and their particular types: chondroitin/dermatan sulfates (CS/DSs), heparan sulfates (HSs) and hyaluronan (HA) was analyzed in healthy pediatric and adolescent population. Design and methods: Urine samples were collected from 95 healthy children. Sulfated GAGs excreted in the urine were quantitated using standardized dye-binding method, while the concentrations of HA were determined by immunoassay. Results: Age-dependent decline in total uGAG excretion (r = −0.686; p b 0.001), resulting from a decrease in particular GAG fractions i.e. CS/DS (r = − 0.757; p b 0.001), HS (r = − 0.401; p b 0.05) and HA (r = − 0.638; p b 0.001), was found in healthy subjects. The observed differences were not gender specific with the exception of HS, in which excretion declines with age in males (r = − 0.501; p b 0.05) and does not change in females. Changes in the distribution pattern of uGAG were also found. CS/DS were the predominant uGAG's fraction, representing from 55% to 76% of the total GAGs. Children up to 3 years excreted more GAGs than older subjects and with a higher proportion of CS/DS and less content of HS. Moreover, the relative contribution of HA was increased twofold in adolescents, aged 15–18, as compared to younger subjects. A negative correlation existed between uGAG excretion and body height, except for HS, for which this relationship was found only in males. Conclusions: Changes in urinary distribution pattern of particular GAG types during physiological human growth and development were found. Evaluation of urinary GAG screening procedures during pathological conditions should be based on the GAG/creatinine ratios with age and gender taken into account. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Age-related remodeling of the extracellular matrix components is an integral part of child development from infancy to adolescence. This process is associated with still not fully elucidated structural and functional changes in the extracellular matrix components including glycosaminoglycans (GAGs) such as chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate/heparin (HS/H), keratan sulfate (KS) and hyaluronan (HA) [1,2]. The increasing importance of urinary GAG (uGAG) excretion has been implicated in clinical practice, including Abbreviations: CS, chondroitin sulfate; CS/DSs, chondroitin/dermatan sulfates; DS, dermatan sulfate; GAGs, glycosaminoglycans; ECM, extracellular matrix; HA, hyaluronan; HS/H, heparan sulfate/heparin; KS, keratan sulfate; PGs, proteoglycans; uGAGs, urinary glycosaminoglycans. ⁎ Corresponding author at: Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Silesia, Jednosci 8, 41-200 Sosnowiec, Poland. Fax: + 48 323641157. E-mail address: [email protected] (K. Komosinska-Vassev).
childhood and developmental disorders. So far, preliminary data on uGAG changes in healthy children were conducted on a small number of participants, with different analytical procedures, and without dividing the children into subgroups according to stage of growth and development [3–7]. Data related to uGAG excretion are especially lacking in children less than 10 years of age. Hence, the aim of the present study was to evaluate the influence of age and gender on the urinary content of total GAGs and their particular fractions in healthy children and adolescent, using a standardized method of GAG measurement.
Material and methods Subjects Studies were carried out on 95 healthy volunteers (51 males and 44 females), aged from 1 to 18 years. Subjects were selected after medical history, physical examination and laboratory analyses. The study
http://dx.doi.org/10.1016/j.clinbiochem.2014.06.012 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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Table 1 Urinary glycosaminoglycan (GAG) concentrations of particular types of GAGs in age-specific subgroups of healthy children. Age groups (years)
Total GAGs
μg/mL
mg/g Cr
μg/mL
mg/g Cr
μg/mL
mg/g Cr
ng/mL
μg/g Cr
1–3 (n = 21)
30.7 (23–37.4) 40.3 (32–50) 40.1 (31.7–63) 37.4 (21.5–44.7) 17.1 (10.9–37.8)
76.4 (56.1–96) 47.2 (34.2–69.8) 41.8 (33.4–53.2)a 23.4 (16.3–30.3)a,b 11.8 (5.1–19.1)a,b,c
22.7 (17.7–35.2) 23.4 (19.7–29.7) 24.1 (16.3–32.3) 25.6 (12.2–29.7) 12.7 (9.5–33.5)
58 (40.3–75.8) 29 (20.6–35.8)a 24.7 (18.4–31.7)a 12.7 (10.5–20.9)a 7.4 (4.3–10.8)a,b,c
6.7 (2.3–10.1) 15.6 (9.4–24.4) 14.5 (11.3–29.7) 11.9 (7.5–15.6) 4.9 (4–8.8)
11 (5.1–21) 15.7 (9.3–34.8) 14.6 (10.2–27) 7.2 (4.4–13.6) 4.1 (1.7–5.6)b,c
12.7 (10.5–13.1) 13.1 (12.2–13.6) 12.9 (12.3–13.3) 12.8 (12.4–13.4) 13.2 (13–13.6)
27.5 (17.6–42.8) 16.3 (10.4–20.8) 12.3 (9.9–17) 9 (6.2–13.4)b 7.8 (5.5–11)a,b,c
4–6 (n = 24) 7–10 (n = 20) 11–14 (n = 16) 15–18 (n = 14)
Glycosaminoglycan types Chondroitin/dermatan sulfates (CS/DS)
Heparan sulfates (HS)
Hyaluronan (HA)
Results are expressed as medians (quartile 1–quartile 3). a p b 0.05, compared to subjects aged 1–3 years. b p b 0.05, compared to subjects aged 4–6 years. c p b 0.05, compared to subjects aged 7–10 years.
