Iron deficiency has been linked to obesity. Hepcidin is the main regulator of iron homeostasis and is higher in obese children compared to controls. To gain insight into the link between obesity and hepcidin, we performed an intervention study in 15 obese children. These children were subjected to a 6-month weight loss program and underwent physical examination and iron status and absorption as well as hepcidin, interleukin-6 and leptin serum levels evaluation at baseline and after the weight loss program. After the program all children reduced their body mass index standard deviation score (BMI SDS) of at least 0.5. We observed a significant decrease in hepcidin (P=0.003) and leptin levels (P=0.005), and a significant increase in iron absorption (P=0.02). A direct correlation between the measure of hepcidin and leptin reduction was observed and this correlation appeared significant (r2=0.33, P=0.003) when adjusted for interleukin-6 and BMI SDS variations. In conclusion, we have shown that, in obese children, BMI reduction is associated with hepcidin reduction, potentially improving iron status and absorption. Implications of these findings could be considered in the management of obese children with poor iron status.
Iron deficiency, in developed countries, is the most common nutritional deficiency and has been linked to obesity in adults and children.1, 2 Hepcidin is a 25 amino-acid peptide, produced mainly by the liver but also by adipose tissue, and is the body's main regulator of systemic iron homeostasis, restricting intestinal iron absorption as well as macrophage iron release.3 Hepcidin expression is increased by inflammation and elevated hepcidin levels have been associated with the anemia of inflammatory disease. In fact, pro-inflammatory cytokines, such as interleukin-6, but also the adipokine leptin, stimulate hepcidin transcription through JAK–STAT3 interactions.4, 5 Three recent studies have shown that, both in obese women and in obese children, serum hepcidin levels were significantly higher compared with normal weight controls and have focused on the hepcidin-mediated inhibition of dietary iron absorption in obese patients.6, 7, 8 To gain insight into the link between obesity and hepcidin, we performed an intervention study in a group of obese children and evaluated if body mass index (BMI) decrease may reduce circulating hepcidin levels, increasing, therefore, iron absorption and improving iron status.
Materials and methods
Fifteen Caucasian obese children and adolescents (eight girls), who have attended the Department of Pediatrics of the Second University of Naples (Italy) between January 2008 and January 2009 to take part in a weight loss program, were enrolled in this study. All these subjects had a BMI exceeding the 95th percentile for age and sex according to reference values,9 with age ranging from 9 to 16 years.
We excluded from the study any children who had obesity caused by genetic syndromes, endocrinological disease or psychiatric disorders, as well as children with blood disorders or chronic inflammatory disease. Procedures followed were in accordance with the Helsinki Declaration of Principles 1975 as revised in 1983. The ethical committee of the Second University of Study of Naples approved the study. Written informed consent and assent were obtained from parents before any procedure.
At baseline, all participants underwent physical examination and iron status and absorption as well as hepcidin, interleukin-6 and leptin serum levels were evaluated. Weight and height were measured. BMI SDS were calculated by using the LMS method.10 Blood samples were drawn after overnight fasting.
Intestinal absorption of iron was studied using the iron loading test based on the increment of iron level 2 h after administrating an iron load.11, 12 Serum iron concentrations were measured at 0800 hours (baseline) and then 120 min after administration of 1 mg kg−1 ferrous sulfate (at 1000 hours). Test was performed after an overnight fast and a solution containing 20 mg ml−1 of ferrous sulfate was used for the loading test. Change in iron concentration (Δiron) was measured by subtracting baseline serum iron from serum iron at 120 min.
Hemoglobin, serum iron, transferrin and ferritin levels were measured and transferrin saturation was calculated as previously described.8 Interleukin-6 and leptin were measured by commercially available enzyme-linked immunosorbent assay kits. The coefficients of variations within assay were 7% for interleukin-6 and 6% for leptin and between assays were 5% for interleukin-6 and 4% for leptin.
