Serum Adiponectin, Body Adiposity and Metabolic Prole in Relation to Obesity among Egyptian Children with Down Syndrome

Children with Down syndrome (DS) have a higher risk for obesity. Adiponectin plays a crucial role in obesity-related cardiometabolic comorbidities. We aimed to determine whether body adiposity indicators, metabolic syndrome (MetS) components, serum adiponectin and insulin resistance indicies are different in "obese-DS" children compared to BMI-Z-score-matched "obese-control" and to clarify the usefulness of serum adiponectin as a biomarker for MetS in DS children. Cross-sectional study included 150 prepubertal children classed into three groups; obese-DS (n = 50), obese-control (n = 50) and normal-weight-control (n = 50). Participants were evaluated for waist-circumference (WC), body adiposity, serum triglycerides, HDL-C, adiponectin and Homeostasis-Model-Assessment of Insulin-Resistance (HOMA-IR). MetS was dened using modied Adult Treatment Panel III-criteria. Obese-DS had signicantly higher WC, %body fat, total-fat mass, trunk-fat mass, trunk/appendicular-FM ratio, triglycerides, insulin and HOMA-IR and signicantly lower HDL-C values compared to obese-control. Higher prevalence of MetS and its components were observed in obese-DS that was evident at younger age. Serum adiponectin was signicantly lower in obese-DS compared with obese-control and in obese-DS children with MetS compared to obese-control with MetS. The decrease in adiponectin with increasing grades of obesity was pronounced in obese-DS. Adiponectin exhibited strong correlations with body adiposity, several MetS components and HOMA-IR in obese-DS. Adiponectin performed better as a biomarker for MetS among obese-DS (AUC = 0.808) than among obese-control (AUC = 0.674). In conclusion, prepubertal obese-DS displayed excess body adiposity with pronounced central fat distribution, atherogenic lipid prole and higher insulin resistance compared to matched obese-control. Adiponectin performed on and adiponectin and to the potential use of as a therapeutic target for MetS in our results reinforce early and to prevent and annual for in


Introduction
Down syndrome (DS) (OMIM #190685) is the most common human autosomal aneuploidy, with an incidence of approximately 1/1000 live births worldwide, resulting from the presence of a supernumerary copy of the human chromosome 21 (HSA21) [1].
Children with DS have an increased prevalence of overweight/obesity secondary to several interrelated physiological and behavioral factors [2]. They also have a high prevalence of obesity-related cardiometabolic comorbidities, add to that obesity may hinder the care of individuals with DS, and implicated in the poor quality of life [2,3].
The term "Metabolic Syndrome" (MetS) have been proposed to describe the clustering of cardiometabolic risk factors (CMRFs), including centeral adiposity, hypertension, dyslipidemia and/or glycemic dysregulation including impaired fasting blood glucose (FBG) or type 2 diabetes mellitus. MetS is a convient clinical tool for identi cation of individuals at risk of developing obesity-related cardiometabolic adverse outcomes [4].
The pathophysiology of MetS is complex and not completely clari ed. In this context, adipose tissue has been described as an endocrine gland producing an imbalance between pro-and anti-in ammatory cytokines that elicits a state of low-grade systemic chronic in ammation that is closely correlate with insulin resistance [5,6].
Adiponectin is one of the most studied adipocytokines that is secreted mainly by differentiated adipocytes and regulates metabolic homeostasis particularly improving insulin sensitivity. In addition, it has anti-atherogenic, anti-in ammatory, anti-apoptotic and cardioprotective characteristics [7,8].
Paradoxically, in several pediatric studies, adiponectin levels were lower in obese children compared with normal-weight children, and even lower in obese children with MetS compared to those without [9,10].
However, data concerning serum adiponectin level in DS children with obesity are limited. Therefore, we aimed to explore for the rst time whether body adiposity indicators, the frequency of MetS and its components, serum adiponectin and insulin resistance indicies as well as the validity of serum adiponectin as a biomarker for identi cation of MetS are different in prepubertal "obese-DS" children compared to age-, sex-and BMI-Z-score-matched "obese-control" children.

