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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 5 2171-2179
Copyright © 2002 by The Endocrine Society

Other Original Articles

IGF-I, IGF Binding Protein (IGFBP)-3, Phosphoisoforms of IGFBP-1, and Postnatal Growth in Very Low Birth Weight Infants

Eero Kajantie, Leo Dunkel, Eeva-Marja Rutanen, Markku Seppälä, Riitta Koistinen, Annikki Sarnesto and Sture Andersson

Hospital for Children and Adolescents (E.K., L.D., A.S., S.A.), Helsinki University Central Hospital, 00029 HUS, Helsinki, Finland; and Department of Obstetrics and Gynecology (E.-M.R., M.S., R.K., S.A.), Helsinki University Central Hospital, 00029 HUS, Helsinki, Finland

Address all correspondence and requests for reprints to: Eero Kajantie, M.D., Hospital for Children and Adolescents, Helsinki University Central Hospital, PL 280, 00029 HUS, Finland. E-mail: . eero.kajantie{at}hus.fi

Abstract

Impaired postnatal growth in very low birth weight (VLBW, <1500 g) infants is per se a major clinical challenge and may also serve as a model in studying the mechanisms of growth retardation in general. This study was undertaken to characterize the role of IGFs and their binding proteins (IGFBPs), key regulators of fetal and infant growth, during the postnatal period in VLBW infants.

Forty-eight VLBW infants (gestational age 27.6 ± 2.2 wk, birth weight 923 ± 257 g) were studied. Blood samples were drawn at 1, 2, 4, and 8 wk of age for measurements of IGF-I, IGFBP-1 (lesser phosphorylated, lpIGFBP-1, and highly phosphorylated, hpIGFBP-1), IGFBP-3, and insulin, simultaneous growth velocities being assessed by a rigorous protocol of repeated, frequent lower leg length and body weight measurements. All regression analyses were adjusted for postnatal age and repeated measurements.

Lower leg growth velocity showed a positive correlation with IGF-I (P = 0.01) and IGFBP-3 (P = 0.03), and weight growth velocity with IGFBP-3 (P = 0.057) and with lpIGFBP-1/hpIGFBP-1 ratio (P = 0.01). Moreover, concurrent glucocorticoid dose showed a negative correlation with both IGFBP-1 isoforms, observable, however, only in samples with high (>10 U/liter) insulin (lpIGFBP-1, P = 0.02; hpIGFBP-1, P = 0.007). In backward multiple regression analysis, the factor remaining significantly associated with lower leg growth velocity (R2 = 0.63) was IGF-I, and factors associated with weight growth velocity (R2 = 0.81) were IGFBP-3 and the lpIGFBP-1/hpIGFBP-1 ratio.

In conclusion, circulating IGF-I and IGFBP-3, and the lpIGFBP-1/hpIGFBP-1 ratio, reflect short-term growth velocity in VLBW infants. lpIGFBP-1 isoforms, abundant in the circulation of these infants, may thus also have properties that are at least less inhibitory, if not promoting, on the growth-stimulating action of IGF-I. Finally, the regulation of IGFBP-1 by glucocorticoids may be divergent in situations with a high or low insulin concentration.

GROWTH RETARDATION DURING the fetal period and infancy may result into deviant body composition, short adult stature, and predispose to various common late-onset disorders such as arteriosclerosis (1, 2). In very low birth weight infants (VLBW, defined as <1500 g), suboptimal postnatal growth constitutes a major clinical challenge. Many VLBW infants have already been growth retarded in utero, and the first postnatal months of these infants are characterized by a long and resource-intensive hospital treatment with frequent severe illness, suboptimal growth, undernutrition, and growth-inhibiting treatments such as glucocorticoids. Therefore, in minimizing the adverse consequences of early preterm birth, in-depth understanding of the mechanisms involved in the regulation of postnatal growth in VLBW infants is essential. Furthermore, VLBW infants may serve as a model useful in studying the mechanisms of different growth-inhibiting conditions not necessarily confined to the immediate postnatal period.

