Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 67–73
ORIGINAL ARTICLE
Non-traditional adipokines in pediatric HIV-related lipodystrophy: a-FABP as a biomarker of central fat accumulation
KALLIOPI THEODORIDOU1,2, ALEXANDRA MARGELI2, VANA SPOULOU1, IRINI BATHRELLOU3, CHRYSANTHI SKEVAKI2, GEORGE P. CHROUSOS1, IOANNIS PAPASSOTIRIOU2 & CHRISTINA KANAKA-GANTENBEIN1 1First
Department of Pediatrics, University of Athens, ‘Aghia Sophia’ Children’s Hospital, Athens, of Clinical Biochemistry, ‘Aghia Sophia’ Children’s Hospital, Athens and 3Laboratory of Nutrition and Clinical Dietetics, Department of Nutrition and Dietetics, ‘Harokopio’ University, Kallithea, Greece
2Department
Abstract Background. Lipodystrophy characterized by adipose tissue redistribution and lipid and glucose metabolism abnormalities, is common among HIV-infected adults and children on highly-active-antiretroviral-therapy (HAART). In a previous study of HIV-infected children, we did not detect insulin resistance, despite a high percentage of body fat redistribution abnormalities. Aim of the study. To investigate the non-traditional adipokines Retinol-binding-Protein-4 (RBP4), neutrophil-gelatinase-associated-lipocalin (NGAL), a-Fatty-Acid-Binding-Protein (a-FABP) and YKL-40 in HIV-infected children on highly-active-antiretroviral-therapy and evaluate their possible association to lipodystrophic changes or insulin resistance. Methods. Seventeen vertically HIV-infected children (mean age: 12.5 years, mean duration of HAART: 5.2 years) and 20 age- and BMI-matched controls were recruited. The HIV-children were re-evaluated after 12 months. RBP4, NGAL, a-FABP and YKL-40 were assessed at study entry and 12 months later and were correlated to body fat content and insulin resistance. Results. RBP4 values were similar at study entry and 12 months later in HIV-children and controls and showed no correlation to body fat or insulin resistance. NGAL was lower in HIV children at study entry but normalized after 12 months with no positive correlation to insulin resistance. a-FABP was positively correlated to body fat content, especially to trunk fat, both at initial evaluation and at follow-up in HIV children and, after prolonged highly-active-antiretroviral-therapy, it was also positively correlated to insulin resistance. Conclusions. This study is the first one to demonstrate that a-FABP could be a useful marker in unraveling central fat accumulation in HIV-infected children on highly-active-antiretroviral-therapy. Large prospective studies are needed to confirm these results. Key Words: Highly-active-antiretroviral-therapy, HIV, a-FABP, insulin resistance, lipodystrophy, NGAL, RBP4 Abbreviations: a-FABP, a-Fatty-Acid-Binding-Protein or FABP4; HAART, highly-active-antiretroviral-therapy; HOMA, HOmeostasis Model Assessment; NGAL, neutrophil-gelatinase-associated-lipocalin; RBP4, Retinol-bindingProtein-4.
Introduction Lipodystrophy (LD) syndrome, characterized by adipose tissue redistribution, dyslipidemia and insulin resistance, and accompanied by an increased risk of cardiovascular morbidity and mortality, is quite common among HIV-infected adults on highly active antiretroviral therapy (HAART) [1,2]. In pediatric patients, lipodystrophy syndrome does
occur [3,4], but its onset is subtle and studies based on clinical criteria have low sensitivity for early detection of such changes [5,6]. We recently evaluated body composition in a cohort of Greek HIV-1-infected children on HAART by Dual Energy X-ray Absorptrometry (DEXA)-based measurements, as described in the Methods section, and observed that a significant percentage of such
Correspondence: Ioannis Papassotiriou, Department of Clinical Biochemistry, ‘Aghia Sophia’ Children’s Hospital, Athens 11527, Greece. Tel/fax: ⫹ 30 213 2013171. E-mail: [email protected]; [email protected] (Received 31 December 2012 ; accepted 23 October 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.859725
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children demonstrated lipodystrophic abnormalities after a long period on HAART; however, no clear-cut insulin resistance was shown in our cohort based on the homeostasis model assessment (HOMA) insulin resistance index [7], while others reported insulin resistance in their HIV-infected children presenting with central fat adiposity [5,8]. Here we aimed to evaluate the possibility of unraveling central fat accumulation or even an underlying insulin resistant state in HIV-infected children on HAART through the investigation of novel cardio-metabolic risk markers, implicated in low grade chronic inflammation and insulin resistance, even in the absence of abnormal HOMA. Among these markers, we have included the retinol binding protein-4 (RBP4) that has been proposed as a major circulating adipokine implicated in systemic insulin resistance [9,10]. In a previous study in obese children and adolescents, we were unable to correlate RBP4 elevations with increasing Body Mass Index (BMI) [11], while others have found such a correlation in childhood and adolescent obesity in both cross-sectional and longitudinal analyses, suggesting a relationship between RBP4, obesity and insulin resistance in children [12]; however, no clear correlation between circulating RBP4 and HOMA could be demonstrated in another study in obese youngsters [13]. Moreover, another adipokine, lipocalin-2 or neutrophil gelatinase-associated lipocalin (NGAL) is reported as a biomarker positively correlated with BMI and insulin resistance [14]. Moreover, a newly discovered adipokine, a-fatty acid binding protein (a-FABP) that has been related to obesity and the metabolic syndrome [15], has been evaluated in HIV-related lipodystrophy in adult HIV patients [16,17]; however, no data exist on its role in pediatric HIV. Finally, another biomarker of low-grade inflammation, YKL-40 (also called human cartilage glycoprotein 39 or CHI3L1) was recently reported to be a marker of obesity and insulin resistance in prepubertal children [18,19]. The aim of this study was to investigate the possible impact of RBP4, NGAL, a-FABP and YKL-40 in HIV-infected children and to examine their possible association to lipodystrophic changes or insulin resistance markers.