protocol was approved by the Ethics Committee of the Medical University of Silesia, Poland. First-morning urine samples were divided into five groups according to the typical pediatric classification of age developmental periods [8]. Laboratory analysis Total sulfated GAGs were quantitated using the Blyscan™ Sulfated Glycosaminoglycan Assay Kit (Biocolor Ltd., United Kingdom), based on the reaction of 1,9-dimethylmethylene blue with sulfated GAGs. For the quantitative measurement of CS/DS, isolation procedure of these glycans was performed. All sulfated GAG measurements were done in a single-run. The intra-assay CVs for total sulfated GAG method was less than 6%. The results of GAGs excreted in the urine sample were expressed as μg/mL and also normalized to creatinine and expressed as uGAG to creatinine ratio (uGAG/g Cr). The urine HA was measured by TECO® Hyaluronic Acid Test Kit provided by TECOmedical AG (Sissach, Switzerland) according to the manufacturer's protocol. The appropriate low and high control samples were used for quality control procedure. The HA determination was completed within one day. The intra-assay variations of this assay was b5.6%. Urinary HA level was normalized to creatinine and expressed in mg/g Cr. Statistical methods Statistical analysis was performed using data analysis software system: StatSoft, Inc. Statistica 10.0. Statistical differences between the five age subgroups were verified by the nonparametric Kruskal–Wallis test. A Mann–Whitney U-test for independent variables was used to compare the differences between male and female subjects. Spearman's rank correlation analysis was used to determine the correlation between variables. p values of less than 0.05 were considered to indicate statistical significance. Results Age-related differences in uGAG excretion in healthy children were found in our study only when the obtained results were expressed as a GAG/creatinine ratio (Table 1). The total amount of uGAG/Cr decreased gradually with advancing age (r = − 0.686; p b 0.001), both in males (r = − 0.746; p b 0.05) and in females (r = − 0.56; p b 0.05). The highest values of total uGAG excretion were found in the youngest group of children. Regression analysis revealed that uGAG/Cr was inversely related to body height (r = − 0.658;
p b 0.001), demonstrating a stronger character in males (r = −0.774; p b 0.001) than in females (r = −0.485; p b 0.05) (Table 2). Qualitative analysis of uGAGs allowed identifying the following GAG fractions: CS/DS, HS and HA. The first one was always the predominant GAG type in all age groups, representing from 55% to 76% of the total GAGs. Strong age-related decline of CS/DS to creatinine ratio was shown in the whole group of participants (r = − 0.757; p b 0.001). There were no significant differences among urinary CS/DS results for males versus females. Moreover, decreasing with age urinary CS/DS excretion has been closely related with body height (r = − 0.723; p b 0.001), both in males (r = − 0.798; p b 0.001) and in females (r = − 0.618; p b 0.05). Further analyzed fraction of sulfated GAGs, HS when normalized to creatinine excretion, represents from 14% of the total GAGs in the youngest study participants to above 30% in older subgroups. Urinary HS changes were manifested as a gradual decrease with age (r = −0.401; p b 0.05) in the whole group of healthy children. When the results were examined by gender, the correlation between age and HS was evident in males only (r = − 0.501; p b 0.05). In males, decreasing with age urinary HS excretion was additionally correlated with body height (r = −0.421; p b 0.05), which was not observed in females. The urinary HA excretion normalized to creatinine showed a continued decrease with age (r = −0.638; p b 0.001). Gender factor had no direct impact on age-related urinary HA excretion. A negative correlation existed between urinary HA levels and body height (r = −0.653; p b 0.001). Additionally, changes in the distribution pattern of particular GAG types presented as a percentage of total GAGs were observed. Children aged 1–3 excreted considerable less heparin sulfates and considerable more CS/DS than did children from the other age subgroups. Proportional amount of urinary HA in teenagers aged 15–18 increased two times as compared to the infancy group.