IGFs are major regulators of growth and organ development. They promote growth by both paracrine end endocrine pathways, their bioavailability being controlled by at least six IGF-binding proteins (IGFBPs). A wide body of evidence ranging from studies in genetically engineered mice (3, 4) and human IGF-I gene mutation (5) to clinical studies in term and preterm infants (6, 7, 8) links this hormonal system with both fetal and childhood growth and specific disorders associated with intrauterine growth restriction such as preeclampsia (6, 9). Importantly, life-long programming of the IGF system by the intrauterine hormonal and nutritional environment has also been suggested to be a key mediator of the link between small size at birth and cardiovascular disease in adulthood (1).

With regard to fetal growth, relevant circulating binding-proteins include IGFBP-3, which extends the half-life of IGF-I, and IGFBP-1 (10). In most clinical studies, circulating IGFBP-1 has been associated with growth inhibition (6, 7, 8, 11). IGFBP-1 circulates, however, in different phosphoisoforms, of which the lesser-phosphorylated have decreased affinity for IGF-I (12, 13) suggesting that lpIGFBP-1 may have a weaker growth-inhibiting or even a growth-promoting effect.

Despite the key role of the IGF system in regulating fetal and infant growth, knowledge on its actions during the postnatal period in VLBW infants remains fragmentary. Therefore, we undertook this study to determine whether circulating concentrations of IGF-I, different IGFBP-1 phosphoisoforms, and IGFBP-3 reflect 1) growth velocity; 2) nutrient intake; and 3) other clinical determinants in VLBW infants during the first 2 months after birth.

Subjects and Methods

Study population

To concentrate the study effort upon infants at a high risk for impaired postnatal growth, we chose a study population of 48 VLBW infants comprised of two arms. All infants weighing below 1000 g (n = 32) were recruited. Of those of a birth weight between 1000 and 1500 g (n = 16), only those with a respiratory distress syndrome necessitating surfactant treatment were recruited because these infants are more susceptible to growth impairment (14). Surfactant treatment was considered to be indicated if the infant was less than 24 h of age and required treatment in a ventilator with either an inspired air oxygen fraction over 40% or a mean airway pressure over 7 cm H2O or an arterial/alveolar oxygen ratio less than 0.24.

The clinical characteristics of the infants are shown in Table 1Go. The energy intake (mean ± SD) was 88 ± 16 kcal/kg·d (wk 1), 100 ± 20 kcal/kg·d (wk 2), 112 ± 24 kcal/kg·d (wk 4), and 131 ± 13 kcal/kg·d (wk 8). Correspondingly, the protein intake was 3.1 ± 0.6 g/kg·d (wk 1), 3.0 ± 0.7 g/kg·d (wk 2), 2.9 ± 0.7 g/kg·d (wk 4), and 3.3 ± 0.3 g/kg·d (wk 8).


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  		Table 1. Patient data

 
Gestational age was recorded by ultrasound during early gestation, and birth weight immediately after birth. To describe intrauterine growth in units adjusted for gestational age, we used a birth weight SD score (SDS) determined with reference to a Finnish newborn population of 74,766 singletons born from 1978 to 1982 (15), with each newborn infant’s relative birth weight expressed in SD (birth weight SDS) units according to gestational age and gender.

Informed consent was obtained from the parents. The study protocol was approved by the Institutional Review Boards of Helsinki University Central Hospital, Helsinki City Maternity Hospital and Jorvi Hospital.

Study protocol

Blood samples. Blood samples were obtained at the age of 1 wk (7–9 d), 2 wk (12–16 d), 4 wk (26–30 d), and 8 wk (53–59 d) through an indwelling arterial catheter, by venipuncture or by heelstick. All samples were drawn at least 2 h after the last peroral feeding except when the infant received continuous feeding through a nasogastric tube. Blood samples drawn during iv insulin treatment (n = 12) were excluded from the data analysis. The lithium heparin or EDTA tubes were spun immediately at 1000 x g for 10 min, and the plasma was separated and frozen at -70 C until analysis.

Clinical follow-up. The study period continued from birth to 9 wk of age. The study setting was the Neonatal Intensive Care Unit of the Hospital for Children and Adolescents at Helsinki University Central Hospital and, when intensive care was no longer required, two local step-down neonatal units. Similar treatment and nutrition guidelines were followed by the clinicians in these units. Three infants died during the study period. Due to blood sample volume constraints or the transfer of the infant to another hospital, some assays and measurements could not be performed at all time points (Fig. 1Go). Individual nutritional intake was calculated daily for energy and protein, and the mean intake of each nutrient during the 7 d before each blood sample was used in the data analysis. Possible postnatal glucocorticoid dose (hydrocortisone or dexamethasone) was recorded for 24 h before each blood sample, and if dexamethasone was used, the dose was transferred into hydrocortisone equivalents by multiplying it by 25. Obstetric data were collected from hospital records.