Patients Seventeen children (7 boys) vertically infected with HIV-1, and followed at ‘Aghia Sophia’ Children’s Hospital, Athens, Greece, that participated in our first study [7] were included. The HIV-infected children were enrolled at a mean age of 12.5 (SD ⫽ 3.98, range: 5–17.5) years, had a mean weight of 45.5 (SD ⫽ 14.16, range: 17.5–64.8) kg, a mean height of 149.8 (SD ⫽ 16.9 range: 109–164) cm and a mean BMI of 19.1 (SD ⫽ 3.55, range: 12.89–25.93) (Table I). At study entry, 6 children were prepubertal and 11 pubertal, whereas during the following 12 months 1 prepubertal child entered puberty. At study entry, all patients were receiving HAART with at least 3 drugs (mean duration, 5.2 years); 64.7% were receiving at least one protease inhibitor (PI). During the 12-month followup period, 1 patient had to change his treatment regimen based on genotype resistance pattern and by the end of the study 58.8% children were on at least one PI. The regimen distribution was as follow: At study entry, 4 out of 17 patients were receiving 2 nucloside reverse transcriptase inhibitors (NRTI), namely AZT ⫹ 3TC/ abacavir and 1 nonnucloside reverse transcriptase inhibitor (NNRTI), namely Efavirenz, 7 out of 17 were on 2 NRTIs and 1 boosted protease inhibitor (PI), namely on Lopinavir/Ritonavir, 5 out of 17 patients were receiving avtiretrovirals of all 3 classes and the remaining one patient was receiving 3NRTIs. During the study period, 4 patients had to change their treatment regimens and at the end of the study 3 children were on 2 NRTIs and 1 NNRTI, 8 on 2 NRTIs and 1 boosted PI, 2 were on HAART of all 3 classes and 4 patient were receiving 3NRTIs. Twenty age- and BMI-matched healthy controls, examined in the Outpatient Clinic of the Division of Endocrinology, Metabolism and Diabetes for thyroid function evaluation, but proven to be healthy and willing to participate in the study served as controls. The study was approved by the Ethics Committee of ‘Aghia Sophia’ Children’s Hospital. Written informed consent was obtained from the parents or guardians of all study subjects before inclusion in the study.
Table I. Demographic characteristics of the study population.
Study design We performed this observational longitudinal study evaluating RBP4, NGAL, a-FABP and YKL-40 in the cohort of HIV-infected children followed in the First Department of Pediatrics, University of Athens. The first measurements were performed at study entry, as already reported, while re-assessment was done after 12 months on further HAART [7].
BMI-SDS Age (years, range) Fasting glucose (mmol/L) Fasting insulin (mIU/L) HOMA index Total cholesterol (mmol/L)
Controls n ⫽ 20
HIV n ⫽ 17
⫺0.11 ⫾ 0.4 6.0–15.0 4.7 ⫾ 0.1 11.6 ⫾ 1.7 1.99 ⫾ 0.4 4.1 ⫾ 0.6
⫺0.19 ⫾ 1.15 5.0–17.0 4.7 ⫾ 0.4 6.7 ⫾ 4.6∗ 1.42 ⫾ 0.95∗ 5.2 ⫾ 1.3∗
∗p ⬍ 0.01. BMI-SDS, Body Mass Index-Standard Deviation Score.
New adipokines in pediatric HIV
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Methods
Statistical analyses
All HIV-infected patients were assessed by the same physician for body weight, height, pubertal status and clinical characteristics of LD, e.g. fat wasting in the extremities, face or buttocks and/or fat accumulation in the abdomen or dorsocervical spine (buffalo hump). Soft tissue composition, i.e. fat mass and bone mineral-free lean mass of the whole body and of sub-regions (arms, legs, and trunk), were determined by Dual Energy X-ray Absorptrometry (DEXA) (model DPX-MD⫹, Lunar Corp., Madison, WI, USA), as previously reported [7]. Blood chemistry, including serum glucose and total cholesterol was performed using Siemens ADVIA 1800 Clinical Chemistry Analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA), while insulin levels were measured with an electrochemiluminescence immunoassay using the automated analyzer Cobas e411 and the Elecsys Insulin Kit (Roche Diagnostics, Basel, Switzerland). All blood chemistry parameters were measured after overnight fasting. Insulin resistance was determined according to the initial formula of the homeostasis model assessment (HOMA) [20]. Serum RBP4 was measured using a sandwich ELISA (Immunodiagnostik AG, Behsheim, Germany), identical in protocol and reagent composition with the ELISA kit from ALPCO Diagnostics (USA) tested by Graham et al. [10]. Plasma samples were diluted so that the absorbance was in the middle of the range of linearity for the assay. The intra- and inter-assay coefficients of variation (CV)s of RBP4 were 5.0 and 9.7%, respectively. Serum lipocalin-2 levels were determined by a solid phase ELISA technique (R&D Systems, Minneapolis, MN, USA). The intra-assay and inter-assay CVs ranged between 3.1 and 4.1% and between 5.6 and 7.9%, respectively. Serum levels of a-FABP were determined using a commercial human enzyme immunoassay kit (BioVendor Laboratory medicine, Inc., Modrice, Czech Republic). The assay was conducted according to the manufacturer’s instructions. The antibodies in human a-FABP ELISA are highly specific for human a-FABP, otherwise called FABR4, with no detectable cross-reactivity to other members of human FABP family, such as liver-FABP (FABP1), intestinal-FABP (FABP2), heart-FABP (FABP3) or epidermal-FABP (FABP5), and also to leptin and adiponectin. The intra- and interassay coefficients of variation (CVs) were less than 5.5% and the sensitivity limit was 0.1 μg/L. Serum YKL-40 levels were measured in duplicate by a commercial two-site sandwich-type enzymelinked immunosorbent assay (Quidel Corporation, San Diego, CA, USA), assessed daily for precision; coefficients of variations were 4–6%.
Values are expressed as mean ⫾ SD, as indicated. Relations between the parameters were investigated both by calculation of Pearson’s and/or Spearman’s correlation coefficients. Statistical significance was set at p ⬍ 0.05. The p values reported are two-tailed. All statistical procedures, as well as Box-Plot values presentations, were performed using the Statgraphics Centurion for Windows program (Graphic Software System, Statpoint Technologies, Inc., Warrenton, Virginia, USA). We used the standardized skewness and standardized kurtosis, to determine whether the sample has a normal distribution. Values of these statistics outside the range of ⫺2 to ⫹2 indicate significant departures from normality, which would tend to invalidate many of the statistical procedures normally applied to this data. These values integrated automatically by the program indicated the parameters needed to transform in either log, reciprocal or square root, as needed.
