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Serum levels of IGF-I reflect endogenous GH secretion and correlate with height velocity in healthy children ((1,2)). In contrast to GH(3), which is secreted in a pulsatile pattern, serum concentrations of total IGF-I show no diurnal fluctuations in adults(4–6). The majority of IGF-I in serum is bound to specific IGF binding proteins, and only a minute fraction of total IGF-I circulates in the unbound form. The concentration of free IGF-I has been reported to be inversely related to IGFBP-1 in fasting serum samples(7–10). Most studies, however, have used cross-sectional designs, and the diurnal pattern of free IGF-I in serum remains to be assessed.

Several methods are available for determination of free IGF-I. Direct ultrashort incubation has been exploited in the hope that specific IGF-I antibodies may primarily react with the unbound IGF-I fraction in serum; however, it has been suggested that some easily dissociable, binding protein-complexed IGF-I may be comeasured(11–13). Ultrafiltration of serum, on the other hand, allows maintenance of equilibrium(8). The aim of the present study was to assess 24-h serum free IGF-I profiles in healthy children using an ultrafiltration method for determination of free IGF-I.

METHODS

Subjects. Seven healthy children without any history of disease were studied during the month of July. Puberty ratings were performed according to the Tanner staging of puberty(14). Height, weight, and body mass SD scores were calculated(15). Demographic characteristics are given in Table 1.

Table 1 Study population characteristics
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The study was performed in accordance with the Helsinki II declaration and was approved by the local ethics committee. Informed consent was obtained from all children and their parents.

Design. An indwelling venous catheter was placed in an antecubital vein at 0730 h. A fasting blood sample was drawn at 0800h, and thereafter samples were drawn at specific times every 20 min until 0800 h the next morning.

Each subject received breakfast at 0815 h, lunch at 1300 h, an ice cream at 1630 h, and dinner at 1900 h. Sleep was permitted from 2200 to 0730 h.

Serum analyses. Blood samples were centrifuged at 4000 rpm for 10 min. The sera were stored at -80°C and batch assayed at the completion of the study. GH was analyzed in 20-min samples, free IGF-I, IGFBP-1, and insulin in 1-h samples, and total IGF-I in 2-h samples.

Total IGF-I was determined by an in-house, noncompetitive TR-IFMA after acid ethanol extraction of serum as previously described(16).

Free IGF-I was separated from bound IGF by ultrafiltration(8). Serum samples were diluted 1:11 in Krebs-Ringer bicarbonate buffer containing 5% human serum albumin (pH = 7.4); 600 µL of the dilution was applied to a YMT-30 ultrafiltration membrane mounted in an MPS-1 supporting device (both Amicon Division, W.R. Grace and Co., Beverly, MA) and centrifuged at 300 × g at 37°C in triplicate. We have previously demonstrated that dilution of serum from normal subjects and subjects with GH deficiency or acromegaly before centrifugation can be done without altering the concentration of free IGF(8). After appropriate dilution of the filtrate the concentrations of free IGF-I were measured directly in the TR-IFMA. The detection limit in serum was 27.5 ng/L. The average intra-assay and interassay CV were 14 and 17%, respectively.

IGFBP-1 was measured by an enzyme-linked immunosorbent assay (Medix Biochemica, Kainainen, Finland).

Insulin was determined by RIA using a polyclonal antibody and recombinant human insulin and 125I-labeled recombinant human insulin as calibrator and tracer (Novo Nordisk, Bagsværd, Denmark). The detection limit of the assay is 3 pmol/L (0.5 µIU/mL). The intra-assay and interassay CV were <5 and <10%, respectively.

GH was analyzed by noncompetitive human GH TR-IFMA on the basis of the dissociation-enhanced lanthanide fluorescence immunoassay principle (Wallac Oy, Turku, Finland).

Statistical analyses. Data were described as percentage of the overall day mean ± SEM in the 24-h profiles and as mean ± SEM in the correlation analyses. The AUC for free IGF-I, IGFBP-1, and GH was calculated according to the trapezoidal rule. To evaluate the 24-h profiles, one-way analysis of variance for repeated measurements was performed, and if significant, Student-Newman-Keuls method for pairwise multiple comparisons was used to identify time points. Correlations were tested by Pearson correlation analysis and expressed by r values. Determination of phase relationships between free IGF-I and IGFBP-1 patterns were performed by calculating cross-correlation matrices. The 5% level of significance was used.

All statistics were performed using the SPSS for Windows version 7.5.1 Statistical Software Package (SPSS Inc., Chicago, IL).

RESULTS

Mean fasting values ± SEM at 0800 h in the first and second morning, respectively, were 1.70 ± 0.36 and 1.78 ± 0.45 µg/L (p = 0.67; free IGF-I), 387.7 ± 77 and 406.9 ± 85.3 µg/L (p = 0.42; total IGF-I), 5.05 ± 1.09 and 4.83 ± 1.05 (p = 0.44; total IGF-I/protein), and 8.53 ± 1.30 and 6.85 ± 0.81 µg/L (p = 0.36; IGFBP-1). Figure 1 shows the 24-h profiles of free IGF-I, total IGF-I, total IGF-I adjusted for serum proteins, IGFBP-1, insulin and GH, respectively. All data are expressed as percent of overall day mean. A statistically significant diurnal variation in serum free IGF-I was seen (p < 0.001), with peak values between 0900 and 1200 h in the morning and a nocturnal decrease with a nadir at 0700 h (p < 0.05), followed by a statistically significant increase at 0800 h (p < 0.05). Concomitantly with the decrease in free IGF-I, an increase in IGFBP-1 was observed between 0200 and 0700 h (p < 0.001). Total IGF-I concentrations varied significantly (p = 0.05) throughout the 24-h study period; however, when the levels of total IGF-I were adjusted for serum proteins, no statistically significant diurnal variations (p = 0.49) were detected.

