Author: Yis, Uluç
Date published: January 1, 2010
In the first year of life, different growth patterns have been identified in breast-fed (BF) and formula-fed (FF) infants. BF infants have accelerated growth rate in the first 36 months of age1·2. However, in the second six months of life, the growth rate of these infants decreases progressively, while it does not decrease to the same extent in the FF group3·4. The differences in growth patterns in these two groups of infants remain poorly understood. The neuroendocrine regulation of food intake is complex and involves the brain receiving both neuronal and hormonal inputs. Gut hormones have been found to be important in appetite regulation5. Stimulating hormones originating from the gastrointestinal system are important in the initiation, cessation and frequency of eating5. Recently, the studies have focused on ghrelin, a novel peptide hormone that is isolated from the rat stomach6"8. It has been concluded that ghrelin stimulates hunger and food intake7.
Ghrelin is a natural antagonist to leptin. Leptin is secreted mainly by adipocytes, decreases food intake and increases metabolic rate8. The central effects of leptin and ghrelin are mediated, at least partly, through inhibition and stimulation, respectively, of the neuropeptide Y/Yl receptor pathway in the arcuate nucleus neurones in the hypothalamus9. Insulin, another hormone, also regulates body weight and energy metabolism.
In this study, our goal was to investigate the effect of various hormones, including ghrelin, leptin and insulin, on growth patterns and feeding characteristics of BF and FF infants during the first six months of life.
Material and Methods
Healthy term babies of approximately three to four months of age (range: 80-135 days) were enrolled in this prospective study. Those babies who were breastfed only formed the BF group and those who were given formula only since birth formed the FF group. None of the babies in either group received supplemental foods before four months of age. Premature or small for gestational age babies and those having infection or immunization 10 days prior to the study were excluded. The study was approved by the Ethical Committee of Dokuz Eylül University Faculty of Medicine, Izmir, Turkey (Project No: 04.KB.SAG.027). Signed informed consent was obtained from legal guardians of each infant.
Birth weight, feeding patterns and bowel movement patterns of the babies were queried via a questionnaire. The questionnaire was performed by face-to-face interview between the investigator (U. Y.) and the caregivers.
At the onset of the study, when the infants were around three-to-four months old, physical examinations including anthropometric measurements [body weight, length, head circumference, and triceps skin fold thickness (TSF) ] were performed and body mass indexes (BMI) were calculated. In addition, venous blood samples of all babies were obtained for plasma leptin, ghrelin, insulin, and glucose measurement between 10:00 a.m. to 12:00 noon after 3-4 hours of fasting. At the same time, pre-feeding breast-milk samples of the mothers of BF infants were obtained for leptin and ghrelin determination.
When the babies were re-evaluated three months later, at around six months of age, timing of the introduction of supplemental foods, feeding characteristics, and daily energy and protein intake only from supplemental foods were queried, and anthropometric measurements were repeated. Blood and breast-milk hormone levels were not studied at that time.
For plasma ghrelin assay, 1-2 ml whole blood was drawn into a centrifuge tube containing 1.8 mg of EDTA plus 500 U of aprotinin per ml of blood (Sigma-Aldrich, Germany) . Samples were transferred on ice, cold centrifuged, and plasma was separated and stored at -700C until the assay.
For serum leptin, insulin and glucose, 3-4 ml whole blood was allowed to clot and then centrifuged at room temperature. The serum samples were transferred and stored at -700C until the assay.
Breast-milk samples were taken into plain test tubes for leptin and to tubes containing 500 U of aprotinin per ml of milk for ghrelin assay. Milk samples were stored at -70°C until the assay. For leptin and ghrelin analyses, milk samples were thawed overnight at 4°C, and whole milk was sonicated for 3-10 second burst with 20-second cooling intervals.
Plasma and breast-milk ghrelin were measured by using a "peptide enzyme immunoassay" kit (Peninsula Laboratories, Inc., Texas, USA) with intra-assay and inter-assay variations of coefficients (CV) of less than 5% and 14%, respectively, sensitivity limit of 0.08-1.0 ng/ml, and measuring range of 0-25 ng/ml.