Discussion Decreasing with age uGAG excretion in healthy children and adolescent volunteers has been demonstrated in our study. uGAG/Cr was a few fold elevated in the youngest group of children, as compared with the older participants. The obtained results consisted of some earlier investigations, which demonstrated decreasing with age uGAG excretion in healthy subjects [3,5,7]. However, these reports did not provide details in the qualitative evaluation of uGAG subclasses. In our study, a distinct age-related decrease in urine CS/DS, HS and HA fractions was found. CS/DS was the prevailing urinary GAG. Changes in the proportional amount of particular GAG types were also presented in our
K. Komosinska-Vassev et al. / Clinical Biochemistry 47 (2014) 1341–1343
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Table 2 Effect of age on urinary excretion of total sulfated glycosaminoglycans (sGAG) and particular GAG fractions i.e. chondroitin/dermatan sulfates (CS/DSs), heparan sulfates (HSs) and hyaluronan (HA) in healthy children. Age
sGAG/creatinine ratio CS–DS/creatinine ratio HS/creatinine ratio HA/creatinine ratio
Body length
Whole population
Male gender
Female gender
Whole population
Male gender
Female gender
−0.686⁎⁎ −0.757⁎⁎ −0.401⁎ −0.638⁎⁎
−0.746⁎ −0.776⁎ −0.501⁎ −0.566⁎⁎
−0.56⁎ −0.704⁎ NS −0.703
−0.658⁎⁎ −0.723⁎⁎
−0.774⁎⁎ −0.798⁎⁎ −0.421⁎ −0.667⁎⁎
−0.485⁎ −0.618⁎ NS −0.651⁎⁎
NS −0.653⁎⁎
Note: Spearman's rank correlation coefficients. ⁎ p b 0.05. ⁎⁎ p b 0.001.
study. Children up to 3 years excreted more GAG than older subjects and with a higher proportion of CS/DS and less content of HS. The observed differences can be explained in part by the fact that during infancy and toddler stage the most marked growth in humans occurs compared to any other stage of child development [2]. Then, rapid metabolic turnover of connective tissue during postnatal development may be the main source of increased urinary CS/DS excretion, especially that CS/DS PGs are the most abundant PGs in the ECM [9]. The close relationship between urinary CS/DS excretion and body height found in our study additionally supports that a great part of this GAG fraction in the urine seems to be of systemic origin. In this study we did not observe significant variation in the relative concentration of urinary CS/DS and HA between males and females. However, sex was an important variable influencing urinary HS levels. Urinary changes of HS are significantly inversely correlated with age only in males, not in females. The cause of this difference seems to involve sex hormones. Endogenous estrogens have been shown in experimental studies to inhibit the synthesis of the ECM molecules by many mesenchymal cell types [10,11]. Such an inhibition would shift PG/ GAG turnover toward degradation, which could explain the increased amount of GAGs excreted in the urine. On the other hand, female sex steroid hormones can also modulate the activity of matrix metalloproteinases, enzymes taking part in the ECM components' degradation [12], consequently affecting the amount and composition of uGAGs excretion. In accordance with some earlier reports, we suggest that urinary HS subfraction seems to mainly result from the kidney PG/GAG metabolism, with some contribution of urinary tract cells and systemic ECM matrix turnover. A close relationship between uHS excretion and body height was found in our study only in males and not in females. Lemos et al. [10] experimental studies have shown that male sex hormones may play a role in a synthesis of renal GAG, mainly HS. As regards another component evaluated in our work, hyaluronan, we found a significantly decreasing level without gender dependent relationship. These results could be related with age developing