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Objective Pregnancy-associated plasma protein-A2 (PAPP-A2) is a metalloproteinase that cleaves IGFBP-3 and IGFBP-5. Human mutations in PAPPA2 result in short stature with a low percentage of free IGF-I. Little is known about PAPP-A2 levels and the regulation of free IGF-I throughout childhood. We examined PAPP-A2 and intact IGFBP-3 levels in childhood and explored associations between PAPP-A2, free and total IGF-I, and total and intact IGFBP-3 and their relationship to the percentage of free to total IGF-I and anthropometric factors. Design Cross-sectional study at a single center. Methods PAPP-A2, free IGF-I, and intact IGFBP-3 levels were measured in childhood (3–18 years old) and an evaluation of the relationship between these proteins and anthropometric factors. Results In 838 children, PAPP-A2 consistently decreased throughout childhood. In contrast, free IGF-I increased. A pubertal peak in free IGF-I was present in females but was less evident in males. Intact and total IGFBP-3 increased throughout childhood; however, intact IGFBP-3 had a more marked rise than total IGFBP-3. Percent free IGF-I decreased with no distinct pubertal peak. PAPP-A2 levels positively correlated with the percent free IGF-I (Male, Female; r = 0.18, 0.38; P r = −0.58, −0.65; P Conclusions This is the first study to describe serum PAPP-A2 and intact IGFBP-3 in children between 3 and 18 years of age. Our correlative findings suggest that PAPP-A2 is an important regulator of the percent free IGF-I which can be a marker of perturbations in the GH/IGF-I axis.

Intentional weight loss may reduce breast cancer risk through lowering levels of circulating free IGF-I but few studies have measured this longitudinally. We determined the effect of weight loss (=5% body weight) over 12 months, using an energy restriction and exercise programme, on an expanded panel of IGF-related peptides amongst 23 weight losing and 46 weight stable or gaining pre-menopausal women at increased risk of breast cancer, BMI (mean ± SD) 29.2 ± 6.2 kg/m2. Fasting measures of total and free IGF-I (ultra-filtration), and IGFBP-1, -2 and -3, body weight, body fat (DXA), intra-abdominal fat (MRI) were assessed at 6 and 12 months. After 12 months, women who lost ≥ 5% of body weight had a significant increase in serum total IGF-I; mean (95% CI difference) 17 (2.3 to 34.0) μg/l, P < 0.05, and IGFBP-2; mean (95% CI ratio) 1.24 (1.06 to 1.46) P < 0.001, compared to weight stable/gaining women. Serum IGFBP-1 tended to increase in weight losers compared to the weight stable/gaining women; mean (95% CI ratio) 1.19 (0.97 to 1.45) P=0.09, whereas IGFBP-3 remained unchanged; mean (95% CI ratio) 1.02 (0.94 to 1.20] P=0.99. Weight loss did not significantly alter serum levels of free IGF-I; mean difference 0.1 (-0.1 to 3.4) μg/l, P=0.21. Increased serum total IGF-I levels, and maintenance of free IGF-I despite increased concentrations of serum IGFBP-1 and -2 with weight loss, does not suggest intentional weight loss with diet and exercise mediates reduced risk through the circulating IGF-axis.

Objectives: To examine an immunoassay for measuring free IGF-I in a saliva specimen (free sIGF-I) and to study the levels in relation to the training conditions comparing young athletes and sedentary females. Design and methods: The analysis was carried out by modifying a commercial kit for plasma matrix to measure the free sIGF-I. The plasma free and total IGF-I fractions, hGH and salivary total proteins were also measured. Saliva and blood specimens were collected from 15 well-trained young female volleyball athletes and from a control group of 14 young sedentary females. Results: The calibration curve to assay free sIGF-I covered the range 0.05–5.00 μg/L. The detection limit was 0.07 μg/L. The within-run and between-run imprecision CVs were 10% and 13% respectively. The average recovery was 88%. Free sIGF-I, measured in 15 athletes and in 14 young sedentary females, was 0.10 ± 0.03 and 0.20 ± 0.05 μg/L respectively ( p Conclusions: There were decreased levels of free sIGF-I in well-trained athletes, compared with sedentary females. This decrease could be related to a greater tissue requirement by the active muscle subjected to intense exercise for several days.