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  		Figure 1. Box plots (median, range, and interquartile values) for IGF-I, IGFBP-3, lpIGFBP-1 (including nonphosphorylated) isoforms, hpIGFBP-1, the lpIGFBP/hpIGFBP-1 ratio, and insulin at different blood sample time points. Number of samples (N) at each time point is shown in parentheses. P values for linear trends are also indicated.

 
Growth velocity. Knee-heel length was recorded with an infant knemometer (FORCE Institutes, Copenhagen, Denmark), a caliper designed to monitor short-term growth velocity in the neonatal intensive care unit. The measurements were performed by a single observer (E.K.) three times a week from birth to 6 wk of age and thereafter two to three times a week. Each measurement consisted of a set of eight recordings, whereof the three first were discarded to minimize the effect of soft tissue compression, a method validated by Gibson et al. (16). Whenever possible, both legs were measured. Specific lower leg growth velocities (mm/d) for each leg at each blood sample time point were calculated by use of linear regression within a time period from a week before to a week after the blood sample. In the data analysis, the mean growth velocity of both legs at one time point was compared with the biochemical and clinical variables of the same individual at the same time-point.

Infant weight was recorded daily as a routine procedure of the neonatal ward that includes the use of the same scales for each infant. Weight growth velocity (g/d) for each blood sample time-point was calculated with linear regression as lower leg growth velocity.

Biochemical assays

IGF-I was determined from plasma samples using IGF-I enzyme-linked immunosorbent assay kit (DSL-10–5600, Diagnostics Systems Laboratories, Inc., Webster, TX). Inter and intraassay coefficients of variations were 4.8–8.8% and 4.5–7.1%, respectively, and the detection limit of the assay is 5 µg/liter.

IGFBP-1. Two monoclonal antibody (MAb)-based immunoenzymometric assays detecting different phosphoisoforms of IGFBP-1 were used. In assay 1, the detecting antibody was MAb 6305 (17) (Medix Biochemica, Kauniainen, Finland), which detects the nonphosphorylated and lesser-phosphorylated isoforms of IGFBP-1 (18). In assay 2, the detecting antibody was MAb 6303 (17), which recognizes the hpIGFBP-1 and lpIGFBP-1. Neither of the assays is specific for nonphosphorylated or highly phosphorylated isoform, because the lesser phosphorylated isoforms can be detected by both assays but the hpIGFBP-1 by assay 2 (MAb 6303) only (18). Assay 1 (MAb 6305) gives higher values compared with those obtained by assay 2 (MAb 6303), if the fluid contains predominantly nonphosphorylated isoforms of IGFBP-1, like amniotic fluid (19). This indicates that MAb 6303 detects phosphorylated rather than all isoforms of IGFBP-1. Correspondingly, compared with assay 1 (MAb 6305), assay 2 (MAb 6303) gives higher values from fluids containing predominantly phosphorylated isoforms. Both assays have been described in detail previously (19, 20).

The detection limits for assay 1 and assay 2 are 0.25 µg/liter and 0.30 µg/liter, respectively. The intra and interassay variations were 3.0% and 6.8% for assay 1, the corresponding variations for assay 2 being 4.6% and 6.4%. In this article, the non- and lesser phosphorylated IGFBP-1 (detected by MAb 6305) are referred to as lpIGFBP-1 and the phosphoisoforms including the highly phosphorylated isoform (detected by MAb 6303) are referred to as hpIGFBP-1.

IGFBP-3 was measured by immunofluorometric assay (21). Interassay and intraassay coefficients of variation were 4.9–11% and 3.6–6.2%, respectively, and detection limit is 0.3 µg/liter.