pro-oxidant/ antioxidant imbalance [13]. Products of PG/GAG degradation influenced by reactive oxygen species consist of smaller fragments which appear in the urine after being filtrated through the glomerulus [12]. Nonsulfated GAG, hyaluronan is readily depolymerized by free radicals to smaller chain length fragments [14], which can finally increase the urine pool of these molecules, as we have shown in the oldest group of study participants. A potential limitation of our study is the small number of samples. The results of our study must be carefully assessed when comparing
them to other populations. Thus, longitudinal race-matched studies with a larger sample size are required to confirm our results. In conclusion, our data indicate that significant urinary quantitative GAG alterations and changes in the distribution pattern occur in healthy pediatric and adolescent population. Chondroitin/dermatan sulfates were predominant urinary GAG fraction and the relative contribution of hyaluronan was increased twofold in adolescents than in younger subjects. Quantitative determination of urinary GAG excretion should be adjusted to age and gender factor and only then it appears to present a valuable, non-invasive diagnostic tool in clinical practice. Conflict of interest statement No conflict of interest. References [1] Bailey AJ. Molecular mechanisms of ageing in connective tissues. Mech Ageing Dev 2001;122:735–55. [2] Sames K. The role of proteoglycans and glycosaminoglycans in aging. In: Karger S, Hamburg AG, editors. 1994. p. 103. [3] Lee E-Y, Kim S-Y, Whang S-K, Ywang K-Y, Yang J-O, Hong S-Y. Isolation, identification and quantification of urinary glycosaminoglycans. Am J Nephrol 2003;23:152–7. [4] Buzzega D, Pederzoli F, Maccari F, Aslan D, Turk M, Volpi N. Comparison of cetylpyridinium chloride and cetyltrimethylammonium bromide extractive procedures for quantification and characterization of human urinary glycosaminoglycans. Clin Chem Lab Med 2010;48:1133–9. [5] de la Cruz Amorós V, Cortés Castell E, Moya M. Urinary excretion of mucopolysaccharides in pediatric and adolescent patients. An Esp Pediatr 1999;50:361–6. [6] Harangi F, Györke Z, Melegh B. Urinary glycosaminoglycan excretion in healthy and stone-forming children. Pediatr Nephrol 1996;10:555–8. [7] Gallegos-Arreola MP, Machorro-Lazo MV, Flores-Martínez SE, Zúñiga-González GM, Figuera LE, González-Noriega A, et al. Urinary glycosaminoglycan excretion in healthy subjects and in patients with mucopolysaccharidoses. Arch Med Res 2000;31:505–10. [8] Krawczyński M. Okresy rozwoju ontogenetycznego człowieka. In: Krawczyński M, editor. Propedeutyka pediatrii. 1st ed. PZWL; 2003. p. 34–5. [9] Thelin MA, Bartolini B, Axelsson J, Gustafsson R, Tykesson E, Pera E, et al. Biological functions of iduronic acid in chondroitin/dermatan sulfate. FEBS J 2013;280:2431–46. [10] Lemos CC, Tovar AM, Guimarães MA, Bregman R. Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure. Braz J Med Biol Res 2013;46:567–73. [11] Maroclo MV, Pereira SD, Sampaio FJ, Cardoso LE. Urinary glycosaminoglycan excretion during the menstrual cycle in normal young women. J Urol 2005;173:1789–92. [12] Singh AK, Chattopadhyay R, Chakravarty B, Chaudhury K. Altered circulating levels of matrix metalloproteinases 2 and 9 and their inhibitors and effect of progesterone supplementation in women with endometriosis undergoing in vitro fertilization. Fertil Steril 2013;100:127–34. [13] Tsukahara H. Biomarkers for oxidative stress: clinical application in pediatric medicine. Curr Med Chem 2007;14:339–51. [14] Moseley R, Waddington RJ, Embery G. Degradation of glycosaminoglycans by reactive oxygen species derived from stimulated polymorphonuclear leukocytes. Biochim Biophys Acta 1997;1362:221–31.