Methods
This is cross-sectional study conducted during the period between January 2019 and June 2020 and included 150 prepubertal children (6-11 years) classi ed into three groups; obese-DS (n = 50), obesecontrol (n = 50) and normal weight-control (n = 50). The study was approved by the local Ethics Committee of Mansoura Faculty of Medicine-Institutional Research Board (Code No. R.20.07.923).
Informed written consent was obtained from parents of all participants.
Obese children were recruited sequentially during their routine visits at Pediatric Endocrinology Outpatients Clinic at Mansoura University Children's Hospital, Mansoura, Egypt. All participants were of the same ethnicity (Caucasian). All DS cases were full trisomy 21 caused by chromosomal nondisjunction. Normal weight-control children were recruited from a primary school at the same locality. Overall, participants belong to middle-class families and none reported participation in organized physical activity. All participants have normal thyroid pro le. Exclusion criteria include; children had syndromic or secondary obesity, those suffering from chronic comorbidities, or receiving medication known to affect the study variables.

Clinical evaluation
A trained nurse measured weight and height of participants according to standardized techniques. Body mass index (BMI) was calculated as weight divided by squared height (kg/m 2 ). Waist circumference (WC), an indicator for central (abdominal) obesity, was measured midway between costal margin and iliac crest at the end of normal expiration. For all participants, height and BMI Z-scores were calculated using reference data for Egyptian children and adolescents [11].
Given that the characteristic phenotype of individuals with DS as having lower stature, the use of absolute BMI values may lead to a bias in assessing the nutritional status in individuals with DS if compared to peers of same chronological age [12]. Therefore, in short-for-age obese-DS children whose height-for-age Z-scores <-2SD, BMI Z-scores were adjusted for height-age (i.e. the age at which the height of a child is at the 50th percentile on height-for-age growth curve) [13].
Systolic and diastolic blood pressure (SBP/DBP) measurements were obtained using conventional mercury sphygmomanometer following standard technique [16]. Pubertal development was determined in all participants using Tanner classi cations. For the purpose of the study, we speci cally included prepubertal children only to investigate the study variables independent of the impact of sex steroids.

Body composition evaluation:
Body composition measurements were obtained by bioimpedance technique using Tanita BC-418MA body composition analyzer (Tanita Corp., Tokyo, Japan). Body adiposity indicators include percentage of total body fat (%BF), total body-fat mass (FM; kg), trunk-FM (kg), a marker of central adiposity, and appendicular-FM (kg) as a sum of the FM in the four limbs that re ect peripheral fat distribution. Trunk FM to appendicular FM ratio was calculated.
Biochemical evaluation: Blood samples were collected in the morning, after a 12-hour overnight fast and centrifuged, then sera were stored at − 20°C until analysis. Fasting lipid pro le including; triglycerides was measured by colorometric kit supplied spinreact (Girona, Spain), and high-density lipoprotein cholesterol (HDL-C) was measured by colorometric kit supplied by Human Diagnostics (Wiesbaden, Germany). Serum adiponectin was determined ELISA using AviBion Human Adiponectin (Acrp30) commercial kits (Orgenium Laboratories Business Unit, Finland). Serum insulin was measured by quantitative sandwich ELISA technique using Human Insulin Kit (ab200011) (abcam, USA).
Insulin resistance was assessed by Homeostasis model assessment of insulin resistance (HOMA-IR) using this formula: HOMA-IR = FBG (mg/dL) x Fasting Insulin (µU/ml)/ 405. A HOMA-IR value greater than 2.5 was the cut-off point to determine insulin resistance in prepubertal children that corresponds to the 90th percentile of healthy children in previous studies [17]. A fasting serum insulin levels > 15 µU/ml has been suggested to be an alternative for evaluating insulin resistance [18]. De nition of metabolic syndrome: Obese children were classi ed as having Mets if they had at least three of the following ve components according to Adult Treatment Panel III (ATP III) criteria modi ed for the pediatric age group [19]; (1) central adiposity (WC ≥ 90th percentile for age and gender); (2) FBG ≥ 100 mg/dL (5.6 mmol/L); (3) triglycerides ≥ 150 mg/dL (1.7 mmol/L); (4) HDL-C ≤ 40 mg/dL (1.03 mmol/L); and (5) SBP and/or DBP ≥ 90th percentile for age, gender and height percentile.

