It is widely believed that the relationship between nutrition and growth is mediated by influences on the hormone-growth factor axis. Growth hormone (GH) growth promoting effects are mediated by serum factors known as insulin-like growth factors (IGF-I and IGF-II) that have been considered to stimulate cellular proliferation and differentiation in a variety of tissues, in both intrauterine and postnatal life (1-3). IGFs circulate linked to binding proteins (IGFBPs) that are thought to mediate IGFs' access to cellular IGF receptors, operating as modulators of their biological effect (4,5). Six IGFBPs molecular forms have been identified. In postnatal life the main IGF-I binding protein is IGFBP-3. Its concentration does not vary throughout the day. Therefore, measurement of IGFBP-3 in a single blood sample obtained at any time of the day is representative.
High GH levels have been found in cord blood and newborn infants, although this hormone does not seem to be necessary for fetal growth (6). On the other hand, IGF-1 and IGFBPs are thought to play an important role during this period. Contrary to what happens during postnatal life, in the fetal period neither IGF-I nor IGFBP-3 synthesis is under GH control (1-4, 7-10).
The presence of IGF-I, IGF-II, and IGFBP, mainly IGFBP-2, has been demonstrated in mature and premature human milk samples (11-13). Lucas et al. (14) showed a beneficial effect of human milk on neurodevelopment in preterm infants. IGF-I and IGFBP-2 together with other hormones and trophic factors present in human milk are thought to influence brain growth and maturation.
The purpose of our study was to analyze the role played by nutrition, IGF-I, and IGFBP-3 on neonatal growth in preterm infants and whether their blood levels are influenced by the type of lactation.
MATERIALS AND METHODS
Two newborn groups at the neonatology service of our hospital were studied, after parental informed consent had been obtained: a control group, including full-term, healthy, appropriate for gestational age (AGA) neonates, and a group of clinically stable preterm AGA infants.
Gestational age, measured in weeks, was estimated from maternal history and according to the Dubowitz test. All infants were biochemically euthyroid and none had any evidence of renal disease. None of the children received aminoglucoside antibiotics, parenteral nutrition, iron, or vitamins during the study period.
Feeding began on the first day of life. Infants were fed every 3 h by intermittent nasogastric gavage or by sucking. According to the feeding pattern, preterm infants were subdivided into two groups. One of them included breast- and bottle-feeding, and the other included those infants fed exclusively with a milk formula [following European Society of Paediatric Gastroenterology and Nutrition (ESPGAN) recommendations] (15). The mean ± SD human milk intake in partially breast-fed preterm infants was 15.8 ± 8.6% of the total milk intake at the first week of postnatal life and 22.1 ± 18.4% of the total milk intake at the third week of postnatal life.
Preterm neonates were separated into two subgroups, according to their gestational age: 28-32 and 33-37 weeks.
Other characteristics of the sample are shown in Table 1.
Serum IGF-I and IGFBP-3 levels were determined in the first and third weeks after birth. First- and third-week samples were obtained from the same AGA preterm infants in 11 cases. These two samples were obtained from different infants in the remaining cases.
Body weight, length, head and arm perimeter, and skin fold were measured on the day of blood extraction. Nude weight was determined using an electronic balance with an accuracy of ±5 g. Crown-heal length was measured in the supine position to the nearest 0.1 cm using a Holtain neonatometer. Occipitofrontal head circumference was determined as the largest measurement taken across the occiput and forehead. Mid-upper arm circumference was recorded on the right limb, as the mean of two successive measurements, and rounded to the nearest 0.1 cm. Skin-fold measurements were made using a Holtain skin-fold caliper from the triceps and subscapular sites. Skinfolds were recorded as the mean of two successive measurements and rounded to the nearest 0.1 mm. The measurements were always taken by the same person (N.M.D.). The coefficient of variance (CV) for repeated measurements on the same neonate was 3.8% for triceps skin-fold and 4.4% for subscapular skin-fold.
Combined intra- and extrauterine Largo's growth curves were used as the reference pattern (16). Increments in weight were calculated as grams gained per kilogram and day. Increments in length and head circumference were calculated as centermeters gained per week.
All the preterm infants received a low-birth weight (LBW) formula. In artificially fed infants the initial volume of formula was between 25 and 35 ml/kg/day during the first day of postnatal life. The volume was increased as tolerated to reach 180-200 ml/kg/day, and macronutrient intake was determined from the volume of feeds retained. Breast-fed LBW infants were fed ad libitum. Infants received their own mother's milk and were weighed twice, before and after every meal, using an electronic scale, to determine the milk intake. Afterward these infants received the necessary volume of a LBW formula to cover their nutritional requirements. The average intake of energy and nutrients was calculated using Anderson's tables of human full-term and preterm milk composition (17). For the administered formula similar data were obtained from the manufactuer (Table 2). Volumes of milk intake were 104 ± 24 and 179 ± 31 ml/kg/days, energy intakes were 72 ± 12 and 124 ± 21 kcal/kg/day, and protein intakes were 1.9 ± 0.4 and 3.3 ± 0.7 g/kg/day, in the first and third weeks of postnatal life, respectively.
Serum samples were stored at -40°C until use. IGFBP-3 was measured by radioimmunoassays (RIA) and IGF-I by RIA after separation of the IGFs from the serum BPs by acid-ethanol extraction, avoiding their interfering with the technique. Kits purchased from Nichols Institute Diagnostics were used in both cases. All samples were assayed in duplicate. For the IGF-I assay the intraassay CV was 3% and the interassay CV was 10%. For the IGFBP-3 assay these values were 5 and 8%, respectively.