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Urinary glycosaminoglycan (uGAG) excretion in healthy pediatric and adolescent population Katarzyna Komosinska-Vassev a,⁎, Dorota Blat a, Paweł Olczyk b, Anna Szeremeta a, Agnieszka Jura-Półtorak a, Katarzyna Winsz-Szczotka a, Katarzyna Klimek c, Krystyna Olczyk a a b c
Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Silesia, Poland Department of Community Pharmacy, Medical University of Silesia, Poland Department of Statistics, Medical University of Silesia, Poland
a r t i c l e
i n f o
Article history: Received 1 December 2013 Received in revised form 9 June 2014 Accepted 10 June 2014 Available online 20 June 2014 Keywords: Urinary glycosaminoglycans Chondroitin/dermatan sulfates Heparan sulfates Hyaluronan Healthy children Extracellular matrix remodeling
a b s t r a c t Objectives: The influence of age and gender factor on the urinary excretion of total glycosaminoglycans (uGAGs) and their particular types: chondroitin/dermatan sulfates (CS/DSs), heparan sulfates (HSs) and hyaluronan (HA) was analyzed in healthy pediatric and adolescent population. Design and methods: Urine samples were collected from 95 healthy children. Sulfated GAGs excreted in the urine were quantitated using standardized dye-binding method, while the concentrations of HA were determined by immunoassay. Results: Age-dependent decline in total uGAG excretion (r = −0.686; p b 0.001), resulting from a decrease in particular GAG fractions i.e. CS/DS (r = − 0.757; p b 0.001), HS (r = − 0.401; p b 0.05) and HA (r = − 0.638; p b 0.001), was found in healthy subjects. The observed differences were not gender specific with the exception of HS, in which excretion declines with age in males (r = − 0.501; p b 0.05) and does not change in females. Changes in the distribution pattern of uGAG were also found. CS/DS were the predominant uGAG's fraction, representing from 55% to 76% of the total GAGs. Children up to 3 years excreted more GAGs than older subjects and with a higher proportion of CS/DS and less content of HS. Moreover, the relative contribution of HA was increased twofold in adolescents, aged 15–18, as compared to younger subjects. A negative correlation existed between uGAG excretion and body height, except for HS, for which this relationship was found only in males. Conclusions: Changes in urinary distribution pattern of particular GAG types during physiological human growth and development were found. Evaluation of urinary GAG screening procedures during pathological conditions should be based on the GAG/creatinine ratios with age and gender taken into account. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Age-related remodeling of the extracellular matrix components is an integral part of child development from infancy to adolescence. This process is associated with still not fully elucidated structural and functional changes in the extracellular matrix components including glycosaminoglycans (GAGs) such as chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate/heparin (HS/H), keratan sulfate (KS) and hyaluronan (HA) [1,2]. The increasing importance of urinary GAG (uGAG) excretion has been implicated in clinical practice, including Abbreviations: CS, chondroitin sulfate; CS/DSs, chondroitin/dermatan sulfates; DS, dermatan sulfate; GAGs, glycosaminoglycans; ECM, extracellular matrix; HA, hyaluronan; HS/H, heparan sulfate/heparin; KS, keratan sulfate; PGs, proteoglycans; uGAGs, urinary glycosaminoglycans. ⁎ Corresponding author at: Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Silesia, Jednosci 8, 41-200 Sosnowiec, Poland. Fax: + 48 323641157. E-mail address: [email protected] (K. Komosinska-Vassev).
childhood and developmental disorders. So far, preliminary data on uGAG changes in healthy children were conducted on a small number of participants, with different analytical procedures, and without dividing the children into subgroups according to stage of growth and development [3–7]. Data related to uGAG excretion are especially lacking in children less than 10 years of age. Hence, the aim of the present study was to evaluate the influence of age and gender on the urinary content of total GAGs and their particular fractions in healthy children and adolescent, using a standardized method of GAG measurement.