Results Viral load and CD4 counts At study entry the mean (range) CD4 cell count and viral load (by reverse-transcriptase-PCR, Amplicor Roche, Alameda, USA) were 876.3 (145.4–1858) cells/mL and 1.05 ⫻ 103 (⬍ 50 ⫺ 1.3 ⫻ 105) vc/ml, respectively. At the end of the follow-up period, mean CD4 counts and viral load were 953.9 cells/ mL and 1.01 ⫻ 103 vc/ml, respectively (p-value ⬎ 0.06 for both CD4 and viral load). Body composition measurements and metabolic abnormalities Body composition changes detected by DEXA in HIV-infected children were previously reported [7]. Although, no significant differences were observed in mean trunk fat (4.4 ⫾ 3.0 vs. 3.8 ⫾ 1.9 kg, p ⬎ 0.201), leg fat (4.0 ⫾ 2.3 vs. 4.5 ⫾ 1.9 kg, p ⬎ 0.108), leg lean (9.8 ⫾ 4.4 vs. 9.8 ⫾ 3.5 kg, p ⬎ 0.910) in HIVinfected children, when compared with expected values derived from controls, fat distribution expressed as trunk/leg fat ratio was significantly higher in patients, indicating central fat accumulation (1.03 ⫾ 0.3 vs. 0.81 ⫾ 0.09, p ⫽ 0.004) [7]. As reported however, there was no difference at study entry and after 12 months for fasting glucose, fasting insulin, and HOMA index between subjects with lipohypertrophy or lipoatrophy [7]. Non-traditional cardiometabolic risk markers in HIV-infected children on HAART The non-traditional adipokines RBP4, NGAL, aFABP and YKL-40 were assessed in the HIV-infected
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Table II. Adipocytokines levels in controls and patients with HIV at baseline and after 1 year of further observation under HAART.
RBP-4 (mg/L) NGAL (μg/L) a-FABP (μg/L) YKL-40 (μg/L)
Controls
Patients baseline
Patients 1 y HAART
22.0⫾0.8 (13.8–31.2) 67.5⫾5.5 (33.5–136.1) 39.5⫾3.9 (18.6–74.5) 38.3⫾2.7 (17.6–57.9)
21.1⫾1.2 (10.0–28.0) 18.8⫾4.6a (4.4–81.7) 19.0⫾3.0a (5.4–54.8) 48.0⫾4.5 (19.4–87.7)
21.3⫾1.6 (13.4–37.4) 68.2⫾22.3b (6.1–377.9) 16.8⫾2.9c (4.0–49.5) 59.7⫾6.9d (18.5–110.9)
Patients at baseline vs. Controls ap ⬍ 0.001 and patients 1 y HAART vs, Patients at baseline bp ⬍ 0.001. Patients 1 y HAART vs. Controls cp ⬍ 0.001. Patients 1 y HAART vs. Controls dp ⫽ 0.003.
children at study entry and after a further 12 months on HAART and were compared to controls’ data that have been collected at study entry (Table II). RBP4 levels were similar at baseline and after further 12 months on HAART in HIV-infected children (20.8 ⫾ 5.1 vs. 21.3 ⫾ 6.3 mg/L, p ⬎ 0.75) and were similar to controls (22.0 ⫾ 3.9 mg/L, p ⬎ 0.53). NGAL levels at study entry were significantly lower in HIV-infected children than controls (18.8 ⫾ 18.5 vs. 67.5 ⫾ 25.7 μg/L, p ⬍ 0.001). After further 12 months on HAART, NGAL levels normalized in HIV-infected children and were not different from controls (68.1 ⫾ 89.1 vs. 67.5 ⫾ 25.7 μg/L, p ⬎ 0.85). a-FABP levels were similar at baseline and after 12 months in HIV-infected children (18.9 ⫾ 12.2 vs.16.7 ⫾ 11.5 μg/L, p ⬎ 0.60), but were significantly lower than controls at both time points (39.5 ⫾ 18.2 μg/L p ⫽ 0.001) (Figure 1). YKL-40 levels in HIV-infected children at study entry were not significantly higher than controls (48.0 ⫾ 18.1 vs. 38.2 ⫾ 12.5 μg/L, p ⬎ 0.06). After a further 12 months on HAART, YKL-40 levels were not significantly different to those at study entry in HIV-infected
Figure 1. Concentration of a-FABP in HIV-infected children at study entry and after 12 months in comparison to controls. Boxes represent the interquartile range; lines inside boxes represent the median value; cross represents mean marker; whiskers represent the lowest and highest observations, respectively.
children but significantly higher than in controls (59.6 ⫾ 27.6 vs. 38.2 ⫾ 12.5 μg/L p ⫽ 0.003). We investigated whether serum concentrations of RBP4, NGAL, a-FABP and YKL-40 could have any correlation to body fat content or insulin resistance markers in HIV- infected children. We found no correlation between percentage of body fat content and specifically trunk fat accumulation and RBP4, neither at the initial evaluation (p ⬎ 0.056) nor after further 12 months (p ⬎ 0.267). Moreover, there was no correlation of RBP4 values and HOMA, neither at study entry (p ⬎ 0.3) nor after 12 months of follow-up (p ⬎ 0.05). NGAL concentration was negatively correlated with trunk fat accumulation both at the initial evaluation (p ⬍ 0.04), as well as after further 12 months on HAART (p ⫽ 0.0001). Furthermore, NGAL correlated negatively with both fasting insulin (p ⬍ 0.0001) and HOMA (p ⬍ 0.0001) at baseline and after a further 12-month observational period on HAART (p-value for insulin at 12 months of follow-up: 0.025 and p ⬍ 0.0001 for HOMA). We found a significant positive correlation between a-FABP and both total body fat content and trunk fat accumulation at the initial evaluation of our HIV-infected children on HAART (p ⫽ 0.0001 for both correlations), that persisted over the 12-month follow-up period (p ⫽ 0.0009 for total body fat, p ⫽ 0.0012 for trunk fat accumulation) (Figure 2). At the initial evaluation, a-FABP correlated neither with fasting insulin (p ⬎ 0.2578), nor with HOMA (p ⬎ 0.36). However, after a further 12 months on HAART, a-FABP correlated both to fasting insulin (p ⫽ 0.0036) and to HOMA (p ⫽ 0.0031). YKL-40 had a borderline positive correlation to trunk fat content at the initial evaluation (p ⫽ 0.05). However, this correlation did not persist after a further 12-month period (p ⫽ 0.91).