For nearly 30 years, the endogenous bioactivity of insulin-like growth factor I (IGF-I) has been estimated by its circulating concentrations of immunoreactive IGF-I, obtained after either removal or inactivation of the IGF-binding proteins (IGFBPs), and today serum/plasma total IGF-I serves as a useful parameter in the diagnosis and clinical control of growth hormone (GH) disorders. Different assays for the measurement of free, unbound IGF-I were introduced more than a decade ago. Nevertheless, this measurement remains controversial, and in daily clinical practice serum total IGF-I has retained its position as the most widely used IGF-related measurement in GH disorders. This review will provide a survey of data on free versus total IGF-I, with particular reference to GH disorders. As it will be clear, there is reasonable clinical evidence to conclude that both in the diagnosis of as well as during treatment of patients with GH disorders, serum/plasma total IGF-I should remain the primary IGF-related measurement. However, in certain patients the inclusion of free IGF-I may be useful and therefore, some guidelines for the inclusion of free IGF-I measurements will be given.

An increasing number of epidemiologic studies are investigating the relationship between serum levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) and risk of cancer, cardiovascular disease, and other diseases. However, little is known regarding the effects of blood specimen processing time on measured levels of total and free IGF-I, and on IGFBP-3, the major binding protein.Two serum separation tubes were collected from each of 12 subjects. One tube was centrifuged as soon as possible following blood collection (a mean of 47 min; range=30-80 min), and serum aliquots were placed into -70 degrees C storage either shortly after centrifugation, or following 2, 4, 10, or 24 h at ambient temperature (measured from the time of blood draw). The second serum separation tube was maintained at ambient temperature for 24h before centrifugation and freezing. Total IGF-I, free IGF-I, and IGFBP-3 levels were determined using commercial enzyme linked immunosorbent assays (ELISAs) commonly employed in epidemiologic studies. The effects of time until centrifugation and freezing on seroassay results were evaluated using generalized estimating equation (GEE) linear regression models and Spearman correlation.Total IGF-I and IGFBP-3 levels did not vary significantly with the amount of time at ambient temperature following centrifugation, even up to 24 h, in blood specimens that were centrifuged soon after collection (all pchi2). However, free IGF-I levels increased significantly with increasing time intervals between centrifugation and freezing in these same specimens (ptrend0.001). Total IGF-I/IGFBP-3 molar ratio, a crude measure of free IGF-I levels, showed no clear association. In blood specimens that were not centrifuged for 24h, total IGF-I, free IGF-I, and IGFBP-3 were each significantly elevated (each pchi2) compared with results in blood specimens that were centrifuged and frozen soon after collection, whereas the total IGF-I/IGFBP-3 molar ratio was decreased pchi2. Nonetheless, all total IGF-I, free IGF-I, IGFBP-3, and total IGF-I/IGBFBP-3 molar ratio values altered by delays in processing were highly correlated with the values in specimens processed as soon as possible (all Spearman rank correlation coefficients 0.84).Total IGF-I and IGFBP-3 can be fairly stably measured in serum with commonly used commercial assays regardless of the interval between blood collection and freezing, up to at least 24 h, as long as centrifugation and serum aliquoting take place shortly after blood collection. Free IGF-I levels, however, increase steadily with the time interval until freezing, even if serum separation has been completed soon after blood collection. Because the altered serum values are highly correlated with the referent values, analysis of total IGF-I, free IGF-I, IGFBP-3, and total IGF-I/IGFBP-3 molar ratio data by quartile might help mitigate concerns regarding the effects of delays in processing time.