Material and methods Subjects Studies were carried out on 95 healthy volunteers (51 males and 44 females), aged from 1 to 18 years. Subjects were selected after medical history, physical examination and laboratory analyses. The study
http://dx.doi.org/10.1016/j.clinbiochem.2014.06.012 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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Table 1 Urinary glycosaminoglycan (GAG) concentrations of particular types of GAGs in age-specific subgroups of healthy children. Age groups (years)
Total GAGs
μg/mL
mg/g Cr
μg/mL
mg/g Cr
μg/mL
mg/g Cr
ng/mL
μg/g Cr
1–3 (n = 21)
30.7 (23–37.4) 40.3 (32–50) 40.1 (31.7–63) 37.4 (21.5–44.7) 17.1 (10.9–37.8)
76.4 (56.1–96) 47.2 (34.2–69.8) 41.8 (33.4–53.2)a 23.4 (16.3–30.3)a,b 11.8 (5.1–19.1)a,b,c
22.7 (17.7–35.2) 23.4 (19.7–29.7) 24.1 (16.3–32.3) 25.6 (12.2–29.7) 12.7 (9.5–33.5)
58 (40.3–75.8) 29 (20.6–35.8)a 24.7 (18.4–31.7)a 12.7 (10.5–20.9)a 7.4 (4.3–10.8)a,b,c
6.7 (2.3–10.1) 15.6 (9.4–24.4) 14.5 (11.3–29.7) 11.9 (7.5–15.6) 4.9 (4–8.8)
11 (5.1–21) 15.7 (9.3–34.8) 14.6 (10.2–27) 7.2 (4.4–13.6) 4.1 (1.7–5.6)b,c
12.7 (10.5–13.1) 13.1 (12.2–13.6) 12.9 (12.3–13.3) 12.8 (12.4–13.4) 13.2 (13–13.6)
27.5 (17.6–42.8) 16.3 (10.4–20.8) 12.3 (9.9–17) 9 (6.2–13.4)b 7.8 (5.5–11)a,b,c
4–6 (n = 24) 7–10 (n = 20) 11–14 (n = 16) 15–18 (n = 14)
Glycosaminoglycan types Chondroitin/dermatan sulfates (CS/DS)
Heparan sulfates (HS)
Hyaluronan (HA)
Results are expressed as medians (quartile 1–quartile 3). a p b 0.05, compared to subjects aged 1–3 years. b p b 0.05, compared to subjects aged 4–6 years. c p b 0.05, compared to subjects aged 7–10 years.
protocol was approved by the Ethics Committee of the Medical University of Silesia, Poland. First-morning urine samples were divided into five groups according to the typical pediatric classification of age developmental periods [8]. Laboratory analysis Total sulfated GAGs were quantitated using the Blyscan™ Sulfated Glycosaminoglycan Assay Kit (Biocolor Ltd., United Kingdom), based on the reaction of 1,9-dimethylmethylene blue with sulfated GAGs. For the quantitative measurement of CS/DS, isolation procedure of these glycans was performed. All sulfated GAG measurements were done in a single-run. The intra-assay CVs for total sulfated GAG method was less than 6%. The results of GAGs excreted in the urine sample were expressed as μg/mL and also normalized to creatinine and expressed as uGAG to creatinine ratio (uGAG/g Cr). The urine HA was measured by TECO® Hyaluronic Acid Test Kit provided by TECOmedical AG (Sissach, Switzerland) according to the manufacturer's protocol. The appropriate low and high control samples were used for quality control procedure. The HA determination was completed within one day. The intra-assay variations of this assay was b5.6%. Urinary HA level was normalized to creatinine and expressed in mg/g Cr. Statistical methods Statistical analysis was performed using data analysis software system: StatSoft, Inc. Statistica 10.0. Statistical differences between the five age subgroups were verified by the nonparametric Kruskal–Wallis test. A Mann–Whitney U-test for independent variables was used to compare the differences between male and female subjects. Spearman's rank correlation analysis was used to determine the correlation between variables. p values of less than 0.05 were considered to indicate statistical significance. Results Age-related differences in uGAG excretion in healthy children were found in our study only when the obtained results were expressed as a GAG/creatinine ratio (Table 1). The total amount of uGAG/Cr decreased gradually with advancing age (r = − 0.686; p b 0.001), both in males (r = − 0.746; p b 0.05) and in females (r = − 0.56; p b 0.05). The highest values of total uGAG excretion were found in the youngest group of children. Regression analysis revealed that uGAG/Cr was inversely related to body height (r = − 0.658;
p b 0.001), demonstrating a stronger character in males (r = −0.774; p b 0.001) than in females (r = −0.485; p b 0.05) (Table 2). Qualitative analysis of uGAGs allowed identifying the following GAG fractions: CS/DS, HS and HA. The first one was always the predominant GAG type in all age groups, representing from 55% to 76% of the total GAGs. Strong age-related decline of CS/DS to creatinine ratio was shown in the whole group of participants (r = − 0.757; p b 0.001). There were no significant differences among urinary CS/DS results for males versus females. Moreover, decreasing with age urinary CS/DS excretion has been closely related with body height (r = − 0.723; p b 0.001), both in males (r = − 0.798; p b 0.001) and in females (r = − 0.618; p b 0.05). Further analyzed fraction of sulfated GAGs, HS when normalized to creatinine excretion, represents from 14% of the total GAGs in the youngest study participants to above 30% in older subgroups. Urinary HS changes were manifested as a gradual decrease with age (r = −0.401; p b 0.05) in the whole group of healthy children. When the results were examined by gender, the correlation between age and HS was evident in males only (r = − 0.501; p b 0.05). In males, decreasing with age urinary HS excretion was additionally correlated with body height (r = −0.421; p b 0.05), which was not observed in females. The urinary HA excretion normalized to creatinine showed a continued decrease with age (r = −0.638; p b 0.001). Gender factor had no direct impact on age-related urinary HA excretion. A negative correlation existed between urinary HA levels and body height (r = −0.653; p b 0.001). Additionally, changes in the distribution pattern of particular GAG types presented as a percentage of total GAGs were observed. Children aged 1–3 excreted considerable less heparin sulfates and considerable more CS/DS than did children from the other age subgroups. Proportional amount of urinary HA in teenagers aged 15–18 increased two times as compared to the infancy group.