Figure 2. Correlation of % trunk fat and a-FABP levels in HIV infected children: baseline black dots, r ⫽ 0.847, p ⫽ 0.0009 and after 1 year empty dots, r ⫽ 0.723, p ⫽ 0.0012.
New adipokines in pediatric HIV Discussion In this study the circulating concentrations of RBP4, NGAL, a-FABP and YKL-40 in HIV-infected children under HAART were assessed and correlated to body fat accumulation, more concretely to trunk fat accumulation, and insulin resistance markers. Serum RBP4 concentrations were similar between pediatric HIV patients at study entry and after 12 months of follow-up and did not differ from controls. There was no correlation of RBP4 values neither with body fat content nor with insulin resistance markers. In agreement with our findings several recent reports have also demonstrated lack of a causal association between insulin resistance and RBP4 levels, questioning the role of RBP4 in unraveling insulin resistance [13,21]. NGAL levels at study entry were significantly lower in HIV-infected children than controls. After further 12 months on HAART, NGAL levels normalized in HIV-infected children and were no longer different from controls. Correlation analysis revealed that NGAL concentrations correlated negatively with trunk fat accumulation at both time points of the study. Furthermore, NGAL correlated negatively with both fasting insulin and HOMA at the initial evaluation and after further 12-months on HAART. In adult HIV-infected patients, NGAL levels were significantly reduced before treatment initiation [22]. During HAART, a pronounced fall in viral load was associated with significant increase in CD4⫹T cell counts accompanied by a gradual and significant increase in NGAL concentrations, reaching levels comparable to those of healthy controls after 12 months of treatment. A further 12-month period on HAART achieved a normalization of NGAL levels in our pediatric cohort. To the best of our knowledge, there are no other pediatric reports investigating NGAL concentrations in HIV-infected children in relation to the time course of normalization of CD4⫹T cell counts and viral load. These data suggest that NGAL concentrations in HIV-infected patients under HAART may be influenced by several factors, such as viral load or immunological status, and do not necessarily reflect an insulin resistance state. In the current study, a-FABP levels were similar at baseline and after 12 months of further treatment in HIV-infected children and were significantly lower than controls at both time points. Correlation analysis depicted a significant positive correlation between a-FABP and body fat content at the initial evaluation of our HIV-infected children on HAART, which persisted over a further 12-month follow-up period. More specifically, there was a positive correlation of a-FABP concentration to percentage of trunk fat at both time points, suggesting that a-FABP concentrations are mainly related to central fat accumulation. At the initial evaluation, a-FABP correlated neither
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to fasting insulin nor to HOMA. However, after the 12-month follow-up period, a-FABP significantly correlated both to fasting insulin and to HOMA. These findings suggest that among the adipokines studied, a-FABP seems to have the most important role in unraveling central fat accumulation especially after prolonged HAART. The positive correlation of a-FABP concentration to insulin resistance markers only after the 12-month observational period, although the children have already been under HAART for a period of 5.2 years necessitates confirmation by large scale prospective studies and may probably suggest that even a longer period may be necessary to unravel a correlation to insulin resistance markers in HIV-infected children on HAART. To the best of our knowledge, there are no other studies investigating a-FABP levels in pediatric HIV-infected populations. In HIV-infected adult patients, a-FABP levels were similar to those of healthy controls. However, among HIV-infected adults, a-FABP levels were significantly higher in those presenting with lipodystrophy and metabolic syndrome than in those without. In agreement with our results, in adult HIV patients a-FABP concentrations correlated positively with HOMA and fasting plasma insulin [16,17]. YKL-40 levels in HIV-infected children at study entry were not significantly different from controls. After a further 12 months on HAART, YKL-40 levels were similar to those at study entry in HIVinfected children but significantly higher than in controls. YKL40 correlated positively to body fat content at the initial evaluation, however, this correlation was lost after a further 12-month period on HAART. There is only one study investigating post mortem YKL-40 levels in the cerebrospinal fluid of patients with AIDS who developed encephalitis and no data exist on the role of YKL-40 in HIVrelated lipodystrophy [23]. One limitation of our study was the lack of DEXA-based body composition measurements in the same cohort of age-matched healthy controls, who provided the comparative data of the cardiometabolic risk markers. However, our previous study investigating the HAART-associated body composition abnormalities in this cohort of HIVinfected children using DEXA-measurements in comparison to a large group of healthy, age-matched controls, revealed a high percentage of lipodystrophic abnormalities in HIV-infected children allowing the assumption that such differences are expected in this group of healthy age-matched controls as well [7]. Another limitation of our study was the relative small number of HIV-infected participants. However, at the pediatric age range, in non-endemic areas of HIV, the number of HIV-infected children is fortunately limited. Our HIV-infected children have been extensively studied regarding viral load,
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biochemical profile, BMI, DEXA-based body composition and non-traditional adipokines, providing the possibility for correlation analysis. We cannot exclude therefore that other significant differences in adipokines between HIV-infected children and healthy controls have been missed because of the small number of subjects included and the inadequate power of the analysis. In conclusion, we previously demonstrated that a high percentage of HIV-infected children on HAART presented lipodystrophic abnormalities, even in the absence of clear-cut insulin resistance. In the current study, for the first time in HIVinfected children we have demonstrated that a novel adipokine, a-FABP, correlates positively with body fat content and more specifically with trunk fat accumulation in HIV-infected children on HAART and might be integrated in the assessment of HIV-related lipodystrophy in pediatric HIV. Largescale prospective studies investigating the concentration of these adipokines in HIV infected children both before HAART initiation and after prolonged HAART would better unravel their role as markers of the HIV-related lipodystrophic state. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Funding was received from Athens University to Dr Ioannis Papassotiriou. The funding source played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication 70/3/9294.