Breast-milk and serum leptin concentrations were analyzed with a "two-site immunoradiometric assay" (IRMA) kit (Diagnostic Systems Laboratories, Ine, Texas, USA) with intra-assay and inter-assay CV of 2.6% to 4.9% and 3.7% to 6.6%, respectively, sensitivity limit of 0.1 ng/ml, and measuring range of 0-120 ng/ml.
Serum insulin levels were measured with a "solid phase enzyme amplified sensitivity immunoassay" kit (BioSource, Belgium), and the results were expressed as µIU/ml. Serum glucose concentrations were measured with a commercially available kit (Roche Diagnostics, Germany) by an auto -analyzer (Roche Diagnostics Modular Analytics D -P, Japan) .
Breastfed and FF infants were compared with respect to anthropometric measurements, feeding and bowel movement characteristics, and hormone levels at three to four months of age. The same comparisons with the exception of hormone levels were repeated at the last visit when they were six months old. In addition, daily energy and protein intake and weight gain were compared at this period of the study. Furthermore, correlation of hormone levels with the evaluated parameters was analyzed. The data were analyzed using software SPSS (version 10.0; SPSS, Chicago, IL). Results were expressed as percentages and as means ± standard deviation (SD). Statistical analyses were carried out by the Wilcoxon signed rank test for difference in two related groups of measures, Mann-Whitney U test in two independent groups of measures, and Spearman and Pearson rank correlation analysis for the relation between two variables. A ? value of <0.05 was considered significant in all analyses.
There were a total of 47 babies (male/female = 25/22), of which, 24 (51%; 13 male; 100±9 days old) were in only BF and 23 (49%; 12 male; 97± 17 days old) in FF groups. There were no differences between the two groups with respect to age, sex, and birth weight (Table I) (p>0.05).
Anthropometric Data, Feeding and Bowel Movement Characteristics
Mean body weight, length, TSF, and postnatal weight gain of babies were higher in the BF group than in the FF group at the first visit at 3-4 months of age (p= 0.017, p= 0.016, p= 0.001, and p= 0.022, respectively; Table I). Frequency and duration of feeding (p= 0.006, p= 0.01, respectively), feeding frequency in the daytime (p= 0.004), and the frequency of bowel movements (p= 0.006) were also higher in the BF than FF group. Nineteen (79.2%) of the BF babies were reported to have bowel movements during feeding. However, timing of the bowel movements was variable in FF babies.
We did not determine any difference between the two groups in relation to body weight, length, and TSF during the second visit at around six months of age. However, mean weight gain between the first and second visits was higher in the FF group compared to the BF group (p= 0.0 13). Difference in the bowel movement pattern between the two groups disappeared at that time (p= 0.088; Table I). Although feeding frequency and duration remained higher in the BF group, daily energy and protein intake from supplemental foods was lower in these babies than in FF babies [363.7± 205.2 (range: 72-730) vs 890.3± 341.3 (range: 440-1700) kcal, ?= 0.001; and 12.5±6.3 (range: 2-25) vs 31.7- 11.8 (17-60) g, p= 0.001, respectively] .
Serum Ghrelin and Leptin Levels and Their Relation to Anthropometric Data, Daily Energy Intake, and Bowel Movement Patterns
Table II shows the results of serum hormonal analyses of babies in the BF and FF groups. In BF babies, mean serum ghrelin was higher than in the FF group. The mean serum leptin, although slightly higher in the BF group, was not significantly different between the two groups. In both groups, serum ghrelin was not correlated with serum leptin, glucose or insulin. On the other hand, there was a positive correlation between serum ghrelin and TSF at the 3rd month of age in the BF group (r= 0.51 1, p= 0.011; Fig. 1), but not in the FF group. Similarly, serum leptin was also correlated with body weight, BMI, TSF, and postnatal weight gain only in BF babies (r= 0.469, p= 0.028; r= 0.564, p< 0.010; r= 0.556, p< 0.007 and r= 0.471, p< 0.027, respectively). On the other hand, as the serum leptin increased, energy intake from supplemental foods at the 6th month of age decreased in the BF group (r= -0.502, p= 0.048; Fig. 2).
Serum Glucose and Insulin Levels in the BF and FF Groups
Mean serum glucose was not different between the two groups (Table II). Although the mean serum insulin concentration in FF babies was higher than in the BF group, the difference did not show statistical significance (Table II).