Discussion Decreasing with age uGAG excretion in healthy children and adolescent volunteers has been demonstrated in our study. uGAG/Cr was a few fold elevated in the youngest group of children, as compared with the older participants. The obtained results consisted of some earlier investigations, which demonstrated decreasing with age uGAG excretion in healthy subjects [3,5,7]. However, these reports did not provide details in the qualitative evaluation of uGAG subclasses. In our study, a distinct age-related decrease in urine CS/DS, HS and HA fractions was found. CS/DS was the prevailing urinary GAG. Changes in the proportional amount of particular GAG types were also presented in our
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Table 2 Effect of age on urinary excretion of total sulfated glycosaminoglycans (sGAG) and particular GAG fractions i.e. chondroitin/dermatan sulfates (CS/DSs), heparan sulfates (HSs) and hyaluronan (HA) in healthy children. Age
sGAG/creatinine ratio CS–DS/creatinine ratio HS/creatinine ratio HA/creatinine ratio
Body length
Whole population
Male gender
Female gender
Whole population
Male gender
Female gender
−0.686⁎⁎ −0.757⁎⁎ −0.401⁎ −0.638⁎⁎
−0.746⁎ −0.776⁎ −0.501⁎ −0.566⁎⁎
−0.56⁎ −0.704⁎ NS −0.703
−0.658⁎⁎ −0.723⁎⁎
−0.774⁎⁎ −0.798⁎⁎ −0.421⁎ −0.667⁎⁎
−0.485⁎ −0.618⁎ NS −0.651⁎⁎
NS −0.653⁎⁎
Note: Spearman's rank correlation coefficients. ⁎ p b 0.05. ⁎⁎ p b 0.001.
study. Children up to 3 years excreted more GAG than older subjects and with a higher proportion of CS/DS and less content of HS. The observed differences can be explained in part by the fact that during infancy and toddler stage the most marked growth in humans occurs compared to any other stage of child development [2]. Then, rapid metabolic turnover of connective tissue during postnatal development may be the main source of increased urinary CS/DS excretion, especially that CS/DS PGs are the most abundant PGs in the ECM [9]. The close relationship between urinary CS/DS excretion and body height found in our study additionally supports that a great part of this GAG fraction in the urine seems to be of systemic origin. In this study we did not observe significant variation in the relative concentration of urinary CS/DS and HA between males and females. However, sex was an important variable influencing urinary HS levels. Urinary changes of HS are significantly inversely correlated with age only in males, not in females. The cause of this difference seems to involve sex hormones. Endogenous estrogens have been shown in experimental studies to inhibit the synthesis of the ECM molecules by many mesenchymal cell types [10,11]. Such an inhibition would shift PG/ GAG turnover toward degradation, which could explain the increased amount of GAGs excreted in the urine. On the other hand, female sex steroid hormones can also modulate the activity of matrix metalloproteinases, enzymes taking part in the ECM components' degradation [12], consequently affecting the amount and composition of uGAGs excretion. In accordance with some earlier reports, we suggest that urinary HS subfraction seems to mainly result from the kidney PG/GAG metabolism, with some contribution of urinary tract cells and systemic ECM matrix turnover. A close relationship between uHS excretion and body height was found in our study only in males and not in females. Lemos et al. [10] experimental studies have shown that male sex hormones may play a role in a synthesis of renal GAG, mainly HS. As regards another component evaluated in our work, hyaluronan, we found a significantly decreasing level without gender dependent relationship. These results could be related with age developing pro-oxidant/ antioxidant imbalance [13]. Products of PG/GAG degradation influenced by reactive oxygen species consist of smaller fragments which appear in the urine after being filtrated through the glomerulus [12]. Nonsulfated GAG, hyaluronan is readily depolymerized by free radicals to smaller chain length fragments [14], which can finally increase the urine pool of these molecules, as we have shown in the oldest group of study participants. A potential limitation of our study is the small number of samples. The results of our study must be carefully assessed when comparing