Data analyses were performed according to the Statistical Package for the Social Sciences (SPSS/PC+). The groups were compared using independent-sample Student's t test and paired-sample t test. Chi-square test with Yate's correction, linear regression, and multiple regression analysis were also used; p < 0.05 was taken as the limit of significance.
Energy, protein, and other nutrient intakes were similar in the two groups of preterm infants studied. No significant differences in IGF-I and IGFBP-3 levels were found between males and females.
At the first week after birth, preterm infants showed lower IGF-I levels in relation to the control group (Table 3).
At the third week of postnatal life, serum IGF-I and IGFBP-3 levels in preterm infants had increased significantly, in relation to the first-week levels (Fig. 1). Preterm infants born before 33 gestational weeks showed significantly lower IGF-I and IGFBP-3 levels in relation to those born between 33 and 37 gestational weeks [mean ± SEM; IGF-I, 16.6 ± 4.4 μg/L (n = 8) vs. 41.9 ± 6.1 μg/L (n = 18), t = 2.6, p < 0.02; IGFBP-3, 401 ± 80 μg/L (n = 6) vs. 701 ± 64 μg/L (n = 16), t = 2.6, p < 0.02].
At the third week of postnatal life, artificially fed preterm infants showed significantly lower serum IGF-I levels than those breast- and bottle-fed [25.4 ± 4.4 μg/L (n = 16) vs. 48.2 ± 9.5 μg/L (n = 10), t = 2.4, p < 0.05] (Fig. 2). The goups did not differ in the other variables studied (Table 4). No significant differences were found in IGFBP-3 levels according to feeding pattern.
When data from all neonates studied were analyzed together, postconceptional age was found to be positively and significantly related to serum IGF-I levels (r = 0.29, n = 87, p < 0.01). Postnatal days were directly related to IGFBP-3 levels (r = 0.46, n = 79, p < 0.001). There was no correlation between postnatal days and serum IGF-I levels. Serum IGF-I and IGFBP-3 levels were directly related (r = 0.59, n = 79, p < 0.001).
In preterm infants there was a positive correlation between protein intake and weight increase between the first and the third weeks of postnatal life (r = 0.57, n = 23, p < 0.01). There was no correlation between weight gain and serum IGF-I levels. Both IGF-I and IGFBP-3 showed a significant correlation with energy and protein intake (Fig. 3).
A multiple regression analysis was performed in preterm infants, taking serum IGF-I levels as a dependent variable and postconceptional age, energy and protein intake, and serum IGFBP-3 levels as independent variables. A significant function was obtained (F = 9.15, significant p = 0.00001) and it was proved that 50% of the variation in IGF-I levels in the neonatal period was explained by these variables. The most predictable variables were energy intake (β = 1.14, significant p = 0.01) and serum IGFBP-3 levels (β = 0.41, significant p = 0.003)
Studies carried out in a variety of animal species have shown that there are few GH receptors in the fetus, while their numbers increase postnatally (18). A decrease in GH BP activity has also been found in neonates (19). Both factors may lead to decreasing endocrine IGF-I production by fetal tissue as a response to GH, and could explain the fact that, in the neonatal period, both serum IGF-I and IGFBP-3 levels were lower than in older children and adults (9,20,21).
In agreement with previous studies (1,22), preterm infants were found to show lower IGF-I levels than the control group at the first week of postnatal life. Subsequently, their concentration increased, and at the third week of postnatal life, differences between groups failed to be significant.
At the third week after birth the lowest IGF-I levels were found in preterm infants born before 33 weeks of pregnancy. In agreement with previous reports (1,23), serum IGF-I levels were found to increase in a direct relationship with postconceptional age.
In the fetal period IGF-I circulates mainly bound to IGFBP-1 and IGFBP-2, whereas IGFBP-3 is the main IGF carrier during postnatal life (24). This could explain the highly significative correlation between them that we found.
In premature infants there was no correlation between weight gain at the hospital and serum IGF-I levels. This could have been due to the short time of follow-up study and small sample size. Nevertheless, in our study both IGF-I and IGFBP-3 showed a significant correlation with energy and protein intake. These data support the hypothesis that, as in children and adults (25,26), IGF-I and IGFBP-3 represent indicators of nutritional status in neonates.
IGF-I, IGF-II, and IGFBP-2 in human milk are significantly higher than in cow's milk. In cow's milk-based infant formulas they have not been detected (11,13,27). The origin of IGFs in human milk has yet to be clarified. It is believed that they could be synthesized by the mammary gland or transferred from the maternal circulation (11-13,28).
Studies carried out in experimental animals have proved that IGF-I in human milk may be able to survive digestion and interact with IGFs receptors in the gastrointestinal tract, where it could enhance intestinal villous growth and close the intestine-blood barrier (29-31). On the other hand, in newborn calves fed milk plus recombinant human IGF-I, an increase in circulating IGF-I has been shown (32). This supports the hypothesis that IGF-I in human milk would be absorbed and could explain the higher IGF-I levels found in breast-fed preterm infants.
Further studies are necessary to confirm our finding that breast-feeding is associated with higher IGF-I levels in preterm newborn infants and could improve neonatal growth or have other long-term biological consequences.
Acknowledgement: Supported by grants from the Foundation Heinz Koch and the University of La Laguna.
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