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School of Dietetics and Human Nutrition, McGill University, Montreal, Canada and * Department of Chemistry and Biochemistry, Concordia University, Montreal, Canada
3To whom correspondence should be addressed. E-mail: kris.koski{at}mcgill.ca.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • birth weight • insulin-like growth factor • fetal growth • somatomedins • amniotic fluid
Amniotic fluid (AF)4 is crucial to fetal health. Aside from protecting the fetus from mechanical and thermal shock, possessing antimicrobial action, and assisting in acid-base balance, AF contains nutritional factors including proteins (1,2). These proteins enter the AF from both maternal and fetal sources (3,4). Fetal swallowing accounts for 80% of AF protein clearance in late gestation (5). Previous studies identified several proteins in human AF (3,6), including a range of growth factors such as insulin-like growth factor (IGF) II and 2 of its binding proteins, IGF BP1 and BP 3 (7,8).
IGF II is considered to be a major modulator of early embryonic and fetal growth (9–11). IGF II promotes differentiation of cells by endocrine (11,12) and autocrine (13) means. IGF II is synthesized by the developing fetal lung in the first trimester (9,14). IGF II has a 3.2-fold greater concentration in early 2nd trimester AF than IGF I (15), which supports the general belief that IGF II predominates over IGF I in human AF from 9 to 12 wk (16); thereafter, it increases rapidly and peaks at 19 wk (17) of gestation. Although less potent than IGF I, IGF II with its later emerging endocrine function can increase glucose uptake (11). The role of IGF II as a key fetal growth factor is further supported by the creation of a growth-deficiency phenotype produced after disruption of IGF II gene expression in mice (18). Human AF IGF II has been less extensively examined, but one study showed that IGF II was not correlated with infant birth weight if gestational age was corrected for, but was correlated with placental weight and the birth weight:placental weight ratio (19).
Evidence also exists demonstrating important regulatory roles for IGF BPs during fetal growth and development. IGF BPs are established regulators of the action of IGFs and can both augment (20) and inhibit (21) IGF effects. IGF BP1, produced primarily by maternal deciduas (15) as well as fetal liver and kidney (21), is considered the most abundant IGF BP in human AF (21); it increases 20-fold from 9 to 12 wk (16). IGF BP1 binds to and modulates the activity of both IGF I and IGF II (13). Interestingly, a relation with birth weight was established for AF IGF BP1 in which high 2nd trimester AF IGF BP1 concentrations were associated with lower infant birth weight (21,22). However, the existence of this relation was disputed in another human study that showed that once gestational age was corrected for, AF IGF BP1 remained strongly correlated with amniotic fluid IGF I and IGF II levels, but not with infant birth weight (19). These authors also concluded that high IGF BP1 did not predict small-for-gestational-age (SGA), but was associated with lower placental weights (19).
In sharp contrast to the possible negative relation of IGF BP1 with fetal growth, there is the suggestion in the literature that IGF BP3, which increases 20-fold from 9 to 12 wk in human amniotic fluid (16), may promote IGF 1 action and be positively associated with fetal growth. Through in utero catheterization and infusion, AF IGF BP3 was also correlated in vivo with late gestational IGF I binding in fetal sheep and the regulated delivery of IGF 1 to the developing fetus late in gestation (23). Recently, lower AF IGF BP3 was associated with intrauterine growth retardation in humans (24).