Statistical analysis
All statistical analyses were performed using SPSS software package version 25.0. Categorical variables were presented as percentages and compared by Chi-square test. Continuous variables were presented as median and interquartile range (IQR) (1st quartile-3rd quartile). Mann-Whitney U test was used to compare medians of two groups. Kruskal-Wallis analysis used to compare medians of three groups followed by Dunn's test to detect pair-wise comparison. Spearman`s rank-order correlation was applied to determine the strength and direction of the linear relationship between adiponectin and CMRFs. The variables found to be signi cantly correlated in univariate regression analysis were introduced in multiple regression analysis using adiponectin as a dependent variable.
Receiver operator characteristic (ROC) curves were constructed to evaluate the validity of serum adiponectin as a biomarker for identi cation of MetS in DS children compared to matched obese-control.
The results were expressed as areas under the curves (AUC) and 95% con dence intervals (CI) and the optimal adiponectin cut-off values for the diagnosis of MetS with relevant speci city and sensitivity were considered at the maximum of Youden index (sensitivity + speci city − 1). The AUCs of obesity-groups were compared using Medcalc statistical software version 19.6.1. Statistical signi cance was set at P < 0.05.
As depicted in Fig. 1, Adiponectin levels were signi cantly lower in obese-DS compared to obese-control in moderate obesity (P < 0.001) and morbid obesity (P = 0.001) classes only. Within obese-DS, serum adiponectin was signi cantly lower in moderate obesity compared to severe obesity classes (P = 0.001) and in severe obesity compared to morbid obesity classes (P = 0.001), while within obese-control, serum adiponectin was signi cantly lower in moderate obesity compared to severe obesity classes (P = 0.001), but no signi cant difference was detected between severe obesity and morbid obesity classes (P = 0.178). Figure 2 demonstrates that the prevalence of MetS was signi cantly higher in obese-DS (56%) compared to obese-control (34%) (P = 0.016). Central adiposity (WC ≥ 90th percentile) was the most prevalent alteration in MetS components in both obesity-groups, that was signi cantly higher in obese-DS compared to obese-control (78% vs. 56%; P = 0.019). Moreover, the frequencies of the remaining altered MetS components were also higher in obese-DS compared to obese-control, but did not reach signi cant levels (P > 0.05). Children with "elevated SBP" were very few in both obesity-groups (16% of obese-DS and 10% of obese-control) and virtually no "elevated DBP" was detected in obesity-groups.
In the context of MetS, obese-DS children with MetS were signi cantly younger in age (P = 0.019) and exhibited signi cantly higher median values of WC (P = 0.037), FM (P = 0.033), trunk-FM (P = 0.004), trunk/appendicular FM ratio (P < 0.001), fasting insulin (P < 0.001), and HOMA-IR (P = 0.007) and exhibited signi cantly lower median adiponectin value (P = 0.010) than obese-control children with MetS ( Table 2).  Table 3 demonstrates the results of correlation analysis. In both obesity-groups, serum adiponectin was inversely correlated with age, BMI, WC, total-FM, trunk-FM, FBG, fasting serum insulin, HOMA-IR, and triglycerides. In obese-DS only, serum adiponectin was inversely correlated with trunk/appendicular FM ratio and was positively correlated with HDL-C. Multiple regression analysis of adiponectin in obesity-groups revealed that the signi cance was only Serum adiponectin seems to perform better for MetS identi cation in obese-DS than in obese-control, although the pairwise comparison of AUCs between the two groups did not reach a signi cant level (P = Page 13/20 0.171) (Fig. 3).