Breast-Milk Ghrelin and Leptin Concentrations and Their Relation to the Anthropometric Data and Bowel Movement Patterns
The mean ghrelin and leptin concentrations in breast-milk in the BF group were 0.8±0.5 (range: 0.2- 2.5) ng/ml and 0.4± 0.2 (range: 0.20.9) ng/ml, respectively Neither breast-milk ghrelin nor breast-milk leptin was correlated with the respective serum ghrelin or leptin, anthropometric data or bowel movement patterns of the infants in the BF group.
Although growth in early childhood is related to many factors, such as the intrauterine environment, birth weight, sex, parental height, and nutrition, the possible effects of other undiscovered regulatory mechanisms on growth have also been suggested1·2·10. Nutrition is one of the most important variables affecting growth, and the importance of nutrition on early childhood growth is further emphasized by the marked differences in growth rates between BF and FF infants during the first three months of life1,2,4,11. The babies in the BF group in our study also showed increased growth rate during the first three to four months of life. The lack of difference in size at birth excludes any initial size-based selection bias in forming the feeding groups.
Our results suggest that differences in growth patterns between BF and FF babies in early infancy may be related to the difference in serum ghrelin levels. Higher serum ghrelin in BF babies may stimulate appetite via faster gastric emptying so that BF babies feed more frequently and for a longer duration. As a result, BF babies grow more rapidly than FF babies during the first three to four months of life.
Additionally, more frequent bowel movements and moving their bowels during breastfeeding may be related to higher serum ghrelin in BF groups12,13. Furthermore, we speculate that breast-milk ghrelin can also contribute to increased gastrointestinal motility via affecting the gastrointestinal system directly.
Controversial relationships between ghrelin and leptin are reported in many studies14,15. Both of these hormones affect the same areas in the central neuronal system. While ghrelin stimulates appetite and food intake, leptin conducts satiety signals and increases energy consumption5. We did not find any correlation between leptin and ghrelin concentrations in our study population. In the literature, the relationship between ghrelin and leptin concentration was mainly studied in pathologic states like anorexia nervosa and obesity16-17. Further studies are needed to better understand the interaction between ghrelin and leptin in different physiologic conditions.
Lower energy intake from supplemental foods in correlation with higher serum leptin in BF babies may be the underlying cause of evident decline in weight gain in BF babies compared to FF babies. In our study, while serum leptin correlated with anthropometric data in BF babies, such correlations were not present in the FF group. FF infants ingested higher energy and protein from supplemental food and their serum ghrelin and leptin were lower than in the BF group. These findings suggest that the body weight regulatory function of leptin was more effective in BF infants. It is suggested that breast-milk leptin and/or high serum leptin concentrations of BF babies are important in food intake and appetite. In our study, the finding of increased fat tissue in BF babies at three months of age may have caused high serum leptin concentrations, which in turn decreases food intake and results in lower weight gain in the second three months of life.
The weakness of our study, on the other hand, is that there was only one spot measurement of ghrelin and leptin levels in BF and FF infants. Although the levels of these hormones seem to be related to feeding and bowel movement characteristics and growth in this population, serial measurement of ghrelin and leptin would be more informative in future studies.
In conclusion, differences in growth patterns between BF and FF babies in early infancy may be related to the higher serum ghrelin in BF babies. Lower energy intake from supplemental foods in correlation with higher serum leptin in BF babies may explain why BF babies show evident decline in weight gain compared to FF babies. BF infants self-regulate their food intake, which is likely controlled by hormonal mechanisms such as leptin and ghrelin. This physiologic state may be disturbed by formula feeding. Long-term prospective studies are needed to reveal the effects of various hormones on feeding patterns and growth from infancy to adulthood.
We thank Dr. A. Pinar Cemeroglu for her contribution to this study and for the preparation of the manuscript. This work was supported by the Scientific Research Project Unit of Dokuz Eylül University.
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Uluç Yis1, Yesim Öztürk1, Ali Roza Cisman2, Sezer Uysal2, Özlem Bekem Soylu1,
Departments of 1Pediatrics, and 2Biochemistry, Dokuz EyIuI University Faculty of Medicine, /zmir, and department of
Pediatrics, Yeditepe University Faculty of Medicine, Istanbul, Turkey