The literature suggests that these 2 AF IGF BPs, present in high concentrations in 2nd trimester AF (17,19), could have opposite effects on fetal growth and development in humans. In a recent publication (25) we demonstrated that AF total protein was inversely associated with infant birth weight. In the current study, we hypothesize that AF IGF BP1, the binding protein found in greatest abundance in amniotic fluid (7,19), would demonstrate this same inverse relation. We also investigated whether IGF BP3 or IGF II was associated with infant birth weight. We studied a large population of pregnant women (n = 543 mother-infant pairs) undergoing age-related amniocentesis (12–20 wk) for genetic testing to establish whether these AF growth factors were associated with infant birth weight. With our large cohort, we provided a more comprehensive multivariate regression analysis model than previously published studies.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Biochemical analysis. Amniotic fluid samples, stored at –80°C, were analyzed for IGF II (n = 388), IGF BP3 (n = 401), and IGF BP1 (n = 543) by ELISA using Diagnostics Systems Laboratories (DSL) kits 10–9100, 10–6600, and 10–7800, respectively. These kits were chosen for the following reasons: the IGF BP1 kit is unaffected by phosphorylation state of IGF BP1, which can result in underestimation of total IGF BP1 (27,28). The IGF II methodology, which uses a modified acidification followed by ethanol precipitation to extract IGF II from its binding proteins where <5% is free, extracts as effectively as size-exclusion gel chromatography in acid (29). Extraction of IGF II ensures that the binding proteins are not permitted to sequester IGF II in the reaction mixture (30,31), thus allowing for more accurate quantification. The IGF BP3 kit is able to detect IGF BP3 despite glycosylation state and whether it has been proteolyzed (32); both of these can complicate detection and quantification (33–36).
Statistical analysis. All analyses were done using SAS Version 8.02. ANOVA and analysis of covariance (ANCOVA) were performed by dividing the study population into subgroups according to clinical classifications of infant gender-corrected birth-weight-for-gestational-age (26) including SGA (<10%), appropriate-for gestational age (AGA; 10–90%), and large-for-gestational-age (LGA; >90%) followed by post-hoc testing using Scheffé’s test (P < 0.05). Data were reported as means ± SEM unless otherwise indicated. Nonlinear data (prepregnancy week, BMI, amniocentesis week, IGF II, and IGF BP3) were square-root transformed. Covariates for analysis included maternal height, prepregnancy weight, ethnicity, parity, and infant gender, due to previously established associations with birth weight (26,37) and amniocentesis week due to its effect on 2nd trimester AF IGF concentrations (16,38,39). Multivariate regression analysis for birth weight was performed on the whole population and within the clinical classifications of fetal growth including those categories based on infant birth weight in grams: low birth weight (LBW; <2500 g), normal birth weight (2500–4000 g) and macrosomia (>4000 g)) and newer percentile categories that correct for infant gender and gestational age (SGA, AGA, and LGA). Significance was established at P < 0.05.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our study population consisted of mothers undergoing age-related amniocentesis (age = 37.8 ± 0.1 y), and it was interesting to note that our older maternal population differed only slightly from the Canadian population at large. Canadian statistics show that birth weight is 3420 ± 466, with 9.7% of the population SGA and 10.0% LGA (40). We had a shift to fewer SGA (5%) because we eliminated prematurity but reported a comparable prevalence of LGA (11%). Maternal smoking behavior showed the correct directional effect (41) but did not enter significantly as a predictor of birth weight due to the low incidence of smoking in our slightly older women (42). Birth weight was only slightly higher than the Canadian norm. We also observed that previously established (37) maternal and fetal predictors of birth weight including maternal height, prepregnancy weight, fetal gestational age, and gender were similar positive predictors in our older population (43).
Second trimester AF IGF BP1 was reported previously to be negatively correlated with birth weight (19,21,22). Our findings strengthen this observation because we controlled for established predictors of birth weight (37). Given that IGF BP1 is one of the most abundant binding proteins in AF (21), it is likely that IGF BP1 contributes to the negative association reported previously for AF total protein and birth weight (25). However, the failure of our findings to replicate the previously reported relation between high IGF BP1 and fetal growth retardation (22) may be explained by the absence of prematurity and the low incidence of LBW in our study population. A recent study (44) indicated that prematurity rather than intrauterine growth retardation drives the inverse relation between IGF BP1 and birth weight. Usually prematurity dominates in SGA infant populations; our study population included no premature infants, supporting the absence of this relation. Moreover, a previous study using 209 mother-infant pairs found that once gestational age was corrected for, AF IGF BP1 remained strongly correlated with AF IGF I and IGF II concentrations, but not with birth weight (19). Like us, they concluded that high AF IGF BP1 did not predict SGA; they associated higher concentrations with lower placental weights (19).