Insulin. Serum insulin was measured by an immunoradiometric assay kit for human insulin (Insulin-Irma, Biosource Technologies, Inc. Europe S.A., Nivelles, Belgium) with the following modifications. Because of the small sample volumes available the samples were diluted 1:2 with the zero standard to get the 50 µl of sample required for the assay. In addition, to increase the sensitivity of the assay, the incubation time of 2 h suggested in the kit protocol was prolonged to 16 h. The intra- and interassay coefficients were 1.0% and 4.0%, respectively, and the detection limit of the assay is 0.1 mU/liter.

Data analysis

IGF-I and IGFBP-3 concentrations, as well as all growth and nutrition data, gave a good fit to the normal distribution at all time points. lpIGFBP-1 and hpIGFBP-1, as well as the lpIGFBP-1/hpIGFBP-1 ratio, did not follow the normal distribution at any time-point, and logarithmic transformation was used. The distribution of the glucocorticoid dose was extremely skewed, with many infants receiving no glucocorticoid and the rest receiving a highly variable dose (mean dose 1.3 mg hydrocortisone equivalents/kg·d, SD 2.6 mg/kg·d, range 0–13.4 mg/kg·d). Therefore, at each blood sample time-point the infants were categorized according to glucocorticoid dose: "0"—no glucocorticoid (n = 73), "1"—glucocorticoid dose below 1.5 mg hydrocortisone equivalents/kg/·d (n = 41), and "2"—glucocorticoid dose over 1.5 mg hydrocortisone equivalents/kg/·d (n = 31).

The data were analyzed by SPSS for Windows, version 8.0.1 (SPSS, Inc., Chicago, IL). Linear regression was used to assess correlation between variables. To take the repeated measures design of the study into account, each regression analysis was adjusted for the individual patient factor, postnatal age, and, if significant, the interaction between postnatal age the independent variable. Adjustment for repeated measurements was performed by creation of dummy variables for each patient (individual patient factor) as described by Glantz (22). Relationships between measurements and pregnancy data (data constant for each subject) were assessed by linear regression separately for each time point. Results of all regression analyses are presented as the regression coefficient (ß), 95% confidence interval (CI) for ß, and P value.

Separate multiple regression models for lower leg and weight growth velocity were created by use of a stepwise backward elimination strategy, working from the largest number of prediction variables to the smallest. Insulin concentrations could not be included in these models because small blood sample volumes allowed their determination only in a subset of study infants. The largest model included all predictive variables with significant univariate correlation (infant sex, gestational age, energy and protein intake, mode of feeding, and glucocorticoid treatment) and, in addition, relative birth weight, the individual patient factor, postnatal age and interactions between postnatal age and each predictor variable. Nonsignificant interactions and variables were deleted one-by-one until the model contained only statistically significant terms. However, a nonsignificant variable was retained as long as it was present with a significant interaction with another term.

Results

IGF-I, IGFBP-1, IGFBP-3, and insulin

Association with postnatal age. The concentrations of IGF-I, IGFBP-3, lpIGFBP-1, and hpIGFBP-1 increased with postnatal age (Fig. 1Go), whereas the lpIGFBP-1/hpIGFBP-1 ratio showed a decreasing trend (Fig. 1Go).

Correlations between the IGF, IGFBP, and insulin concentrations. With IGF-I as the dependent variable, there was a positive correlation with IGFBP-3 (ß = 0.0186; 95% CI 0.00130 to 0.0241; P < 0.0001), and negative correlations with lpIGFBP-1 concentration (ß = -22.0; 95% CI-32.8 to -11.2; P < 0.0001, after logarithmic transformation) and lpIGFBP-1/hpIGFBP-1 ratio (ß = -15.5; 95% CI-23.3 to -7.8; P = 0.0002, after logarithmic transformation). No other significant correlations were observed.

Growth velocity

Growth velocity and postnatal age. The mean lower leg and weight growth velocities increased with postnatal age (Fig. 2Go).



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  		Figure 2. Box plots (median, range, and interquartile values) for growth velocity at different blood sample time points. Number of measurements (N) at each time point is shown in parentheses. P values for linear trends are also indicated.

 
Growth velocity, IGF, IGFBP, and insulin. Lower leg growth velocity showed a positive correlation with IGF-I and with IGFBP-3 (Table 2Go) but not with insulin, lpIGFBP-1, hpIGFBP-1, or the lpIGFBP-1/hpIGFBP-1 ratio. Similarly, weight growth velocity showed a positive correlation with IGFBP-3 and with the lpIGFBP-1/hpIGFBP-1 ratio (Table 2Go) but no correlation with absolute lpIGFBP-1 or hpIGFBP-1 concentrations, IGF-I, or insulin.