Serum hepcidin-25 (the mature, active form of the peptide) measurements were performed by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry.13 Peptide spectra were generated on a Microflex LT matrix-enhanced laser desorption/ionization TOF MS platform (Bruker Daltonics, Billerica, MA, USA). Serum hepcidin-25 concentrations are expressed as nmol l−1. The lower limit of detection of this method was 0.5 nM; average coefficients of variation were 2.7% (intra-run) and 6.5% (inter-run).
All patients consumed a nutritionally balanced (50% carbohydrate, 30% fat and 20% protein) self-selected diet of common foods (60% of the recommended dietary energy allowances for age and sex) and underwent lifestyle modifications. They followed a program based on physical exercise, and behavioral therapy, including individual psychological care of the child and his or her family.14 After 6 months anthropometric and biochemical evaluations were repeated.
Difference before and after the weight loss program was evaluated with the paired Student's t-test.
In the same children, hepcidin and leptin variations (that is, Δ hepcidin and Δ leptin, respectively) were calculated subtracting serum concentrations evaluated after the weight loss program from baseline concentrations. A multiple regression analysis including interleukin-6 and BMI standard deviation score (SDS) variations as covariates was used to correlate Δ hepcidin with Δ leptin. Both measures were log-transformed before performing the analysis.
Data are expressed as means and s.d. We considered P<0.05 as statistically significant. Stat-Graphics 3.0 Centurion XV software (Werrenton, VA, USA) for Windows was used for all the statistical analyses.
After the weight loss program all children reduced their BMI SDS of at least 0.5. We observed a significant decrease in hepcidin (P=0.003) and leptin levels (P=0.005) and a significant increase in iron absorption (P=0.02) (Table 1). Furthermore, although not statistically significant, a trend toward an increase in serum iron and transferrin saturation levels was found.
Finally, a direct correlation between Δ hepcidin and Δ leptin levels was observed and this correlation appeared significant (r2=0.33, P=0.003) when adjusted for interleukin-6 and BMI SDS variations (Figure 1).
The major finding of this study is that weight loss among obese children is associated with lower hepcidin concentrations and a significant improvement in iron absorption. In fact, we have shown that in obese children decrease of BMI reduces circulating hepcidin levels and increases iron absorption. This should, at least potentially, improve iron status. These results support the data reported in some articles showing that obese patients have higher circulating hepcidin concentrations compared to normal weight subjects and corroborate the idea that the poor iron status frequently observed in obese children may be due to the hepcidin-mediated inhibition of dietary iron absorption.6, 7, 8 Recently, in a group of obese women subjected to restrictive bariatric surgery, in agreement with our results, after weight loss, decreased serum hepcidin and improved functional iron status has been shown.15 We also observed that change in hepcidin concentrations was associated with change in leptin independently of BMI. Therefore, among obese children subjected to a weight loss program, the extent of leptin reduction corresponded to the extent of hepcidin reduction. Leptin has been shown to stimulate hepcidin m-RNA production in a similar manner as interleukin-6.5 Therefore, the possibility exists that, in the context of obesity, leptin may exert a major effect in modulating hepcidin levels, appearing important in linking obesity and hepcidin with the iron status. This is in agreement with the lack of correlation between serum hepcidin and interleukin-6, recently reported in obese women.7 Leptin interacts both with pathways in the central nervous system and through direct peripheral mechanisms. Central, hypothalamic resistance to the leptin anorexigenic actions is characteristic of obesity.16 It likely results from defects in the hypothalamic neural circuitry that regulates energy homeostasis16 and/or in leptin transport into the brain.17 The supposed action of leptin on liver hepcidin production is a typical peripheral action that should not be affected by the mechanisms producing central leptin resistance.
In conclusion, we have shown that, in 15 obese children, a significant BMI decrease allows hepcidin reduction and, consequently, improves iron status and absorption. Implications of these findings could be considered in the management of obese children with poor iron status.
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This work was supported by grant from Ministero dell’Università, PRIN 06/08 (to LP and EMDG).
The authors have nothing to disclose with the exception of DWS who is a co-founder and medical director of the ‘Hepcidinanalysis.com’, an initiative that aims to serve the scientific and medical community with high-quality hepcidin-25 measurements.
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