AF IGF BP1 was also not associated with fetal birth weight in our LGA or macrosomic subpopulations. Previous findings showed elevations in plasma IGF BP1 in food-deprived or malnourished rats (45). Our mothers had prepregnancy BMIs > 20 (82%); 28% were overweight/obese and likely were not fasting or malnourished. Previous studies showed that elevated plasma insulin inhibits hepatic IGF BP1 production early in gestation (46), and that lowered growth hormone results in overproduction of IGF BP1 in humans (47). Although growth hormone was not measured in our study, we did observe that AF insulin concentrations were greater in LGA compared with AGA infants (data not shown). Moreover, considering the lower concentrations of AF IGF BP1 in LGA compared with AGA, we suggest that, as with plasma, a similar inverse relation between higher insulin and lower IGF BP1 exists in AF.
In our study, we demonstrated a positive predictive relation in vivo between AF IGF BP3 and fetal growth in LGA and macrosomic infants. Previous in vitro findings using animal and human cells hinted that IGF BP3 may potentiate IGF action (20,48). Our finding represents the first in vivo report of an association of AF IGF BP3 (12–20 wk) with birth weight. Previous studies in plasma established that growth hormone levels are inversely proportional to IGF BP1 (47) and directly proportional to IGF BP3 (49,50). It was shown that IGF BP3 synthesis is growth hormone dependent (51). Although we did not observe higher AF IGF BP3 across our 3 birth weight categories, literature findings suggest that our positive association between AF IGF BP3 and birth weight in our stepwise regression might be linked with elevated AF concentrations of growth hormone (49). Thus, only larger fetuses would be exposed to both decreased AF IGF BP1 and increased AF IGF BP3. This relation requires further investigation, and measurement of AF growth hormone is proposed.
Last, we showed that AF IGF II was negatively associated with infant birth weight in LGA and macrosomic infants. The previous literature showed that IGF II is the primary growth factor during early fetal development (10,11,24); plasma levels are normally positively associated with fetal growth (10). It is known that IGF II binds to both IGF BP1 and IGF BP3 (52); its greatest association is for IGF BP3 and its lowest dissociation is for IGF BP1 (52). Because total AF IGF II concentrations were essentially the same across the birth weight and gender-corrected birth-weight-for-gestational-age categories, it would seem that IGF BP1 in these groups might play an intervening role in the observed inverse relation between AF IGF II and infant birth weight in macrosomic and LGA infants. Specifically, decreased IGF BP1 occurs with elevated AF insulin (46), which was observed in AF from the macrosomic and LGA infants. The increased levels of insulin in AF at this stage in development may lead to increased competitive binding with IGF II receptors, thus leading to generalized tissue downregulation of receptors. This would subsequently lead to increased levels of IGF II remaining in AF. Until results of an assay that can distinguish between IGF II bound to one protein or another are reported, no conclusive mechanism can be described. However, the underlying mechanism for the negative association with early concentrations of AF IGF II and birth weight for infants classified as LGA and macrosomic at term does warrant further investigation.
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FOOTNOTES |
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2 Funded in part by Natural Sciences and Engineering Research Council of Canada.
4 Abbreviations used: AF, amniotic fluid; AGA, appropriate-for-gestational-age; ANCOVA, analysis of covariance; IGF insulin-like growth factor; IGF BP, IGF binding protein; LBW, low birth weight; LGA, large-for-gestational-age; SGA, small-for-gestational-age.
Manuscript received 7 January 2005. Initial review completed 1 March 2005. Revision accepted 16 April 2005.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Pitkin R. M., Reynolds W. A. Fetal ingestion and metabolism of amniotic fluid protein. Am. J. Obstet. Gynecol. 1975;123:356-363.[Medline]
2. Lev R., Orlic D. Protein absorption by the intestine of the fetal rat in utero. Science (Washington, DC). 1972;177:522-524.