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New adipokines in pediatric HIV [20] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [21] Kotnik P, Fischer-Posovszky P, Wabitsch M. RBP4: a controversial adipokine. Eur J Endocrinol 2011;165: 703–11. [22] Landrø L, Damås JK, Flo TH, Heggelund L, Ueland T, Tjønnfjord GE, Espevik T, Aukrust P, Frøland SS. Decreased
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serum lipocalin-2 levels in human immunodeficiency virus-infected patients: increase during highly active antiretroviral therapy. Clin Exp Immunol 2008;152:57–63. [23] Bonneh-Barkay D, Bissel SJ, Wang G, Fish KN, Nicholl GC, Darko SW, Medina-Flores R, Murphey-Corb M, Rajakumar PA, Nyaundi J, Mellors JW, Bowser R, Wiley CA. YKL-40, a marker of simian immunodeficiency virus encephalitis, modulates the biological activity of basic fibroblast growth factor. Am J Pathol 2008;173:130–43.
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ORIGINAL ARTICLE
Non-traditional adipokines in pediatric HIV-related lipodystrophy: a-FABP as a biomarker of central fat accumulation
KALLIOPI THEODORIDOU1,2, ALEXANDRA MARGELI2, VANA SPOULOU1, IRINI BATHRELLOU3, CHRYSANTHI SKEVAKI2, GEORGE P. CHROUSOS1, IOANNIS PAPASSOTIRIOU2 & CHRISTINA KANAKA-GANTENBEIN1 1First
Department of Pediatrics, University of Athens, ‘Aghia Sophia’ Children’s Hospital, Athens, of Clinical Biochemistry, ‘Aghia Sophia’ Children’s Hospital, Athens and 3Laboratory of Nutrition and Clinical Dietetics, Department of Nutrition and Dietetics, ‘Harokopio’ University, Kallithea, Greece
2Department
Abstract Background. Lipodystrophy characterized by adipose tissue redistribution and lipid and glucose metabolism abnormalities, is common among HIV-infected adults and children on highly-active-antiretroviral-therapy (HAART). In a previous study of HIV-infected children, we did not detect insulin resistance, despite a high percentage of body fat redistribution abnormalities. Aim of the study. To investigate the non-traditional adipokines Retinol-binding-Protein-4 (RBP4), neutrophil-gelatinase-associated-lipocalin (NGAL), a-Fatty-Acid-Binding-Protein (a-FABP) and YKL-40 in HIV-infected children on highly-active-antiretroviral-therapy and evaluate their possible association to lipodystrophic changes or insulin resistance. Methods. Seventeen vertically HIV-infected children (mean age: 12.5 years, mean duration of HAART: 5.2 years) and 20 age- and BMI-matched controls were recruited. The HIV-children were re-evaluated after 12 months. RBP4, NGAL, a-FABP and YKL-40 were assessed at study entry and 12 months later and were correlated to body fat content and insulin resistance. Results. RBP4 values were similar at study entry and 12 months later in HIV-children and controls and showed no correlation to body fat or insulin resistance. NGAL was lower in HIV children at study entry but normalized after 12 months with no positive correlation to insulin resistance. a-FABP was positively correlated to body fat content, especially to trunk fat, both at initial evaluation and at follow-up in HIV children and, after prolonged highly-active-antiretroviral-therapy, it was also positively correlated to insulin resistance. Conclusions. This study is the first one to demonstrate that a-FABP could be a useful marker in unraveling central fat accumulation in HIV-infected children on highly-active-antiretroviral-therapy. Large prospective studies are needed to confirm these results. Key Words: Highly-active-antiretroviral-therapy, HIV, a-FABP, insulin resistance, lipodystrophy, NGAL, RBP4 Abbreviations: a-FABP, a-Fatty-Acid-Binding-Protein or FABP4; HAART, highly-active-antiretroviral-therapy; HOMA, HOmeostasis Model Assessment; NGAL, neutrophil-gelatinase-associated-lipocalin; RBP4, Retinol-bindingProtein-4.
Introduction Lipodystrophy (LD) syndrome, characterized by adipose tissue redistribution, dyslipidemia and insulin resistance, and accompanied by an increased risk of cardiovascular morbidity and mortality, is quite common among HIV-infected adults on highly active antiretroviral therapy (HAART) [1,2]. In pediatric patients, lipodystrophy syndrome does
occur [3,4], but its onset is subtle and studies based on clinical criteria have low sensitivity for early detection of such changes [5,6]. We recently evaluated body composition in a cohort of Greek HIV-1-infected children on HAART by Dual Energy X-ray Absorptrometry (DEXA)-based measurements, as described in the Methods section, and observed that a significant percentage of such
Correspondence: Ioannis Papassotiriou, Department of Clinical Biochemistry, ‘Aghia Sophia’ Children’s Hospital, Athens 11527, Greece. Tel/fax: ⫹ 30 213 2013171. E-mail: [email protected]; [email protected] (Received 31 December 2012 ; accepted 23 October 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.859725
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children demonstrated lipodystrophic abnormalities after a long period on HAART; however, no clear-cut insulin resistance was shown in our cohort based on the homeostasis model assessment (HOMA) insulin resistance index [7], while others reported insulin resistance in their HIV-infected children presenting with central fat adiposity [5,8]. Here we aimed to evaluate the possibility of unraveling central fat accumulation or even an underlying insulin resistant state in HIV-infected children on HAART through the investigation of novel cardio-metabolic risk markers, implicated in low grade chronic inflammation and insulin resistance, even in the absence of abnormal HOMA. Among these markers, we have included the retinol binding protein-4 (RBP4) that has been proposed as a major circulating adipokine implicated in systemic insulin resistance [9,10]. In a previous study in obese children and adolescents, we were unable to correlate RBP4 elevations with increasing Body Mass Index (BMI) [11], while others have found such a correlation in childhood and adolescent obesity in both cross-sectional and longitudinal analyses, suggesting a relationship between RBP4, obesity and insulin resistance in children [12]; however, no clear correlation between circulating RBP4 and HOMA could be demonstrated in another study in obese youngsters [13]. Moreover, another adipokine, lipocalin-2 or neutrophil gelatinase-associated lipocalin (NGAL) is reported as a biomarker positively correlated with BMI and insulin resistance [14]. Moreover, a newly discovered adipokine, a-fatty acid binding protein (a-FABP) that has been related to obesity and the metabolic syndrome [15], has been evaluated in HIV-related lipodystrophy in adult HIV patients [16,17]; however, no data exist on its role in pediatric HIV. Finally, another biomarker of low-grade inflammation, YKL-40 (also called human cartilage glycoprotein 39 or CHI3L1) was recently reported to be a marker of obesity and insulin resistance in prepubertal children [18,19]. The aim of this study was to investigate the possible impact of RBP4, NGAL, a-FABP and YKL-40 in HIV-infected children and to examine their possible association to lipodystrophic changes or insulin resistance markers.