Discussion
Adiponectin has attracted tremendous scienti c interest as a candidate biomarker of MetS in childhood obesity [9,10,20,21], and as a therapeutic target for obesity [22], owing to pediatric studies reported that after weight reduction, adiponectin increases with associated improvement in insulin sensitivity [23,24].
It has been hypothesized that DS could be a "metabolic disease", in which trisomy of HSA21 has a key role in altered energy metabolism that is strongly related to dysregulation of insulin and insulin signaling pathways [25]. Moreover, all the body adiposity indicators such as BMI, WC, waist to height ratio, %BF, and FM were reported to be signi cantly higher in obese-DS children and adolescents compared to ageand sex-matched normal-weight controls [26][27][28].
In the current study, obese-DS children exhibited signi cantly excess body adiposity with profound central fat distribution as indicated by higher WC, trunk-FM, and trunk/appendicular FM ratio in obese-DS compared to age-and BMI Z-score-matched obese-controls which point to the higher risk for MetS in obese-DS.
Practically, the frequency of MetS was signi cantly higher in the obese-DS (56%) compared to obesecontrol (34%). Central adiposity was the most prevalent alteration in MetS components in both obesitygroups, that was signi cantly higher in obese-DS compared to obese-control (78% vs. 56%), while the frequencies of the remaining altered MetS components were also higher in obese-DS compared to obesecontrol, but did not reach signi cant levels. Interestingly, fasting serum insulin and HOMA-IR values as well as the frequencies of insulin resistance indices including hyperinsulinemia (fasting insulin ≥ 15 µU/ml) and HOMA-IR (> 2.5) were signi cantly higher in obese-DS compared to obese-control.
Considerable progress has been made in the clari cation of the central role of insulin resistance as a keyelement linking obesity and CMRFs clustering, and the potential mechanisms leading to insulin resistance development in obese children [29]. Previuos studies in obese adolescents have indicated that insulin resistance is related to a particular centeral fat distribution and ectopic fat accumulation [30,31].
In this context, obese-DS with MetS were signi cantly younger in age and exhibited pronounced central fat distribution and higher fasting serum insulin and HOMA-IR values than obese-control with MetS. Data regarding the circulating adiponectin level in DS children are scarce [26,32,33]. Tenneti et al. [32] reported a lower adiponectin levels in normal weight children with DS. Similar ndings were detected by Gutierrez-Hervas et al. [26] in adolescents with DS with variable degree of adiposity. Corsi et al. [33] found that adiponectin levels were signi cantly higher in three different age cohorts of normal-weight DS individuals including children (n = 23; age 2-14 years), adults (n = 14; age 20-50 years) and elders (n = 13; age > 60 years) compared to healthy controls. Moreover, they reported age-related increments in adiponectin levels among DS individuals, where elders with DS had signi cantly higher adiponectin levels compared to the younger and middle age counterparts.
Interestingly, individuals with DS represent a still unsolved biological/clinical paradox. Although an increment of classical biochemical risk factors for atherosclerosis should induce an elevated risk of atherosclerosis in normal-weight DS individuals, DS has been considered an atheroma-free model [33], with evident low risk of atherosclerosis-related morbidity and mortality in healthy subjects with DS during adulthood and senility [34]. Reasons of this discrepancy remain obscure. Corsi et al. [33] suggested that elevated adiponectin level in healthy adults and elders with DS may play a protective role against atherosclerosis by regulating endothelial activation.
This study showed for the rst time that serum adiponectin level was signi cantly lower in prepubertal obese-DS compared to obese-controls, despite being matched for age-and BMI Z-score. We also detected a decreasing trend in adiponectin concentrations with increasing the severity of obesity in both obesity groups that was more pronounced in obese-DS than in obese-control. Interestingly, obese-DS with MetS also exhibited signi cantly lower median adiponectin value than obese-control with MetS.
The observed high degree of discordance between obese-DS and matched obese-control in respect to body adiposity indicators, the frequency of MetS and its components, serum adiponectin and insulin resistance indices point to intrinsic idiosyncratic factors other than traditional risk factors may contribute to the increased risk for cardiometabolic disorders in subjects with DS that are closely related to trisomy of HSA21.
As to the association between serum adiponectin level and CMRFs, serum adiponectin was inversely correlated with age, BMI, WC, total-FM, trunk-FM, FBG, fasting serum insulin, HOMA-IR, and triglycerides and was positively correlated with HDL-C in obese-DS. These associations might related to the effect of adiponectin on metabolic homeostasis whereas adiponectin enhances fatty acids and triglycerides catabolism, promotes glucose uptake by skeletal muscle and increasing serum HDL-C level by its action on hepatic lipase activity [7,8].
Interestingly, central obesity was the predominant CMRFs for MetS and strongly correlated with low adiponectin levels, thus evident needs to motivate individuals with DS to participate and adherence to regular and consistent exercise programs to reduce abdominal obesity as prophylactic and as a treatment for MetS.
Previous studies conducted on non-DS children and adolescents with obesity reported variable and nonconsistent associations between adiponectin level and various CMRFs [35][36][37][38]. It has been presumed that the variation in genetic backgrounds in different ethnicity are major factors in determining the level of adiponectin expression and its association with various CMRFs [39,40].
For clinical practice, we explored the validity of serum adiponectin level for the diagnosis of MetS in obese-groups, the results of ROC curve analysis showed that adiponectin seems to perform better in the diagnosis of MetS in obese-DS (AUC = 0.808) than in obese-control (AUC = 674), with lower cut-off value in obese-DS compared to obese-control (4.8 µg/ml vs. 5.5 µg/ml, respectively). Our results were comparable to the reports from studies conducted on non-DS obese children and adolescents of different ethnicity. The optimal cut-off values of serum adiponectin for diagnosis of MetS among Japanease children was 6.65 µg/ml (AUC = 0.672) [41], while among Chinese children was 4.5 ug/ml for boys (AUC = 0.697) and was 5.2 ug/ml for girls (AUC = 0.689) [42], and among Italian children was 10.9 mg/dl (AUC = 0.72) [43].
The principal limitation of the current study is the cross-sectional design, which precludes us to identify the causal direction between adiponectin concentrations and CMRFs in DS children. However, as strength, our study is the rst to explore body adiposity, MetS components, insulin resistance indices, and serum adiponectin level among prepubertal obese-DS children in comparison to age-and BMI-matched obesecontrols. Our results collectively suggest that low adiponectin level seems to be an important mediator in the development of MetS in DS children as have been suggested in individuals without DS [44].

