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The gap between overweight and obesity status in children - (STROBE-compliant article)

Abstract Overweight might represent only the early stage of obesity or it might act as a trigger of self-awareness turning into an ideal chance for preventing further obesity development. The aim of this study was to assess the differences between overweight and obese children in terms of anthropometric, low-grade systemic inflammation, liver impairment and atherosclerotic risk. We performed a study on 132 children aged between 5 and 18 years, divided according to the BMI into 2 groups: group 1 to 76 obese children, and group 2 to 56 overweight children, assessing anthropometric, laboratory and elastography parameters. We obtained significantly higher values of anthropometric parameters in obese children versus overweight ones. We found higher levels of leukocytes, lymphocytes, AST, ALT, and E median (P = .0345, P = .0103, P < .0001, P = .0008 and P < .0001) in the obese group as compared to the overweight one. BMI was positively correlated with neutrophils, NLR, ESR, glycemia, anthropometric parameters, and E median (P = .0007/<.0001/.0018/.0044/<.0001/<.0001/<.0001/<.0001/<.0001/.0204); and negatively with lymphocytes and HDL-cholesterol (r = −0.2747/−0.2181, P = .0116/.0120). Our study underlined significant differences between overweight and obese children in terms of inflammatory status and liver impairment suggesting that the risk is directly related to the increase in BMI.


Introduction
Overweight and obesity, 2 major current public health problems worldwide are defined as excessive or abnormal fat accumulation with negative impact on human's wellbeing. According to the reports of the World Health Organization, obesity has almost tripled since 1975 with more than 1.9 billion adults diagnosed with obesity, of which over 650 million suffering from overweight. [1] The incidence of these 2 conditions increased considerably in pediatric patients affecting 40 million children below 5 years of age in 2018. [1] This report is truly alarming taking into account the fact that childhood obesity persists into adulthood. It is a well-documented fact that obesity is a preventable condition and overweight might play a dichotomous role in the development of obesity. Thus, overweight might represent only the early stage of obesity or it might act as a trigger of self-awareness turning into an ideal chance for preventing further obesity development. It is also true that obesity development is determined by both genetic and environmental or "obesogenic" factors, [2] which are interrelated and the latter ones can be modified.
The burden of obesity is expressed by the wide-range of complications affecting both the quality and life length, such as cardiovascular, hepatic or metabolic ones. [3] Part of these complications were hypothesized to be a result of the wellaccepted low-grade inflammatory status associated to obesity, which was also proved in pediatric patients. [4] The hypothesis that adipose tissues serves as both the trigger and contributor to systemic inflammation might be a reliable explanation for the relationship between specific organ and systemic inflammation in obesity through activation of immune cells and the inflammatory processes expressed by immune cells within different specific tissues. [3] Thus, it seems that a malfunction in terms of immune activity at the level of adipose tissue involving a transient infiltration and binding of neutrophils to the adipocytes within the abdominal fat. [5] Multiple studies showed that otherwise healthy patients with obesity express increased levels of different leukocyte subclasses and elevated inflammatory biomarkers, such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) proving the strong association between obesity and subclinical systemic inflammation independently of the age. [4,6] It was also proved that adipose tissue owns the capacity to secrete different substances required for particular biological functions among which leptin, adiponectin, resistin, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). [7] Moreover, preadipocytes were also proved to contribute to an increased cytokine production. [8] Therefore, the association between obesity and low-grade systemic inflammation with further lifethreatening implications is an incontestable fact.
As a result of increasing obesity incidence, non-alcoholic fatty liver disease (NAFLD) is currently considered the most frequent chronic hepatic condition, its incidence reaching up to 15% in developed countries. [9] In case of children with overweight, its prevalence varies between 2.6% to 9.6%, whereas in those with obesity it rises up to 44%. [10][11][12] NAFLD might have a benign course, but it can also result in non-alcoholic steatohepatitis with life-threatening complications such as liver fibrosis, cirrhosis, malignant conditions or organ failure in case of long-term obesity persistence. [13] Thus, the assessment of liver inflammation through laboratory parameters and elastography methods is essential in children with overweight and obesity for a proper management.
Atherosclerosis is a major cardiovascular risk factor whose onset was established to occur during childhood in case of children diagnosed with obesity. [14] Unfortunately, due to the lack of awareness, it usually remains undiagnosed in young ages, but it persists into adulthood. [14,15] Atherosclerotic plaques are a result of the association between increased levels of low-density lipoprotein cholesterol (LDL-chol) and low levels of high-density lipoprotein cholesterol (HDL-chol). [16] Moreover, dyslipidemia, defined as hypertriglyceridemia and low HDL-cholesterol levels might led to metabolic complications in case of obese patients. [17] Lipid profile parameters, such as total cholesterol (Chol), HDL-cholesterol, LDL-cholesterol and triglycerides must be included in the routine assessment of overweight and obese children in order to establish an early diagnosis of atherosclerosis.
The aim of this study was to assess the differences between overweight and obese children in terms of anthropometric parameters, low-grade systemic inflammatory status, liver impairment and atherosclerotic risk in order to highlight the potential opportunity provided by overweight to prevent the further development of obesity, and especially its long-term associated complications.

