Journal of the American College of Nutrition Adolescent Life-Event

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Adolescent Life-Event Stress in Boys Is Associated with Elevated IL-6 and Hepcidin but Not Hypoferremia a

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Little Flower Augustine MSc , Krishnapillai Madhavan Nair PhD FAMS , Sylvia Fernandez Rao b

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PhD , Mendu Vishnuvardhana Rao PhD , Punjal Ravinder PhD , Nagalla Balakrishna PhD , d

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Avula Laxmaiah MBBS MPH & Shahnaz Vazir PhD a

Division of Micronutrient Research, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania Hyderabad, Andhra Pradesh, INDIA b

Behavioral Sciences, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania. Hyderabad, Andhra Pradesh, INDIA. c

Biostatics, National Institute of Nutrition, Indian Council of Medical Research, JamaiOsmania. Hyderabad, Andhra Pradesh, INDIA. d

Community Studies, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania. Hyderabad, Andhra Pradesh, INDIA. Published online: 10 Oct 2014.

To cite this article: Little Flower Augustine MSc, Krishnapillai Madhavan Nair PhD FAMS, Sylvia Fernandez Rao PhD, Mendu Vishnuvardhana Rao PhD, Punjal Ravinder PhD, Nagalla Balakrishna PhD, Avula Laxmaiah MBBS MPH & Shahnaz Vazir PhD (2014) Adolescent Life-Event Stress in Boys Is Associated with Elevated IL-6 and Hepcidin but Not Hypoferremia, Journal of the American College of Nutrition, 33:5, 354-362, DOI: 10.1080/07315724.2013.875417 To link to this article: http://dx.doi.org/10.1080/07315724.2013.875417

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Original Research

Adolescent Life-Event Stress in Boys Is Associated with Elevated IL-6 and Hepcidin but Not Hypoferremia Little Flower Augustine, MSc, Krishnapillai Madhavan Nair, PhD, FAMS, Sylvia Fernandez Rao, PhD, Mendu Vishnuvardhana Rao, PhD, Punjal Ravinder, PhD, Nagalla Balakrishna, PhD, Avula Laxmaiah, MBBS, MPH, Shahnaz Vazir, PhD

Downloaded by [Indian Council of Medical Res] at 23:08 26 November 2014

Division of Micronutrient Research (L.F.A., K.M.N., P.R.), and Behavioral Sciences (S.F.R., S.V.), Biostatistics (M.V.R., N.B.), Community Studies (A.L.), National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania Hyderabad, Andhra Pradesh, INDIA Key words: inflammation, stress, IL-6, hepcidin, iron, adolescents, path analysis, India Objective: The link between stress-related increases in inflammatory markers, hepcidin, and iron status are poorly understood, especially in developing countries like India. The aim of the study was to examine the relationship between adolescent life-event stress (ALES), inflammatory markers, and its association with hepcidin and biomarkers of iron status among adolescent boys. Methods: Data pertaining to a subsample of 145 participants from a cross-sectional, school-based study recruiting 370 adolescent boys aged 15–19 years, from 5 schools in Hyderabad, India, were analyzed. Stress was assessed using the ALES scale, psychological distress by the General Health Questionnaire-2 (GHQ-12), and approach and avoidance coping using the Coping Strategies Scale. Biomarkers of iron and concentrations of other micronutrients, hepcidin, IL-6 and C-reactive protein (CRP) in plasma were analyzed. Data were subjected to regression, path analyses, and analysis of covariance (ANCOVA). Results: ALES was a significant predictor of interleukin (IL)-6 (β = 0.196, p = 0.012), CRP (β = 0.217, p = 0.010), and log hepcidin (β = 0.228, p = 0.006). Hepcidin correlated significantly (p < 0.001) with IL-6 (r = 0.344) and CRP (r = 0.370) but not with the biomarkers of iron status. Path analysis showed that the model had an acceptable fit, with a root mean square error of approximation of 0.019, 90% confidence interval (CI) of 0.00–0.074, comparative fit index of 0.988, chi-square p = 0.393, and chi-square/df of 1.053. Conclusions: Adolescent life-event stress is associated with elevated IL-6 and hepcidin concentration but not hypoferremia. These findings may help in iron supplementation programs for tackling anemia.