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  		Table 2. Statistically significant regression equations with either lower leg or weight growth velocity as dependent variables and each of the IGF system data as independent variables

 
Infant sex

Sex and growth velocity. There was no difference in growth velocity between male and female infants.

Sex, IGF, IGFBP, and insulin. Compared with male infants, female infants had higher concentrations of IGF-I (all postnatal ages combined. Mean ± SD 22.2 ± 2.0 vs. 17.3 ± 1.1 µg/liter, P = 0.03) and IGFBP-3 (all postnatal ages combined: 1254 ± 69 vs. 1022 ± 49 µg/liter, P = 0.007). No sex difference was observed in the insulin or IGFBP-1 data.

Pregnancy data

The pregnancy data are summarized in Table 1Go.

Gestational age. Compared with infants born before 28 wk of gestation (n = 27), infants born thereafter (n = 21) had higher lower leg growth velocity on wk 2 (P = 0.006) and wk 4 (P = 0.03) and higher weight growth velocity on wk 2 (P = 0.004) and wk 4 (P < 0.0001). With regard to the IGF and IGFBP data, the only significant differences were noted at wk 2, when infants born after 28 wk of gestation had higher IGF-I (P = 0.04) and hpIGFBP-1 (P = 0.03) concentrations than those born before. Moreover, insulin concentrations were higher in infants born before 28 wk of gestation at wk 2 (P = 0.001), wk 4 (P < 0.0001), and wk 8 (P = 0.0002).

Intrauterine growth retardation. Relative birth weight showed no correlation with growth velocity of the IGF system data at any time point. At wk 2, but not at any other time point, a positive correlation was observed between insulin concentration and relative birth weight (P = 0.008).

Infant nutrition

Nutrition and growth velocity. In the total data-set, adjusted for postnatal age and repeated measures, the correlations between nutrient (energy or protein) intake and either lower leg or weight growth velocity were not statistically significant. However, when the data were analyzed separately for each time point, positive correlations between energy intake and lower leg growth velocity were found at wk 2 (P = 0.04) and at wk 4 (P = 0.02), between energy intake and weight growth velocity at wk 2 (P = 0.004) and at wk 4 (P = 0.0004). In addition, protein intake showed a positive correlation with lower leg growth velocity at wk 4 (P = 0.01). The mode of feeding (total or partial iv compared with total oral nutrition) had no effect on lower leg or weight growth velocity.

Nutrition, IGF, IGFBP, and insulin. Neither energy nor protein intake showed any correlation with IGF-I, IGFBP-3, insulin, lpIGFBP-1, hpIGFBP-1, or the lpIGFBP-1/hpIGFBP-1 ratio, when adjusted for postnatal age and repeated measurements. However, compared with total oral nutrition, total or partial iv nutrition was associated with lower IGF-I (P = 0.03) and lpIGFBP-1 (P = 0.04) as well as higher insulin (P = 0.06) but not with any difference in IGFBP-3, hpIGFBP-1, or the lpIGFBP-1/hpIGFBP-1 ratio. Within blood samples obtained during total oral feeding, feeding by continuous nasogastric tube infusion, compared with bolus feeding, was associated with higher insulin (P = 0.004) but no differences in the IGF and IGFBP data.

Glucocorticoid treatment

Glucocorticoid treatment and growth velocity. With increasing glucocorticoid dose, a statistically significant decrease in lower leg growth velocity was observed at wk 1 (P = 0.03), wk 2 (P = 0.005), and wk 4 (P = 0.02), and a corresponding decrease in weight growth velocity at wk 2 (P = 0.01), wk 4 (P < 0.0001), and wk 8 (P = 0.02).

Glucocorticoid treatment, IGF, and IGFBP in the total study population. Increasing glucocorticoid dose was associated with lower concentrations of lpIGFBP-1 (P = 0.002) and hpIGFBP-1 (P < 0.0001), as well as with lower IGFBP-3 (P = 0.02), but no association was observed with the IGF-I concentration or the lpIGFBP-1/hpIGFBP-1 ratio.