Study sample selection
We performed a prospective, cross-sectional study of 132 children aged between 5 and 18 years, admitted to a Pediatric Tertiary Hospital in Romania, from April 2017 to January 2020. Taking into account the inclusion criteria, our study sample was divided according to the body mass index (BMI) into 2 groups: group 1, comprising 76 children with obesity (BMI P ≥95 for children with obesity), and group 2, the overweight group, consisting of 56 children with BMI percentile (P ≥ 85 and <95). [18,19] Thus, the inclusion criteria comprised overweight/ obese children due to poor dietary habits, age between 5 and 18 years, with at least 1-year history of excessive weight, other-wise healthy, with no previous attempts to lose weight and who performed only routine physical activity during school and play time. The exclusion criteria were: age below 5 years, monogenic and secondary obesity, infectious pathologies, chronic diseases or other conditions with inflammatory status, children with obesityrelated complications, incomplete data, and children whose parents did not sign the informed consent form. Children were either referred to our clinic for nutritional status assessment by the general practitioner or endocrinology specialist, or they were brought for a routine medical consult upon their parents'/ caregivers' choice. All children were assessed on a one-day chart system as they had no complications and did not require longer hospitalization.
Initially, we performed a thorough anamnesis and clinical exam to rule out those that did not fulfill our inclusion and exclusion criteria. Afterwards, the laboratory parameters assessed in all subjects included: complete cellular blood count (CBC), ESR, liver inflammation (aspartate aminotransferase -AST and alanine aminotransferase -ALT), Cholesterol (Chol), HDL-chol, LDL-chol, triglycerides (TG), and glycemia. The neutrophils/lymphocytes (NLR) and platelets/lymphocytes ratios (PLR) were calculated from the CBC, by dividing the neutrophil count and platelet count, respectively, to the lymphocyte 1.
2.1.1. Anthropometric parameters. All anthropometric measurements were performed by a single trained person and included the following: weight (kg), height (cm), abdominal perimeter (at the mid-point between the lower rib margin and the upper iliac spina), bitrochanteric perimeter (between the 2 trochanters of the femur), medium upper arm circumference (MUAC, at the midpoint between the shoulder and elbow tips with the use of a tape measure calibrated in centimeters) and tricipital skin thickness (TST, on the posterior face of the arm, using a thickness caliper). Body weight was measured with a daily calibrated scale with a ± 10 g error. A daily calibrated pedometer was used for the assessment of height with a SD (0.1-cm error). The assessment of liver stiffness or liver elasticity (E) was performed by 2D-SWE method, with a Logiq S8 General Electrics Device (General Electric Healthcare, Wauwatosa, WI, USA), using a C1-6 convex probe. The software generated a region of interest, which was positioned at approximately 2 cm under the Glisson's capsule. In order to provide correct measurements, the color map had to be over 50% homogeneous. All children were examined after a fasting period of approximately 6 hours, without sedation. The measurements for each child lasted approximately 20 minutes, being performed by a highly experienced physician with over 10 years experience in pediatric ultrasound, and 4 years in elastography. The informed consent was signed by all parents/caregivers on behalf of their children prior to their inclusion in the study. Each child was explained all the study steps according to the agerelated level of understanding, and we obtained their assent before the inclusion in the study. The approval of the Ethics Committee of the "G.E. Palade" University of Medicine, Pharmacy, Sciences and Technology of Târgu Mureş was granted for this study (No 329/November 17th, 2017), and the study strictly complied to the Helsinki Declaration principles.