Patients Seventeen children (7 boys) vertically infected with HIV-1, and followed at ‘Aghia Sophia’ Children’s Hospital, Athens, Greece, that participated in our first study [7] were included. The HIV-infected children were enrolled at a mean age of 12.5 (SD ⫽ 3.98, range: 5–17.5) years, had a mean weight of 45.5 (SD ⫽ 14.16, range: 17.5–64.8) kg, a mean height of 149.8 (SD ⫽ 16.9 range: 109–164) cm and a mean BMI of 19.1 (SD ⫽ 3.55, range: 12.89–25.93) (Table I). At study entry, 6 children were prepubertal and 11 pubertal, whereas during the following 12 months 1 prepubertal child entered puberty. At study entry, all patients were receiving HAART with at least 3 drugs (mean duration, 5.2 years); 64.7% were receiving at least one protease inhibitor (PI). During the 12-month followup period, 1 patient had to change his treatment regimen based on genotype resistance pattern and by the end of the study 58.8% children were on at least one PI. The regimen distribution was as follow: At study entry, 4 out of 17 patients were receiving 2 nucloside reverse transcriptase inhibitors (NRTI), namely AZT ⫹ 3TC/ abacavir and 1 nonnucloside reverse transcriptase inhibitor (NNRTI), namely Efavirenz, 7 out of 17 were on 2 NRTIs and 1 boosted protease inhibitor (PI), namely on Lopinavir/Ritonavir, 5 out of 17 patients were receiving avtiretrovirals of all 3 classes and the remaining one patient was receiving 3NRTIs. During the study period, 4 patients had to change their treatment regimens and at the end of the study 3 children were on 2 NRTIs and 1 NNRTI, 8 on 2 NRTIs and 1 boosted PI, 2 were on HAART of all 3 classes and 4 patient were receiving 3NRTIs. Twenty age- and BMI-matched healthy controls, examined in the Outpatient Clinic of the Division of Endocrinology, Metabolism and Diabetes for thyroid function evaluation, but proven to be healthy and willing to participate in the study served as controls. The study was approved by the Ethics Committee of ‘Aghia Sophia’ Children’s Hospital. Written informed consent was obtained from the parents or guardians of all study subjects before inclusion in the study.
Table I. Demographic characteristics of the study population.
Study design We performed this observational longitudinal study evaluating RBP4, NGAL, a-FABP and YKL-40 in the cohort of HIV-infected children followed in the First Department of Pediatrics, University of Athens. The first measurements were performed at study entry, as already reported, while re-assessment was done after 12 months on further HAART [7].
BMI-SDS Age (years, range) Fasting glucose (mmol/L) Fasting insulin (mIU/L) HOMA index Total cholesterol (mmol/L)
Controls n ⫽ 20
HIV n ⫽ 17
⫺0.11 ⫾ 0.4 6.0–15.0 4.7 ⫾ 0.1 11.6 ⫾ 1.7 1.99 ⫾ 0.4 4.1 ⫾ 0.6
⫺0.19 ⫾ 1.15 5.0–17.0 4.7 ⫾ 0.4 6.7 ⫾ 4.6∗ 1.42 ⫾ 0.95∗ 5.2 ⫾ 1.3∗
∗p ⬍ 0.01. BMI-SDS, Body Mass Index-Standard Deviation Score.
New adipokines in pediatric HIV
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Methods
Statistical analyses
All HIV-infected patients were assessed by the same physician for body weight, height, pubertal status and clinical characteristics of LD, e.g. fat wasting in the extremities, face or buttocks and/or fat accumulation in the abdomen or dorsocervical spine (buffalo hump). Soft tissue composition, i.e. fat mass and bone mineral-free lean mass of the whole body and of sub-regions (arms, legs, and trunk), were determined by Dual Energy X-ray Absorptrometry (DEXA) (model DPX-MD⫹, Lunar Corp., Madison, WI, USA), as previously reported [7]. Blood chemistry, including serum glucose and total cholesterol was performed using Siemens ADVIA 1800 Clinical Chemistry Analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA), while insulin levels were measured with an electrochemiluminescence immunoassay using the automated analyzer Cobas e411 and the Elecsys Insulin Kit (Roche Diagnostics, Basel, Switzerland). All blood chemistry parameters were measured after overnight fasting. Insulin resistance was determined according to the initial formula of the homeostasis model assessment (HOMA) [20]. Serum RBP4 was measured using a sandwich ELISA (Immunodiagnostik AG, Behsheim, Germany), identical in protocol and reagent composition with the ELISA kit from ALPCO Diagnostics (USA) tested by Graham et al. [10]. Plasma samples were diluted so that the absorbance was in the middle of the range of linearity for the assay. The intra- and inter-assay coefficients of variation (CV)s of RBP4 were 5.0 and 9.7%, respectively. Serum lipocalin-2 levels were determined by a solid phase ELISA technique (R&D Systems, Minneapolis, MN, USA). The intra-assay and inter-assay CVs ranged between 3.1 and 4.1% and between 5.6 and 7.9%, respectively. Serum levels of a-FABP were determined using a commercial human enzyme immunoassay kit (BioVendor Laboratory medicine, Inc., Modrice, Czech Republic). The assay was conducted according to the manufacturer’s instructions. The antibodies in human a-FABP ELISA are highly specific for human a-FABP, otherwise called FABR4, with no detectable cross-reactivity to other members of human FABP family, such as liver-FABP (FABP1), intestinal-FABP (FABP2), heart-FABP (FABP3) or epidermal-FABP (FABP5), and also to leptin and adiponectin. The intra- and interassay coefficients of variation (CVs) were less than 5.5% and the sensitivity limit was 0.1 μg/L. Serum YKL-40 levels were measured in duplicate by a commercial two-site sandwich-type enzymelinked immunosorbent assay (Quidel Corporation, San Diego, CA, USA), assessed daily for precision; coefficients of variations were 4–6%.