INTRODUCTION

have failed to find an association between interleukin (IL)-6, C-reactive protein (CRP), and hepcidin, despite the existence of a good relationship between hepcidin and biomarkers of iron status [5–7]. Contrary to the above observations, a large-scale study from Mexico reported that, on a comparable iron intake, the obese women and children had a significantly lower serum iron concentration and higher CRP compared to controls [8]. The findings pointed toward the influence of inflammation on dietary iron absorption, which could hamper the iron supplementation programs to control iron deficiency in Mexico. Though

The role of inflammation in iron homeostasis is proposed to be mediated through internalization, ubiquitination, and subsequent lysosomal degradation of ferroportin by hepcidin in enterocytes, hepatocytes, and macrophages leading to hypoferremia [1]. This has been established in chronic morbidities such as multiple myelomas and Hodgkin’s lymphoma [2–4] but not in mild inflammatory conditions. Studies in mild inflammatory states, among older adults, pediatric refugees, and obese individuals,

Address correspondence to: Dr. K. Madhavan Nair, Scientist ‘F’ & Head, Micronutrient Research, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania, Hyderabad 500 007, Andhra Pradesh, INDIA. E-mail: [email protected] The abstract was presented at the 44th Annual National Conference of the Nutrition Society of India, Sri Venkateswara University, NH 205, Prakasam Nagar Colony, Tirupathi, AP, India, November 16–17, 2012. Abbreviations: ALESS = Adolescent Life-Event Stress Scale, BMI = body mass index, CFI = comparative fit index, CMIN = chi-square, CRP = C-reactive protein, GHMC = Greater Hyderabad Municipal Corporation, GHQ-12 = General Health Questionnaire version 12, PPS = probability proportionate to size, RIA = radioimmunoassay, RMSEA = root mean square error of approximation, SES = socioeconomic status, SLI = standard of living index, sTfR = soluble transferrin receptor.

Journal of the American College of Nutrition, Vol. 33, No. 5, 354–362 (2014) C American College of Nutrition Published by Taylor & Francis Group, LLC 354


Hepcidin and IL-6 in Life-Event Stress the determinants are poorly understood, studies have shown the possibility of a systemic low-grade inflammatory state in countries like India, where iron-deficiency anemia is a public health problem [9–11]. Psychological stress may contribute to low-grade inflammation and thereby changes in iron status. Induction of hypoferremia through the IL-6–hepcidin axis has been shown in 3-day and 7-day exposure to repeated psychosocial stress in rats using the communication box paradigm [12–14]. The observed changes reversed after injecting anti-IL-6 antibody, establishing the causality. No attempts have been made to date in humans along these lines except for finding an association between IL-6 and acute psychological stress, where the increases were transient [15,16]. Because iron deficiency and anemia are widespread globally and have a multifactorial etiology [17], it was thought that determining whether there is any association between psychological stress, inflammation, and iron status was an innovative research question. We considered adolescence for exploring the relationship, because adolescents are stress prone and are vulnerable to developmental and emotional changes during this period of remarkable growth [18]. Because estradiol is known to inhibit IL-6 [19], we considered adolescent boys as an ideal target group for establishing the association. Therefore, the hypothesis was that adolescent life-event stress is associated with hypoferremia mediated through the IL-6–hepcidin axis among adolescent boys. Fig. 1. Flowchart of study population.

MATERIALS AND METHODS The study was cross-sectional in design. The sample size of 353 was fixed, assuming a stress prevalence of 40% [20], α of 95%, 20% relative precision, 80% power, and 20% dropout. The blood sample available from a subsample of 145 was sufficient to detect a correlation of 0.2 between the study variables. The study was approved by the Institutional Ethics Committee in 2007. Other related approvals from the schools were completed in 2007–2008. The setting for the study was Greater Hyderabad Municipal Corporation, Hyderabad, India. All singlegender boys’ schools (n = 5) were covered, including schools from the central (n = 2), southern (n = 2), and northern (n = 1) regions. The study was carried out during July 2009 and completed in January 2010. Written consent was obtained from the participants and their parents. Those consented were screened through clinical examination. The history of acute infection in the past 15 days was collected using a checklist. Apparently healthy students without any diagnosed hormonal abnormalities, congenital anomalies, or chronic illnesses and those who were not currently taking medication or any multivitamin or mineral tablets for the past year were selected. Probability proportionate to size of school was the sampling method used. Enrollment was carried out using random selection from the list of eligible students from each

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

school. The study camp lasted for 5 school days in a particular school and thereafter shifted to the next school and therefore followed a rolling pattern. To minimize the influence of acute stress associated with school examinations, the study was carried out when there were no terminal examinations. Anthropometry and administration of psychological scales were performed in 370 students. A subsample of 146 participants was randomly selected to provide blood samples (Fig. 1).