Statistical analysis
The statistical analysis comprised elements of descriptive statistics such as mean, median and standard deviation, but also inferential statistics ones. The Shapiro-Wilk test was used for the determination of analyzed data series distribution. The Student t test (parametric test for unpaired data) and Mann-Whitney (non-parametric test for unpaired data) were used for the comparison of means and medians. The power of association between variables was measured by the Pearson correlation test. For multiple correlations we applied the Dunn's multiple comparisons test.
We chose a significance threshold of 0.05 for p value, and we used the utilitarian Graph Pad Prism trial variant for the statistical analysis.

Results
Our study pointed that children included in the obese group had a significantly lower mean age (10.39 ± 3.379 years) as compared to the overweight ones (12.98 ± 3.090 years) (P < .0001), without any difference between the gender (P = .0261) ( Table 1). Concerning the children's area of residence, we found no difference between the 2 groups in terms of rural or urban area (P = .6536). Children with obesity were found to have similar birth weight, 3.301 ± 0.5347 kg, to the overweight ones, 3.351 ± 1.299 kg (P = .4122). The current weight was higher in the obese group (60.59 ± 22.93 kg) in comparison to the overweight 1 (57.12 ± 14.42 kg), but without statistical significance (P = .5965), while the height was significantly higher in the overweight group (155.9 ± 14.95 cm) vs the obese 1 (146.3 ± 19.17 cm), (P = .0021). Analyzing the weight of the parents, we observed Table 1 The descriptive analysis of the demographic and laboratory parameters in the obese group vs overweight group.

Parameters
Obese group (n = 76) Mean ± SD (Median) Overweight group (n = 56) Mean ± SD (Median) P value  that the weight of both parents was significantly higher in case of children with obesity vs overweight ones (P = .0040, and P = .0090, respectively). In terms of anthropometric parameters, we noticed significantly higher values of BMI percentile (P < .0001), BMI z score (P < .0001), MUAC (P = .0008), TST (P = .0317), abdominal perimeter (P < .0001) and bitrochanteric perimeter (P = .0103) in children with obesity in comparison to those with overweight. Regarding the assessed laboratory parameters, our findings revealed significantly increased levels of leukocytes, lymphocytes, AST and ALT in the obese group as compared to the overweight 1 (P = .0345, P = .0103, P < .0001, and P = .0008). Assessing the liver stiffness, we noticed that children with obesity presented significant higher values of E median by 2D-SWE (4.25 ± 0.38 kPa) as compared to the overweight ones (3.60 ± 0.412 kPa) (P < .0001).
The values of all parameters mentioned above are described in Table 1.

The impact of pubertal stages on the assessed parameters
In terms of pubertal status (different in girls and boys as following: for girls prepubertal 5 to 10 years, pubertal 11 to 14 years and postpubertal 15 to 18 years, and for boys: prepubertal 5 to 11 years, pubertal 12 to 16 years and postpubertal 17 to 18 years), we observed according to the Kruskal-Wallis test a significant statistical difference for BMI (P < .0001) between the3 groups as following: 23.63 ± 3.79 kg/m 2 for prepubertal status, 26.66 ± 4.59 kg/m 2 for pubertal status and 27.55 ± 3.39 kg/m 2 for postpubertal status. Applying the Dunn's multiple comparisons test, we observed a significant difference for BMI between prepubertal and pubertal status (P < .01), as well as between prepubertal and postpubertal status (P < .001), and between prepubertal and postpubertal status (P < .01).
Taking into account these 3 main age groups, we assessed the paraclinical, anthropometric and elastography parameters in obese versus overweight children.

Discussions
The reports of the WHO showed during the last decades that obesity and overweight present an increasing incidence in children independently of the age and socioeconomical level. [1] Moreover, it was proved that their prevalence tends to be higher in smaller ages. [1] Our findings are alarming proving that the obese children included in our study are significantly younger as compared to the overweight ones. Table 3 The demographic analysis of the two groups according to the prepubertal period.