Values are expressed as mean ⫾ SD, as indicated. Relations between the parameters were investigated both by calculation of Pearson’s and/or Spearman’s correlation coefficients. Statistical significance was set at p ⬍ 0.05. The p values reported are two-tailed. All statistical procedures, as well as Box-Plot values presentations, were performed using the Statgraphics Centurion for Windows program (Graphic Software System, Statpoint Technologies, Inc., Warrenton, Virginia, USA). We used the standardized skewness and standardized kurtosis, to determine whether the sample has a normal distribution. Values of these statistics outside the range of ⫺2 to ⫹2 indicate significant departures from normality, which would tend to invalidate many of the statistical procedures normally applied to this data. These values integrated automatically by the program indicated the parameters needed to transform in either log, reciprocal or square root, as needed.
Results Viral load and CD4 counts At study entry the mean (range) CD4 cell count and viral load (by reverse-transcriptase-PCR, Amplicor Roche, Alameda, USA) were 876.3 (145.4–1858) cells/mL and 1.05 ⫻ 103 (⬍ 50 ⫺ 1.3 ⫻ 105) vc/ml, respectively. At the end of the follow-up period, mean CD4 counts and viral load were 953.9 cells/ mL and 1.01 ⫻ 103 vc/ml, respectively (p-value ⬎ 0.06 for both CD4 and viral load). Body composition measurements and metabolic abnormalities Body composition changes detected by DEXA in HIV-infected children were previously reported [7]. Although, no significant differences were observed in mean trunk fat (4.4 ⫾ 3.0 vs. 3.8 ⫾ 1.9 kg, p ⬎ 0.201), leg fat (4.0 ⫾ 2.3 vs. 4.5 ⫾ 1.9 kg, p ⬎ 0.108), leg lean (9.8 ⫾ 4.4 vs. 9.8 ⫾ 3.5 kg, p ⬎ 0.910) in HIVinfected children, when compared with expected values derived from controls, fat distribution expressed as trunk/leg fat ratio was significantly higher in patients, indicating central fat accumulation (1.03 ⫾ 0.3 vs. 0.81 ⫾ 0.09, p ⫽ 0.004) [7]. As reported however, there was no difference at study entry and after 12 months for fasting glucose, fasting insulin, and HOMA index between subjects with lipohypertrophy or lipoatrophy [7]. Non-traditional cardiometabolic risk markers in HIV-infected children on HAART The non-traditional adipokines RBP4, NGAL, aFABP and YKL-40 were assessed in the HIV-infected
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Table II. Adipocytokines levels in controls and patients with HIV at baseline and after 1 year of further observation under HAART.
RBP-4 (mg/L) NGAL (μg/L) a-FABP (μg/L) YKL-40 (μg/L)
Controls
Patients baseline
Patients 1 y HAART
22.0⫾0.8 (13.8–31.2) 67.5⫾5.5 (33.5–136.1) 39.5⫾3.9 (18.6–74.5) 38.3⫾2.7 (17.6–57.9)
21.1⫾1.2 (10.0–28.0) 18.8⫾4.6a (4.4–81.7) 19.0⫾3.0a (5.4–54.8) 48.0⫾4.5 (19.4–87.7)
21.3⫾1.6 (13.4–37.4) 68.2⫾22.3b (6.1–377.9) 16.8⫾2.9c (4.0–49.5) 59.7⫾6.9d (18.5–110.9)
Patients at baseline vs. Controls ap ⬍ 0.001 and patients 1 y HAART vs, Patients at baseline bp ⬍ 0.001. Patients 1 y HAART vs. Controls cp ⬍ 0.001. Patients 1 y HAART vs. Controls dp ⫽ 0.003.
children at study entry and after a further 12 months on HAART and were compared to controls’ data that have been collected at study entry (Table II). RBP4 levels were similar at baseline and after further 12 months on HAART in HIV-infected children (20.8 ⫾ 5.1 vs. 21.3 ⫾ 6.3 mg/L, p ⬎ 0.75) and were similar to controls (22.0 ⫾ 3.9 mg/L, p ⬎ 0.53). NGAL levels at study entry were significantly lower in HIV-infected children than controls (18.8 ⫾ 18.5 vs. 67.5 ⫾ 25.7 μg/L, p ⬍ 0.001). After further 12 months on HAART, NGAL levels normalized in HIV-infected children and were not different from controls (68.1 ⫾ 89.1 vs. 67.5 ⫾ 25.7 μg/L, p ⬎ 0.85). a-FABP levels were similar at baseline and after 12 months in HIV-infected children (18.9 ⫾ 12.2 vs.16.7 ⫾ 11.5 μg/L, p ⬎ 0.60), but were significantly lower than controls at both time points (39.5 ⫾ 18.2 μg/L p ⫽ 0.001) (Figure 1). YKL-40 levels in HIV-infected children at study entry were not significantly higher than controls (48.0 ⫾ 18.1 vs. 38.2 ⫾ 12.5 μg/L, p ⬎ 0.06). After a further 12 months on HAART, YKL-40 levels were not significantly different to those at study entry in HIV-infected
Figure 1. Concentration of a-FABP in HIV-infected children at study entry and after 12 months in comparison to controls. Boxes represent the interquartile range; lines inside boxes represent the median value; cross represents mean marker; whiskers represent the lowest and highest observations, respectively.
children but significantly higher than in controls (59.6 ⫾ 27.6 vs. 38.2 ⫾ 12.5 μg/L p ⫽ 0.003). We investigated whether serum concentrations of RBP4, NGAL, a-FABP and YKL-40 could have any correlation to body fat content or insulin resistance markers in HIV- infected children. We found no correlation between percentage of body fat content and specifically trunk fat accumulation and RBP4, neither at the initial evaluation (p ⬎ 0.056) nor after further 12 months (p ⬎ 0.267). Moreover, there was no correlation of RBP4 values and HOMA, neither at study entry (p ⬎ 0.3) nor after 12 months of follow-up (p ⬎ 0.05). NGAL concentration was negatively correlated with trunk fat accumulation both at the initial evaluation (p ⬍ 0.04), as well as after further 12 months on HAART (p ⫽ 0.0001). Furthermore, NGAL correlated negatively with both fasting insulin (p ⬍ 0.0001) and HOMA (p ⬍ 0.0001) at baseline and after a further 12-month observational period on HAART (p-value for insulin at 12 months of follow-up: 0.025 and p ⬍ 0.0001 for HOMA). We found a significant positive correlation between a-FABP and both total body fat content and trunk fat accumulation at the initial evaluation of our HIV-infected children on HAART (p ⫽ 0.0001 for both correlations), that persisted over the 12-month follow-up period (p ⫽ 0.0009 for total body fat, p ⫽ 0.0012 for trunk fat accumulation) (Figure 2). At the initial evaluation, a-FABP correlated neither with fasting insulin (p ⬎ 0.2578), nor with HOMA (p ⬎ 0.36). However, after a further 12 months on HAART, a-FABP correlated both to fasting insulin (p ⫽ 0.0036) and to HOMA (p ⫽ 0.0031). YKL-40 had a borderline positive correlation to trunk fat content at the initial evaluation (p ⫽ 0.05). However, this correlation did not persist after a further 12-month period (p ⫽ 0.91).