Glucocorticoid treatment, IGF, and IGFBP in subgroups of samples with low and high insulin. To assess whether the effect of glucocorticoids on IGFBP-1 synthesis is dependent on insulin concentration (23), we performed a subgroup analysis in two groups of blood samples, one with a low (below 10 mU/liter; n = 42), and another with a high (above 10 mU/liter; n = 47) insulin concentration (56 samples having insufficient volume for insulin measurement). Increasing glucocorticoid dose appeared to remain significantly associated with lower lpIGFBP-1 (P = 0.02) and hpIGFBP-1 (P = 0.007) only in the high insulin subgroup, the associations in the low insulin subgroup being statistically nonsignificant (Fig. 3Go). By contrast, no correlation between glucocorticoid dose and IGF-I, IGFBP-3, or the lpIGFBP-1/hpIGFBP-1 ratio was seen in these two subgroups.



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  		Figure 3. Box plots (median, range, and interquartile values) for lpIGFBP-1/hpIGFBP according to glucocorticoid treatment dose and insulin concentration of the sample. P values for negative linear trends are indicated.

 
Multiple regression models

Multiple regression models, created to assess which variables are most predictive of lower leg and weight growth velocity, are summarized in Table 3Go. Adjusted for postnatal age and the repeated measures design, higher lower leg growth velocity remained significantly associated only with higher IGF-I, and higher weight growth velocity only by higher IGFBP-3 and higher lpIGFBP-1/hpIGFBP-1 ratio. All other variables and interactions (see Table 3Go) were excluded from these models as nonsignificant.


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  		Table 3. Multiple regression models with lower leg and weight growth velocities as dependent variables, adjusted for postnatal age and the repeated measures design

 
Discussion

The aim of this study was to assess how circulating IGF-I, IGFBP-1, and IGFBP-3 concentrations relate to short-term growth velocity, nutrition, and glucocorticoid treatment in postnatal VLBW infants. We found that higher growth velocity was associated with concurrent higher IGF-I concentration and, with regard to weight growth velocity, also higher IGFBP-3 concentration and lpIGFBP-1/hpIGFBP-1 ratio, even after adjustment for postnatal age and other potential confounders (Tables 2Go and 3Go).

The IGF-I and IGFBP-3 concentrations in fetal circulation rise throughout mid-late gestation (6, 8, 24), reach a plateau during infancy (25, 26), continue to increase during childhood, and peak at puberty (11, 27). Although this increasing overall pattern is opposite to that of growth velocity, short-term alterations in growth velocity like those during puberty are reflected by corresponding changes in IGF-I and IGFBP-3 (11, 27). Together with data from cord serum studies (6, 24) and follow-up studies in postnatal preterm infants (25, 28), our results (Fig. 1Go) suggest that after the birth of VLBW infant, IGF-I and IGFBP-3 concentrations fall by 1 wk of age after which they gradually rise. These alterations indeed follow nicely the concurrent changes occurring in clinical condition, nutrition and growth velocity (Fig. 2Go).

The role of the IGF system in fetal as well as childhood growth is supported by a wide body of evidence from laboratory and clinical studies. In mice, knockout of the IGF-I or IGF-I receptor (3), as well as IGFBP-1 gene overexpression (4), results in pre- and postnatal growth retardation, which is also a feature in human IGF-I gene deletion (5). In both preterm and term infants, low birth weight is associated with low cord serum IGF-I (6, 7, 8) and IGFBP-3 (6, 7) and high IGFBP-1 (6, 7, 8). During puberty, changes in Tanner stage, reflective of growth velocity, are associated with changes in IGF-I and IGFBP-3 (11, 27), and, inversely, in IGFBP-1 (11) concentrations.

However, there has been uncertainity about the role of circulating IGF-I and IGFBPs in preterm and term infants during postnatal growth. Most studies have assessed these factors as predictors of catch-up growth after intrauterine growth restriction over a period of months after birth, with some studies reporting an association between catch-up growth and higher IGF-I (29, 30, 31) and IGFBP-3 (29) and some failing to show any associations (32).