Conclusions
The current study highlights for the rst time that pre-pubertal obese-DS children displayed excess body adiposity with pronounced central fat distribution, atherogenic lipid pro le and higher insulin resistance as well as higher prevalence of MetS and its altered components compared to matched obese-control. Serum adiponectin level was signi cantly lower in obese-DS children than in obese-controls and was even lower in obese-DS with MetS compared to obese-control with MetS. Pronounced decrease in adiponectin values with increasing the severity of obesity were detected in obese-DS children.
Adiponectin exhibited strong correlations with body adiposity, several MetS components and HOMA-IR and performed better as a potential biomarker for identi cation of MetS among obese-DS children than in obese-controls.

Recommendations
Although adiponectin is not a measure routinely done and is an expensive assay that preclude its use in clinical settings at this time, our results pave the way for future prospective studies to explore the impact of different cytogenetic types, and pubertal stages on CMRFs and serum adiponectin and to evaluate the potential use of adiponectin as a therapeutic target for MetS in high-risk populations childhood obesity. In clinical practice, our results reinforce early counseling for DS children regarding healthy nutrition and active lifestyle to prevent early-onset adiposity and annual screening for CMRFs in youths with DS, particularly with current evident increase in their life expectancy.

Declarations
Funding source: The authors did not receive funding support from any agency or organization for the research, authorship, and/or publication of the submitted work.