Figure 2. Correlation of % trunk fat and a-FABP levels in HIV infected children: baseline black dots, r ⫽ 0.847, p ⫽ 0.0009 and after 1 year empty dots, r ⫽ 0.723, p ⫽ 0.0012.
New adipokines in pediatric HIV Discussion In this study the circulating concentrations of RBP4, NGAL, a-FABP and YKL-40 in HIV-infected children under HAART were assessed and correlated to body fat accumulation, more concretely to trunk fat accumulation, and insulin resistance markers. Serum RBP4 concentrations were similar between pediatric HIV patients at study entry and after 12 months of follow-up and did not differ from controls. There was no correlation of RBP4 values neither with body fat content nor with insulin resistance markers. In agreement with our findings several recent reports have also demonstrated lack of a causal association between insulin resistance and RBP4 levels, questioning the role of RBP4 in unraveling insulin resistance [13,21]. NGAL levels at study entry were significantly lower in HIV-infected children than controls. After further 12 months on HAART, NGAL levels normalized in HIV-infected children and were no longer different from controls. Correlation analysis revealed that NGAL concentrations correlated negatively with trunk fat accumulation at both time points of the study. Furthermore, NGAL correlated negatively with both fasting insulin and HOMA at the initial evaluation and after further 12-months on HAART. In adult HIV-infected patients, NGAL levels were significantly reduced before treatment initiation [22]. During HAART, a pronounced fall in viral load was associated with significant increase in CD4⫹T cell counts accompanied by a gradual and significant increase in NGAL concentrations, reaching levels comparable to those of healthy controls after 12 months of treatment. A further 12-month period on HAART achieved a normalization of NGAL levels in our pediatric cohort. To the best of our knowledge, there are no other pediatric reports investigating NGAL concentrations in HIV-infected children in relation to the time course of normalization of CD4⫹T cell counts and viral load. These data suggest that NGAL concentrations in HIV-infected patients under HAART may be influenced by several factors, such as viral load or immunological status, and do not necessarily reflect an insulin resistance state. In the current study, a-FABP levels were similar at baseline and after 12 months of further treatment in HIV-infected children and were significantly lower than controls at both time points. Correlation analysis depicted a significant positive correlation between a-FABP and body fat content at the initial evaluation of our HIV-infected children on HAART, which persisted over a further 12-month follow-up period. More specifically, there was a positive correlation of a-FABP concentration to percentage of trunk fat at both time points, suggesting that a-FABP concentrations are mainly related to central fat accumulation. At the initial evaluation, a-FABP correlated neither
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to fasting insulin nor to HOMA. However, after the 12-month follow-up period, a-FABP significantly correlated both to fasting insulin and to HOMA. These findings suggest that among the adipokines studied, a-FABP seems to have the most important role in unraveling central fat accumulation especially after prolonged HAART. The positive correlation of a-FABP concentration to insulin resistance markers only after the 12-month observational period, although the children have already been under HAART for a period of 5.2 years necessitates confirmation by large scale prospective studies and may probably suggest that even a longer period may be necessary to unravel a correlation to insulin resistance markers in HIV-infected children on HAART. To the best of our knowledge, there are no other studies investigating a-FABP levels in pediatric HIV-infected populations. In HIV-infected adult patients, a-FABP levels were similar to those of healthy controls. However, among HIV-infected adults, a-FABP levels were significantly higher in those presenting with lipodystrophy and metabolic syndrome than in those without. In agreement with our results, in adult HIV patients a-FABP concentrations correlated positively with HOMA and fasting plasma insulin [16,17]. YKL-40 levels in HIV-infected children at study entry were not significantly different from controls. After a further 12 months on HAART, YKL-40 levels were similar to those at study entry in HIVinfected children but significantly higher than in controls. YKL40 correlated positively to body fat content at the initial evaluation, however, this correlation was lost after a further 12-month period on HAART. There is only one study investigating post mortem YKL-40 levels in the cerebrospinal fluid of patients with AIDS who developed encephalitis and no data exist on the role of YKL-40 in HIVrelated lipodystrophy [23]. One limitation of our study was the lack of DEXA-based body composition measurements in the same cohort of age-matched healthy controls, who provided the comparative data of the cardiometabolic risk markers. However, our previous study investigating the HAART-associated body composition abnormalities in this cohort of HIVinfected children using DEXA-measurements in comparison to a large group of healthy, age-matched controls, revealed a high percentage of lipodystrophic abnormalities in HIV-infected children allowing the assumption that such differences are expected in this group of healthy age-matched controls as well [7]. Another limitation of our study was the relative small number of HIV-infected participants. However, at the pediatric age range, in non-endemic areas of HIV, the number of HIV-infected children is fortunately limited. Our HIV-infected children have been extensively studied regarding viral load,
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biochemical profile, BMI, DEXA-based body composition and non-traditional adipokines, providing the possibility for correlation analysis. We cannot exclude therefore that other significant differences in adipokines between HIV-infected children and healthy controls have been missed because of the small number of subjects included and the inadequate power of the analysis. In conclusion, we previously demonstrated that a high percentage of HIV-infected children on HAART presented lipodystrophic abnormalities, even in the absence of clear-cut insulin resistance. In the current study, for the first time in HIVinfected children we have demonstrated that a novel adipokine, a-FABP, correlates positively with body fat content and more specifically with trunk fat accumulation in HIV-infected children on HAART and might be integrated in the assessment of HIV-related lipodystrophy in pediatric HIV. Largescale prospective studies investigating the concentration of these adipokines in HIV infected children both before HAART initiation and after prolonged HAART would better unravel their role as markers of the HIV-related lipodystrophic state. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Funding was received from Athens University to Dr Ioannis Papassotiriou. The funding source played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication 70/3/9294.
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