The discrepancies in the previous studies could partly be explained by possible rapid changes in growth velocity and difficulties in obtaining reliable measurements, common problems when studying newborn, especially preterm infants. We have attempted to overcome these problems by using a rigorous protocol of frequent, precise and accurate measurements of both lower leg length and total body weight. Lower leg length measured with an infant knemometer repeatedly by the same observer gives an accurate and specific estimate of skeletal growth velocity during periods as short as 1 to 2 wk (33). It is virtually unaffected by changes in hydration (16, 33), and even the negative lower leg growth velocities occasionally observed have been reproducible (34, 35). Therefore, the combined use of frequent lower leg and weight recordings, the latter taking into account also soft tissue growth, is probably closest to the "gold standard" in measuring short-term growth velocity in preterm infants. With this method, we have now shown that circulating IGF-I, IGFBP-3, as well as a the ratio of lpIGFBP to hpIGFBP-1, reflect prevailing short-term growth velocity in VLBW infants (Tables 2Go and 3Go). Although the individual differences in the concentrations remain relatively large and may limit the use of these factors in everyday clinical practice, they are likely to be informative for instance in research protocols when following how various treatments affect short-term growth velocity.

In the fetus, IGF-dependent growth is primarily controlled by the glucose-insulin axis which allows rapid response to nutritional fluctuations, while the effect of GH is lesser albeit not negligible (36). Birth involves a rapid step in the development from the relatively GH-resistant state of the fetus, with high GH levels and a small number of GH receptors, to the fully active GH-IGF-I axis during childhood growth (36). However, high postnatal GH levels in preterm infants (37) have suggested a degree of GH resistance comparable to the fetal period to continue after preterm birth. Therefore, while our findings clearly confirm that growth in postnatal VLBW infants is IGF-I dependent, the hormonal regulation of the IGF system in these infants remains an interesting issue for further study.

A general note must be taken regarding the type of data analysis used in this study, the design of which is based on repeated measurements of the same subjects. When analyzing data from such studies, it is essential to adjust the correlations to exclude the potential confounding effects of advancing postnatal age and repeated measurements of same subjects (22). Such an adjustment, however, may reduce the power of the analysis to detect weak or moderate effects, and results obtained from such analyses are inevitably fairly conservative estimates. A single statistically nonsignificant adjusted correlation, such as between weight growth velocity and IGF-I in our study, should thus not be interpreted solely as evidence on lack of effect. It may rather reflect the inability of this type of a data analysis, in some instances, to distinguish between the hypothesized effect and the effect of the adjustment for potential confounders, in this case particularly postnatal age.

Unexpectedly, we found no association between either energy or protein intake and any of the postnatal-age-adjusted IGF system data. However, as a result of the relatively strict nutrition guidelines applied in our neonatal units, the interindividual variation in nutrient intake at each time point of our study was considerably low. It is therefore possible that in our study population changes in the IGF data attributed to increasing postnatal age actually in part reflect concurrent increase in mean nutrient intake. This possibility is also supported by previous studies which report clear relationships between IGF-I, IGFBP-3, and nutrient intake in as well postnatal preterm infants (28, 38) as in children and adults (39). On the other hand, the lack of correlation between nutrient intake and IGFBP-1 in our study is not surprising because of the large minute-to-minute variability in IGFBP-1 concentrations (40).

A novel finding in this study was the presence of both hpIGFBP-1 and lpIGFBP-1 in the plasma of VLBW infants after birth, with the proportion of the lesser-phosphorylated isoforms decreasing with increasing postnatal age (Fig. 1Go). The origin of different phosphoisoforms of IGFBP-1 in the circulation of these infants is not known. Fetal liver, which in midgestation contains all IGFBP-1 phosphoisoforms (41), is the most likely candidate, although a contributing role of fetal kidney, containing primarily the nonphosphorylated isoform (41), or other tissues cannot be excluded. Differences in the IGF-I-binding affinity of IGFBP-1 phosphoisoforms may explain the apparently conflicting results of IGFBP-1 either inhibiting (42) or stimulating (43) IGF-I action. Compared with lesser-phosphorylated IGFBP-1, the highly phosphorylated isoform has 4- to 10-fold higher affinity for IGF-I (12, 13) and is thus believed to be a more potent inhibitor of IGF-I action. Almost all of the IGFBP-1 in the serum of nonpregnant adults is highly phosphorylated (13), whereas lesser-phosphorylated isoforms are present, in variable proportions, in amniotic fluid (12, 44) and in the serum of mid-gestation fetuses (12), prepubertal children (45), and pregnant women (18, 46), i.e. in conditions associated with tissue growth. Therefore, it has been suggested that the lesser-phosphorylated isoforms of IGFBP-1 could also have growth-promoting effects. Our finding of a positive association between the ratio of lpIGFBP-1 isoforms to hpIGFBP-1 and concurrent weight growth velocity (Tables 2Go and 3Go) is, to our knowledge, the first clinical observation to support the hypothesis that, compared with hpIGFBP-1, circulating lpIGFBP-1 is, at least, less inhibitory on the growth-promoting action of IGF-I.