Figure 1
figure 1

Diurnal concentrations of free IGF-I, total IGF-I, insulin, IGFBP-1, and GHAUC (AUC over 1 h), and total IGF-I adjusted for serum proteins, in 7 healthy children expressed as percentage of overall day mean ± SEM.

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Inverse relationships were observed between 24-hAUC free IGF-I and 24-hAUC IGFBP-1 (r = -0.94, p = 0.002) and between fasting free IGF-I and fasting IGFBP-1 (r = -0.86, p = 0.01). Twenty-four-hourAUC GH correlated with fasting free IGF-I (r = 0.78, p = 0.04), 24-hAUC free IGF-I (r = 0.80, p = 0.03), fasting total IGF-I (r = 0.77, p = 0.04), and 24-hAUC total IGF-I (r = 0.77, p = 0.04). Finally, fasting free IGF-I correlated with 24-hAUC free IGF-I (r = 0.94, p = 0.002).

To determine phase relationship between free IGF-I and IGFBP-1, and free IGF-I and insulin, cross-correlation analyses were performed. These analyses, however, did not show any consistent patterns.

DISCUSSION

In our method for determination of free IGF-I, serum is diluted before ultrafiltration is performed. Dilution will obviously lead to a relative increase in free IGF-I owing to dissociation of bound IGF-I. However, for dilution up to 20 times there is no increase in the absolute concentration of free IGF-I, i.e. the increase in absolute amounts of free IGF-I is counter-balanced by dilution. This is paralleled by experiences from measuring free thyroid hormones. Serum can be diluted 200 times without changing the absolute concentration of free thyroid hormones(17). We have previously demonstrated by immunoanalysis and by Western ligand blotting that the ultra-filtrate contains no trace of IGFBP and that the recovery of free IGF-I approximates 100%(8). The ultrafiltrate obtained from 0 to 40 min has a very low concentration of IGF-I, probably because of absorption to the ultrafiltration membrane, whereas there is no change in the concentration of ultrafiltered IGF-I from 40 to 130 min of ultrafiltration, i.e. the equilibrium between free and bound IGF-I is undisturbed throughout ultrafiltration. We routinely use the ultrafiltrate obtained from 40 to 100 min of ultrafiltration for measurement of free IGF-I(8). Although no statistical variation in fasting free IGF-I could be detected in the present study, preliminary data in adults have revealed an intra-individual variation of 15-20% in fasting free IGF-I, which is within the variation of the measurement error of the assay (unpublished data).

More than 99% of IGF-I in serum is bound to specific binding proteins. Approximately 90% is bound to IGFBP-3 and the acid-labile subunit in the ternary complex(8,18). It has been suggested that free IGF-I may be the biologically active part of circulating IGF-I(8). The ternary complex ensures that a constant pool of IGF-I is available to the tissues, and IGFBP-3 seems to play a role in regulating serum concentrations of total IGF-I. Furthermore, the half-life of circulating free IGF-I is approximately 10 min, whereas the half-life of the bound form is approximately 20 h. In the present study we adjusted for the concentrations of proteins to eliminate any possible influence on total IGF-I from increased plasma volume during the night leading to dilution and thereby a decrease in total IGF-I(19). The changes in plasma volume during the 24-h study period, however, were found not to affect total IGF-I concentrations, and our observations of no diurnal variations in total IGF-I or in IGFBP-3 are in line with studies in adults(4–6).

So far, IGFBP-1 seems to be the only IGF binding protein showing a circadian variation(20,21). IGFBP-1 is regulated primarily by insulin-mediated suppression of hepatic IGFBP-1 synthesis and increased clearance(22,23). The suppression of IGFBP-1 may be maximal when serum insulin exceeds 70-90 pmol/L(24). That is in agreement with the observed inverse relationship between IGFBP-1 and insulin in the present study.

It has been suggested that IGFBP-1 may be an important inhibitor of IGF-I bioactivity in vitro(25) and in vivo(26–28). In agreement with the present findings, a nocturnal decrease in IGF bioactivity occurring concomitantly with an increase in IGFBP-1 has been observed(25). Furthermore, an increase in IGFBP-1 binding activity closely paralleling a nocturnal increase in IGFBP-1 has been found, and it has been suggested that the decrease in free IGF-I may be caused by binding of free IGF-I to IGFBP-1(18,25). Confirming findings of an inverse relationship between fasting IGFBP-1 and fasting free IGF-I in studies in adults(8,9) and in children(29,30), fasting and 24-hAUC free IGF-I levels in the present study showed a statistically significant inverse correlation with fasting and 24-hAUC IGFBP-1 levels, suggesting a relation to overnight fasting. However, free IGF-I levels may not only be influenced by IGFBP-1. Surprisingly, the increase in free IGF-I between 0700 and 0800 h occurred before the decrease in IGFBP-1. This is in accord with indications that the response in total IGF-I serum levels to GH stimulation seems to be relatively slow. Increases in total serum IGF-I have been observed 6-8 h after the administration of GH in GH-deficient adults, reaching peak values 5-7 h later(31,32). Administration of GH appears not to be associated with acute responses in free IGF-I levels in adults with GH deficiency(33). Thus, the possibility remains that the observed increase in free IGF-I in the morning in normal children may be related to the nocturnal GH surge. However, to further elucidate the mechanisms regulating free IGF-I concentrations, studies applying sleep deprivation or continuous glucose infusion may be needed.

In conclusion, circulating free IGF-I exhibits a nocturnal decrease and an increase in the morning in healthy children. The diurnal secretion of free IGF-I correlates with GH and is inversely related to IGFBP-1. The metabolic significance of these findings needs further study.