Objective: Different facts suggest that the insulin growth factor (IGF)/ insulin growth factor-binding protein (IGFBP) system may be regulated by factors other than growth hormone. It has been proposed that, in healthy subjects, free IGF-I plays a role in glucose metabolism. The role of free IGF-I in glucose homeostasis in insulin resistance is poorly understood. This study was undertaken to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF-I and IGFBP-1 in obese and non-obese subjects. Research Methods and Procedures: Nineteen lean and 24 obese subjects were investigated. A frequently sampled intravenous glucose tolerance test was performed. Free IGF-I and IGFBP-1 were determined at 0, 19, 22, 50, 100, and 180 minutes. Results: Basal free IGF-I levels tended to be higher and IGFBP-1 lower in obese than in lean subjects. IGFBP-1 levels inversely correlated with basal insulin concentration. To determine the effects of insulin on the availability of free IGF-I and IGFBP-1, changes in their plasma concentrations were measured during a frequently sampled intravenous glucose tolerance test. After insulin administration, a significant suppression of free IGF-I at 22% was observed in lean subjects. In contrast, plasma-free IGF-I levels remained essentially unchanged in the obese group. The differences between both groups were statistically significant at 100 minutes (p < 0.01) and 180 minutes (p < 0.05). Serum IGFBP-1 was suppressed to a similar extent in both groups. Discussion: These data suggest that the concentrations of free IGF-I and IGFBP-1 are differentially regulated by obesity. Obesity-related insulin resistance leads to unsuppressed free IGF-I levels.

The goals of this study were to determine whether the long-term training regimens experienced by competitive collegiate swimmers would result in altered levels of total and free serum insulin-like growth factor I (IGF-I) as well as IGF-binding proteins (BP) IGFBP-1 and -3. Two male (Teams 1M and 2M) and one female (Team 2F) teams were studied at the start of training, after 2 mo of training, after 4 mo (2–4 mo had the highest volume of training), after 5 mo (near the end of tapering; only for Team 1M), and several days after training was over. For Team 1M, total IGF-I concentrations were increased by 76% after 4 mo and were subsequently maintained at this level. Total IGF-I responses were more variable for Teams 2F and 2M. Free IGF-I levels were increased nearly twofold for all teams at 2 mo and were maintained or increased further with subsequent training. Only the levels of free IGF-I for Team 1M returned to pretraining values after training had ended. Training had little effect on IGFBP-1 levels. For all teams, serum IGFBP-3 was elevated by 4 mo of training (for Team 2F it was increased at 2 mo) by 30–97% and remained at these higher levels thereafter. The ratio of total IGF-I to IGFBP-3 was not increased by training in any group. These data indicate that serum levels of total and free IGF-I and total IGFBP-3 can be increased with intense training and maintained with reduced training (tapering). The findings show that changes in free IGF-I levels are not accounted for by alterations in the total IGF-I/IGFBP-3 complex or in IGFBP-3 levels and indicate that there are other important determinants of free IGF-I.

Insulin‐like growth factors (IGFs) and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. Clinical and epidemiological studies have indicated that measuring IGFs and IGFBPs in blood has potential implications in assessing growth‐related abnormalities and risks of certain types of cancer. To facilitate the application, we reported a large collection of reference ranges of IGFs and IGFBPs in normal population and evaluations of these molecules in serum and plasma as well as the impact of freeze‐thaw cycles on the measurement. IGF‐I, IGFBP‐3 and ALS showed a similar pattern of change associated with age. Levels of these molecules were low at birth and increased with age through puberty. After puberty the levels declined slowly with age. Overall, IGF‐I, IGFBP‐3 and ALS were slightly higher in females than in males. Free IGF‐I accounted for about 1% of the total IGF‐I and its variation with age was similar to total IGF‐I. IGF‐II levels were also increased with age from birth to puberty, but became stable after puberty. There was little difference in IGF‐II levels between genders. IGFBP‐2 levels declined with age from birth to puberty. Levels of IGFBP‐6 in contrast were increased with age. These IGF binding proteins were higher in males than in females. IGFs, IGFBP‐3 and ALS were 5‐10% higher in serum than in plasma. IGFBP‐2 and IGFBP‐6 differed substantially between serum and plasma. Freeze‐thaw treatment up to five cycles had little impact on plasma levels of IGFs and IGFBP‐3. Our observations suggest that levels of IGFs and their binding proteins are varied with age, gender, and types of specimen and that these variations need to be taken into consideration when IGFs and their binding proteins are utilized in clinic and research. J. Clin. Lab. Anal. 13:166–172, 1999. © 1999 Wiley‐Liss, Inc.