3. Queenen J. Amniotic fluid proteins, amniotic fluid amino acids and their clinical significance. Eskes T. eds. Amniotic Fluid: Research and Clinical Application. 2nd ed. 1978:187-208 Elsevier/North-Holland Biochemical Press New York, NY. .
4. Jauniaux E., Gulbis B., Hyett J., Nicolaides K. H. Biochemical analyses of mesenchymal fluid in early pregnancy. Am. J. Obstet. Gynecol. 1998;178:765-769.[Medline]
5. Gitlin D., Kumate J., Morales C., Noriega L., Arevalo N. The turnover of amniotic fluid protein in the human conceptus. Am. J. Obstet. Gynecol. 1972;113:632-645.[Medline]
6. Drohse H., Christensen H., Myrhoj V., Sorensen S. Characterisation of non-maternal serum proteins in amniotic fluid at weeks 16 to 18 of gestation. Clin. Chim. Acta. 1998;276:109-120.[Medline]
7. Chard T. Insulin-like growth factors and their binding proteins in normal and abnormal human fetal growth. Growth Regul. 1994;4:91-100.[Medline]
8. Merimee T. J., Grant M., Tyson J. E. Insulin-like growth factors in amniotic fluid. J. Clin. Endocrinol. Metab. 1984;59:752-755.[Abstract]
9. Stiles A. D., D’Ercole A. J. The insulin-like growth factors and the lung. Am. J. Respir. Cell Mol. Biol. 1990;3:93-100.
10. Constancia M., Hemberger M., Hughes J., Dean W., Ferguson-Smith A., Fundele R., Stewart F., Kelsey G., Fowden A., Sibley C., Reik W. Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature (Lond.). 2002;417:945-948.[Medline]
11. Sara V. R., Hall K. Insulin-like growth factors and their binding proteins. Physiol. Rev. 1990;70:591-614.
12. Pahlman S., Meyerson G., Lindgren E., Schalling M., Johansson I. Insulin-like growth factor I shifts from promoting cell division to potentiating maturation during neuronal differentiation. Proc. Natl. Acad. Sci. U.S.A. 1991;88:9994-9998.
13. Jones J. I., Clemmons D. R. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev. 1995;16:3-34.[Medline]
14. Brice A. L., Cheetham J. E., Bolton V. N., Hill N. C., Schofield P. N. Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus. Development. 1989;106:543-554.[Abstract]
15. Kubota T., Kamada S., Taguchi M., Aso T. Determination of insulin-like growth factor-2 in feto-maternal circulation during human pregnancy. Acta Endocrinol. 1992;127:359-365.
16. Nonoshita L. D., Wathen N. C., Dsupin B. A., Chard T., Giudice L. C. Insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and proteolyzed IGFBP-3 in embryonic cavities in early human pregnancy: their potential relevance to maternal-embryonic and fetal interactions. J. Clin. Endocrinol. Metab. 1994;79:1249-1255.[Abstract]
17. Chard T., Blum W. F., Brunjes J., Campbell D. J., Wathen N. C. Levels of insulin-like growth factor-binding protein-2 and insulin-like growth factor-II in maternal serum, amniotic fluid and extraembryonic coelomic fluid at 9–20 weeks of pregnancy. J. Endocrinol. 1994;142:379-383.[Abstract]