The most important predictor of the IGFBP-1 concentration was the dose of glucocorticoids, a higher dose being associated with lower lpIGFBP-1 and hpIGFBP-1. At first, this may seem surprising because the IGFBP-1 gene promoter region contains a glucocorticoid-inducible element (23), and for instance in children cortisol and IGFBP-1 measured throughout the day show a close positive correlation as well during fasting (47) as in physiologic conditions (48). However, there are also contrasting reports. When healthy adult men are given a high dose of dexamethasone, IGFBP-1 levels decrease (49). Moreover, in cord plasma of preterm infants, IGFBP-1 concentrations are inversely correlated with the number of maternal antenatal betamethasone treatments given (6). One possible explanation for this discrepancy is provided by an in vitro finding showing that the dexamethasone-induced increase in IGFBP-1 promoter activity is inhibited by simultaneous application of insulin (23). Therefore, we measured insulin concentrations in a subset of the blood samples and found that the association between glucocorticoid dose and IGFBP-1 was indeed profoundly different according to the insulin concentration, the negative correlation between glucocorticoid dose and IGFBP-1 being observable only in the group of samples with a high insulin concentration (Fig. 3Go). This finding supports the idea that the regulation of IGFBP-1 synthesis by glucocorticoids is dependent on the prevailing insulin concentration.

Female infants had higher IGF-I and IGFBP-3 concentrations than males, the difference increasing with postnatal age. With regard to IGFBP-3, this has been observed before in postnatal preterm and term infants (25) and in children before and during puberty (11). Whether this phenomenon has any biological significance during the postnatal period is not known.

We conclude that, in VLBW infants, serum concentrations of IGF-I and IGFBP-3, as well as the lpIGFBP-1/hpIGFBP-1 ratio, reflect concurrent short-term growth velocity. The finding provides further support that circulating IGFs and IGFBPs are important regulators of postnatal growth in these infants. Different IGFBP-1 phosphoisoforms are abundant in the circulation of these infants, and lesser-phosphorylated IGFBP-1 may have properties that are at least less inhibitory on growth-promoting activity of IGF-I. In addition, the regulation of IGFBP-1 synthesis by glucocorticoids may be divergent in situations with a high or low insulin concentration.

Acknowledgments

We are indebted to the infants who participated in the study and their families. The personnel of the Neonatal Intensive Care Unit of the Hospital for Children and Adolescents and the neonatal wards of Helsinki City Maternity Hospital and Jorvi Hospital, as well as the laboratory personnel of these hospitals, are gratefully acknowledged for invaluable help in recruiting patients and collecting blood samples. Dr Visa Honkanen made helpful suggestions regarding the data analysis. Kaija Aspholm, Annikki Koistinen, Kristiina Nokelainen, Anne Salonen, Marita Suni, and Marjatta Vallas provided excellent technical assistance.

Footnotes

This work was supported by grants from Acadamy of Finland, Finska läkaresällskapet, the Federation of Finnish Life and Pension Insurance Companies, the Foundation for Pediatric Research, the Helsinki University Central Hospital Research Fund, the Paulo Foundation, University of Helsinki, Wiipurilaisen osakunnan stipendirahastot, and The Yrjö Jahnsson Foundation.

Abbreviations: CI, Confidence interval; hpIGFBP, highly phosphorylated IGFBP; IGFBP, IGF binding protein; lpIGFBP, lesser phosphorylated IGFBP; MAb, monoclonal antibody; SDS, SD score; VLBW, very low birth weights.

Received September 6, 2001.

Accepted January 16, 2002.

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