Socioeconomic Status and Anthropometry Socioeconomic data such as caste, family size, number of rooms, and education of parents were collected using a pretested questionnaire [21]. Information on assets was collected using a checklist and the standard of living index (SLI) was calculated using 18 household assets and the weighted scores were used as a proxy for economic status [22]. Body weight was taken without shoes and heavy clothing, using a calibrated weighing scale with a precision of 100 g (Seca, Hanover, MD). A portable anthropometric rod was used for measuring height to the nearest 0.1 cm using standard procedures (Galaxy Scientifics, Hyderabad, AP, India). Waist circumference was measured with a fiber-reinforced plastic tape to the nearest 0.1 cm at the point, midway between the lowest margin of the

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Hepcidin and IL-6 in Life-Event Stress ribs and the iliac crest [23]. The biceps, triceps, subscapular, and supra-iliac skinfold thickness was measured on the nondominant side of the body using a Harpenden skinfold caliper to the nearest 0.2 mm (CMS Instruments, London). Percentage of body fat was calculated according to a formula described elsewhere [24–26].


Stress-Related Variables The behavioral parameters studied were adolescent life events, psychological distress, and coping. The test material assessing these were pretested in this age group as described earlier [20]. The 40-item Adolescent Life Event Stress Scale, which is a culture-adapted version of the Social-Readjustment Scale developed for Indian adolescents, was used [27, 28]. This scale is used to assess the stress due to life events experienced by the participants in the past year scored on a dichotomous “yes” or “no” scale. The Cronbach’s alphas for controllable and uncontrollable events were found to be 0.84 and 0.93, respectively. The 12-item General Health Questionnaire (GHQ-12) is a self report measure of psychological morbidity or psychological distress experienced during the past week and has been validated in Indian population [29, 30]. The GHQ is scored on a 0–3 Likert scale. Subsequently, bimodal scoring (0–0–1–1) was used with the 2 least symptomatic answers scoring 0 and the 2 most symptomatic answers scoring 1. The 50-item coping strategies scale was used to assess the methods of coping applied by the students in response to a stressor [31]. This test applied a 5-point scale (0–4) classified into 5 major coping strategies based on combinations of operation and orientation of the coping behavior. The scale consisted of 36 approach–avoidance items with 15 items designed to assess approach coping and 21 statements assessing avoidance coping strategies. The split-half reliability reported for the scale was 0.78 for approach and 0.69 for avoidance and the test–retest reliability was 0.92.

Laboratory Analyses Ten milliliters of blood was collected after a 12-hour fasting into heparinized vacutainers using iron-free syringes within a day of administration of the psychological scales. The blood was processed and plasma was stored at −20◦ C until analysis. Hemoglobin analysis was done on whole blood on the same day using the cyanmethemoglobin method [32]. Ascorbic acid was estimated in plasma using the α-α bipyridyl micromethod [33] within 3 hours of sample collection. Iron, soluble transferrin receptor (sTfR), and ferritin were estimated in plasma. Ferritin was analyzed using an in-house sandwich enzyme-linked immunosorbent assay (ELISA) system with minimum detectable limit of 1 μg/L [34]. Iron was estimated using the method recommended by International Committee for Standardization in Hematology [32]. The in-house methods for ferritin and serum iron have been routinely under the external quality check with

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the VITAL-EQA program (laboratory number 34) by the Centers for Disease Control (CDC, Atlanta, GA). sTfR was analyzed using sandwich ELISA (R&D Systems, Inc., Minneapolis, MN). Plasma folate and vitamin B12 were analyzed using a dual RIA kit (Siemens Inc., Los Angeles, CA). Determination of plasma retinol was carried out by high-performance liquid chromatography (Thermo Finnigan HPLC Systems, Hertz, UK) [35], which is also part of the external validation of the CDC. IL-6 (Quantikine Human IL-6 assay kit, R&D systems Inc.) and CRP (Alpha Diagnostic International, San Antonio, TX) were measured in a previously unthawed aliquot of plasma. The lowest concentration assigned for each sample was the same as the minimum detectable limit of 0.7 ng/L for IL-6 and 0.3 μg/L for CRP, instead of zero. Hepcidin was measured using ELISA (hepcidin-25) based on competitive binding assay (DRG International Inc., GmbH, Mountainside, NJ). The assay range was 3.9–140 μg/L with an intraassay variation of