Preterm newborns developing retinopathy of prematurity (ROP) and bronchopulmonary dysplasia (BPD) show persistently low levels of insulin-like growth factor-I (IGF-I) in sera. They also present higher free IGF-I concentrations in epithelial lining fluids (ELFs) and lung tissues. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase that dissociates three binding proteins from the active form of IGF-I, namely free IGF-I. The present study analyzes the ELF concentrations of free IGF-I, PAPP-A, and their ratios in preterm newborns developing or not BPD, defined as O2 dependence at 36 wk postmenstrual age. Bronchoalveolar lavage fluids of 41 infants (34 without and 7 with BPD) were analyzed on the 2nd and 4th day after birth. Infants developing BPD showed increased ELF free IGF-I and decreased PAPP-A concentrations on both days 2 and 4 compared with newborns without BPD. A nonsignificant trend between these 2 days was observed for free IGF-I (increasing) and PAPP-A (decreasing). On the same days, the free IGF-I-to-PAPP-A ratio was always significantly higher in patients developing BPD. These differences were more significant than those of IGF-I or PAPP-A when individually evaluated. A multivariate analysis confirmed the significance for free IGF-I on day 4, whereas the ratio was confirmed on both days 2 and 4. The same ratio was significantly correlated with some indexes of disease severity, such as hours of oxygen administration, days of hospitalization, and ROP severity scores. Finally, the ratio between ELF free IGF-I and PAPP-A appears to be a useful marker for lung injury of premature newborns.

Background/Aims: To investigate changes in free insulin-like growth factor I (IGF-I) and IGF-binding protein 1 (IGFBP-1) complexed IGF-I during human pregnancy. Methods: Overnight fasting serum was obtained in a longitudinal design from 11 women with non-complicated pregnancy at gestation weeks 6–10, 16–20, 24–28 and 35–38 and, for comparison, 5 weeks post-partum. All samples were analyzed for total and free IGF-I and IGF-II, IGFBP-3 and IGFBP-3 proteolysis, total and non-phosphorylated (np-) IGFBP-1, and IGFBP-1 complexed IGF-I. Results: Total IGF-I was increased in late pregnancy (week 35–38) (p < 0.001), whereas free IGF-I was significantly increased by 77% already at week 6–10 (p = 0.004) and by 140% (p = 0.002) at week 34–38, when compared to post-partum levels. At weeks 16–20 and 24–28, levels of free IGF-I were not significantly different from post-partum levels. Significant IGFBP-3 proteolysis was detectable from week 6–10 and throughout pregnancy (p < 0.05). Total and np-IGFBP-1 were significantly increased from 16–20 weeks of pregnancy (both p < 0.05) and IGFBP-1 complexed IGF-I was increased 2-fold from week 16–20 and throughout pregnancy (p < 0.05). However, the saturation of IGFBP-1 remained constant at 27–29% during the study. Conclusion: We found evidence of increased free IGF-I and increased IGF-I in binary complexes during pregnancy, possibly caused by IGFBP-3 proteolysis and decreased ternary complex formation.

In simple obesity, spontaneous and stimulated growth hormone (GH) secretions are diminished. However, this diminished GH secretion does not result in decreased somatic growth in obese children. Although the increased insulin level, low insulin-like growth factor binding protein (IGFBP)-1 and the resulting increase of bioavailability of insulin-like growth factor I (IGF-I) have been suggested as being involved, the exact mechanism has not yet been established. We investigated serum IGF-I, free IGF-I, IGFBP-1, IGFBP-3 and insulin levels in 36 obese and 39 non-obese healthy children. Insulin and IGFBP-3 were significantly higher in the obese group than in the control group (p < 0.05, p = 0.001, respectively). IGF-I, free IGF-I, free IGF-I/IGF-I and IGFBP-1 levels in the obese children were not significantly different from those in the control group. A positive correlation was found between body mass index (BMI) and IGF-I in the obese children (r = 0.30, p = 0.05). IGFBP-3 levels correlated positively with IGF-I (r = 0.44, p < 0.005), and free IGF-I levels (r = 0.37, p = 0.05) in the obese children. A negative correlation was found between IGFBP-1 and insulin levels (r = -0.30, p = 0.05) in the obese children. We concluded that normal growth in obese children might be maintained through normal IGF-I and increased IGFBP-3 levels, which are stimulated by increased insulin levels or nutritional factors or by increased responsiveness to GH.

THE IGF-I/IGFBP-3 RATIO INCREASES DURING NORMAL AND PRECOCIOUS PUBERTY: AN INDEX OK FREE IGF-I?