them to other populations. Thus, longitudinal race-matched studies with a larger sample size are required to confirm our results. In conclusion, our data indicate that significant urinary quantitative GAG alterations and changes in the distribution pattern occur in healthy pediatric and adolescent population. Chondroitin/dermatan sulfates were predominant urinary GAG fraction and the relative contribution of hyaluronan was increased twofold in adolescents than in younger subjects. Quantitative determination of urinary GAG excretion should be adjusted to age and gender factor and only then it appears to present a valuable, non-invasive diagnostic tool in clinical practice. Conflict of interest statement No conflict of interest. References [1] Bailey AJ. Molecular mechanisms of ageing in connective tissues. Mech Ageing Dev 2001;122:735–55. [2] Sames K. The role of proteoglycans and glycosaminoglycans in aging. In: Karger S, Hamburg AG, editors. 1994. p. 103. [3] Lee E-Y, Kim S-Y, Whang S-K, Ywang K-Y, Yang J-O, Hong S-Y. Isolation, identification and quantification of urinary glycosaminoglycans. Am J Nephrol 2003;23:152–7. [4] Buzzega D, Pederzoli F, Maccari F, Aslan D, Turk M, Volpi N. Comparison of cetylpyridinium chloride and cetyltrimethylammonium bromide extractive procedures for quantification and characterization of human urinary glycosaminoglycans. Clin Chem Lab Med 2010;48:1133–9. [5] de la Cruz Amorós V, Cortés Castell E, Moya M. Urinary excretion of mucopolysaccharides in pediatric and adolescent patients. An Esp Pediatr 1999;50:361–6. [6] Harangi F, Györke Z, Melegh B. Urinary glycosaminoglycan excretion in healthy and stone-forming children. Pediatr Nephrol 1996;10:555–8. [7] Gallegos-Arreola MP, Machorro-Lazo MV, Flores-Martínez SE, Zúñiga-González GM, Figuera LE, González-Noriega A, et al. Urinary glycosaminoglycan excretion in healthy subjects and in patients with mucopolysaccharidoses. Arch Med Res 2000;31:505–10. [8] Krawczyński M. Okresy rozwoju ontogenetycznego człowieka. In: Krawczyński M, editor. Propedeutyka pediatrii. 1st ed. PZWL; 2003. p. 34–5. [9] Thelin MA, Bartolini B, Axelsson J, Gustafsson R, Tykesson E, Pera E, et al. Biological functions of iduronic acid in chondroitin/dermatan sulfate. FEBS J 2013;280:2431–46. [10] Lemos CC, Tovar AM, Guimarães MA, Bregman R. Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure. Braz J Med Biol Res 2013;46:567–73. [11] Maroclo MV, Pereira SD, Sampaio FJ, Cardoso LE. Urinary glycosaminoglycan excretion during the menstrual cycle in normal young women. J Urol 2005;173:1789–92. [12] Singh AK, Chattopadhyay R, Chakravarty B, Chaudhury K. Altered circulating levels of matrix metalloproteinases 2 and 9 and their inhibitors and effect of progesterone supplementation in women with endometriosis undergoing in vitro fertilization. Fertil Steril 2013;100:127–34. [13] Tsukahara H. Biomarkers for oxidative stress: clinical application in pediatric medicine. Curr Med Chem 2007;14:339–51. [14] Moseley R, Waddington RJ, Embery G. Degradation of glycosaminoglycans by reactive oxygen species derived from stimulated polymorphonuclear leukocytes. Biochim Biophys Acta 1997;1362:221–31.