18. Stewart C. E., Rotwein P. Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol. Rev. 1996;76:1005-1026.
19. Verhaeghe J., Coopmans W., van Herck E., van Schoubroeck D., Deprest J. A., Witters I. IGF-I, IGF-II, IGF binding protein 1, and C-peptide in second trimester amniotic fluid are dependent on gestational age but do not predict weight at birth. Pediatr. Res. 1999;46:101-108.[Medline]
20. Conover C. A., Clarkson J. T., Bale L. K. Factors regulating insulin-like growth factor-binding protein-3 binding, processing, and potentiation of insulin-like growth factor action. Endocrinology. 1996;137:2286-2292.[Abstract]
21. Chevallier B., Lagarde A., Degrelle H., Belaisch-Allart J., Giraudet P., Gallet J. P. Insulin-like growth factor binding protein 1 level in amniotic fluid: correlation with birth weight. Biol. Neonate. 1998;73:404-406.[Medline]
22. Hakala-Ala-Pietila T. H., Koistinen R. A., Salonen R. K., Seppala M. T. Elevated second-trimester amniotic fluid concentration of insulin-like growth factor binding protein-1 in fetal growth retardation. Am. J. Obstet. Gynecol. 1993;169:35-39.[Medline]
23. Bloomfield F. H., Breier B. H., Harding J. E. Fate of (125)I-IGF-I administered into the amniotic fluid of late-gestation fetal sheep. Pediatr. Res. 2002;51:361-369.[Medline]
24. Oliver M. H., Bloomfield F. H., Harding J. E., Breier B. H., Bassett N. S., Gluckman P. D. The maternal, fetal and postnatal somatotrophic axes in intrauterine growth retardation. Biochem. Soc. Trans. 1999;27:69-73.[Medline]
25. Tisi D. K., Emard J. J., Koski K. G. Total protein concentration in human amniotic fluid is negatively associated with infant birth weight. J. Nutr. 2004;134:1754-1758.
26. Kramer M. S., Platt R. W., Wen S. W., Joseph K. S., Allen A., Abrahamowicz M., Blondel B., Breart G. A new and improved population-based Canadian reference for birth weight for gestational age. Pediatrics. 2001;108:E35.
27. Westwood M., Gibson J. M., Davies A. J., Young R. J., White A. The phosphorylation pattern of insulin-like growth factor-binding protein-1 in normal plasma is different from that in amniotic fluid and changes during pregnancy. J. Clin. Endocrinol. Metab. 1994;79:1735-1741.[Abstract]
28. Khosravi M. J., Diamandi A., Mistry J. Immunoassay of insulin-like growth factor binding protein-1. Clin. Chem. 1997;43:523-532.
29. Powell D. R., Rosenfeld R. G., Baker B. K., Liu F., Hintz R. L. Serum somatomedin levels in adults with chronic renal failure: the importance of measuring insulin-like growth factor I (IGF-I) and IGF-II in acid-chromatographed uremic serum. J. Clin. Endocrinol. Metab. 1986;63:1186-1192.[Abstract]
30. Lee P. D., Rosenfeld R. G. Clinical utility of insulin-like growth factor assays. Pediatrician. 1987;14:154-161.[Medline]
31. Hintz R. L. The somatomedins. Adv. Pediatr. 1981;28:293-317.[Medline]
32. Diamandi A., Mistry J., Krishna R. G., Khosravi J. Immunoassay of insulin-like growth factor-binding protein-3 (IGFBP-3): new means to quantifying IGFBP-3 proteolysis. J. Clin. Endocrinol. Metab. 2000;85:2327-2333.
33. Giudice L. C., Farrell E. M., Pham H., Lamson G., Rosenfeld R. G. Insulin-like growth factor binding proteins in maternal serum throughout gestation and in the puerperium: effects of a pregnancy-associated serum protease activity. J. Clin. Endocrinol. Metab. 1990;71:806-816.[Abstract]
34. Hossenlopp P., Segovia B., Lassarre C., Roghani M., Bredon M., Binoux M. Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy. J. Clin. Endocrinol. Metab. 1990;71:797-805.[Abstract]
35. Lamson G., Giudice L. C., Rosenfeld R. G. A simple assay for proteolysis of IGFBP-3. J. Clin. Endocrinol. Metab. 1991;72:1391-1393.[Abstract]
36. Gargosky S. E., Pham H. M., Wilson K. F., Liu F., Giudice L. C., Rosenfeld R. G. Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepancies between radioimmunoassay and ligand blotting. Endocrinology. 1992;131:3051-3060.[Abstract]
37. Kramer M. S. Determinants of low birth weight: methodological assessment and meta-analysis. Bull. WHO. 1987;65:663-737.[Medline]
38. Wathen N. C., Wang H. S., Cass P. L., Campbell D. J., Chard T. Insulin-like growth factor-1 and insulin-like growth factor binding protein-1 in early human pregnancy. Early Hum. Dev. 1992;28:105-110.[Medline]
39. Lewitt M. S., Scott F. P., Clarke N. M., Baxter R. C. Developmental regulation of circulating insulin-like growth factor-binding proteins in normal pregnancies and in pre-eclampsia. Prog. Growth Factor Res. 1995;6:475-480.[Medline]
40. Wen S. W., Kramer M. S., Platt R., Demissie K., Joseph K. S., Liu S., Sauve R. Secular trends of fetal growth in Canada, 1981 to 1997. Paediatr. Perinat. Epidemiol. 2003;17:347-354.[Medline]
41. Muscati S. K., Koski K. G., Gray-Donald K. Increased energy intake in pregnant smokers does not prevent human fetal growth retardation. J. Nutr. 1996;126:2984-2989.
42. Kramer M. S., Morin I., Yang H., Platt R. W., Usher R., McNamara H., Joseph K. S., Wen S. W. Why are babies getting bigger? Temporal trends in fetal growth and its determinants. J. Pediatr. 2002;141:538-542.[Medline]
43. Kramer M. S. Intrauterine growth and gestational duration determinants. Pediatrics. 1987;80:502-511.
44. Cutfield W. S., Regan F. A., Jackson W. E., Jefferies C. A., Robinson E. M., Harris M., Hofman P. L. The endocrine consequences for very low birth weight premature infants. Growth Horm. IGF Res. 2004;14(suppl. A):S130-S135.
45. Matsukawa T., Inoue Y., Oishi Y., Kato H., Noguchi T. Up-regulation of upstream stimulatory factors by protein malnutrition and its possible role in regulation of the IGF-binding protein-1 gene. Endocrinology. 2001;142:4643-4651.
46. Lee P. D., Jensen M. D., Divertie G. D., Heiling V. J., Katz H. H., Conover C. A. Insulin-like growth factor-binding protein-1 response to insulin during suppression of endogenous insulin secretion. Metabolism. 1993;42:409-414.[Medline]
47. Norrelund H., Frystyk J., Jorgensen J. O., Moller N., Christiansen J. S., Orskov H., Flyvbjerg A. The effect of growth hormone on the insulin-like growth factor system during fasting. J. Clin. Endocrinol. Metab. 2003;88:3292-3298.
48. Conover C. A. Regulation and physiological role of insulin-like growth factor binding proteins. Endocr. J. 1996;43(suppl.):S43-S48.
49. Bowers C. Y., Granda R., Mohan S., Kuipers J., Baylink D., Veldhuis J. D. Sustained elevation of pulsatile growth hormone (GH) secretion and insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), and IGFBP-5 concentrations during 30-day continuous subcutaneous infusion of GH-releasing peptide-2 in older men and women. J. Clin. Endocrinol. Metab. 2004;89:2290-2300.
50. Lange M., Qvortrup K., Lander Svendsen O., Flyvbjerg A., Nowak J., Petersen M. M., Olgaard K., Feldt-Rasmussen U. Abnormal bone collagen morphology and decreased bone strength in growth hormone-deficient rats. Bone. 2004;35:178-185.[Medline]
51. Bowsher R. R., Lee W. H., Apathy J. M., O’Brien P. J., Ferguson A. L., Henry D. P. Measurement of insulin-like growth factor-II in physiological fluids and tissues. I. An improved extraction procedure and radioimmunoassay for human and rat fluids. Endocrinology. 1991;128:805-814.[Abstract]
52. Wong M. S., Fong C. C., Yang M. Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins. Biochim. Biophys. Acta. 1999;1432:293-301.[Medline]
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