Prepubertal period (n = 58) Parameters
Obese group (n = 45) Mean ± SD (Median) Overweight group (n = 13) Mean ± SD (Median) P value  Adipose tissue has been proved to be not only a source of inflammation, but also a target of inflammatory processes. [5] Thus, adipose tissue owns both a synthesis function and a secretion 1, being hypothesized that the secreted products act as chemoattractants and activators of different immune cells, such as monocytes or polymorphonuclear cells. [20] It is well-accepted now that the peripheral blood of otherwise healthy overweight or obese individuals express a subclinical inflammatory status. [6] A previous study of our team showed significantly higher levels of leukocytes, platelets and lymphocytes in overweight/obese children as compared to normal weight ones. [4] It was documented that lymphocyte count reflect better the nutritional body status and general stress, while the neutrophil one is rather related to the systemic inflammation in obese patients. [21] In addition, the degree of obesity seems to be better expressed by neutrophil count. [22] Except for the CBC parameters, childhood overweight and obesity was also associated with higher levels of certain adipokines, such as leptin, IL 6 and TNF alpha, which further sustain the early-stage inflammatory status associated to obesity in pediatric patients. [23] NLR was defined as an immune response marker related to chronic inflammation associated to different pathologies, such as cardiovascular, autoimmune or infectious ones, but also complicated obesity. [21,24,25] Older studies suggested that patients with uncomplicated obesity represent a particular subgroup expressing an early stage of systemic inflammation similar to the overweight ones. [26] Contrariwise, our study found significant differences in terms of leukocytes and lymphocytes between obese and overweight children, with significantly higher levels in those with obesity. Taking into account that white blood cells were proven to be related with the development of metabolic syndrome, [27] our study might suggest that this risk increases proportionally with the accumulation of fat tissue. Moreover, a direct correlation was underlined between the increase in BMI and leukocyte, neutrophil, lymphocyte and platelet counts. [28] Our findings sustain the previously mentioned ones since we also found a positive correlation between BMI and neutrophils, NLR and ESR. Thus, we may definitely confirm the strong relationship between neutrophils and obesity degree. Moreover, we might emphasize that systemic inflammation associated to excessive weight gain is directly related to the amount of fat tissue and it definitely worsens once the BMI overpasses the limit of overweight. Taking into account the previous findings of our team in terms of childhood obesity, early inflammatory status related to pediatric obesity is an alarming proven risk factor for both short-and long-term life-threatening complications and its effective control is possible only in the context of increased awareness. Contrariwise, we noticed a significant negative correlation between BMI and lymphocytes suggesting indeed that lymphocyte count might be a better indicator of nutritional status and general stress rather than obesity severity. Despite the lack of significant difference between overweight and obese children in terms of NLR, we found a strong positive dependence between this marker and the increase in BMI suggesting that chronic inflammation is most-likely to be present in the context of obesity and not overweight. This statement is further sustained by Table 4 The demographic analysis of the two groups according to the pubertal period.

Pubertal period (n = 55) Parameters
Obese group (n = 26) Mean ± SD (Median) Overweight group (n = 29) Mean ± SD (Median) P value  the negative significant correlation between BMI and HDL suggesting that in case of proper interventions once BMI decreases, the children will no longer be exposed to this cardiovascular risk factor and in exchange they might even be protected due to a subsequent increase in HDL. Anthropometric parameters are important indicators of fat accumulation and they have been positively correlated with weight even from the time of birth. [29] Their importance in assessing excessive weight gain was proved in different population, among which pregnant women with excessive gestational weight gain, [29] but also children with obesity. [30] Additionally, waist circumference was shown to be positively correlated with CBC parameters, among which leukocytes, lymphocytes, neutrophils, platelets and medium platelet volume, [31] suggesting a potential relationship between anthropometric parameters and obesity associated inflammatory status. Similarly, our study underlined significant differences between overweight and obese children regarding all anthropometric parameters underlining a positive correlation between these parameters and BMI.
Liver steatosis or fibrosis due to excessive weight gain is a welldefined condition since non-alcoholic fatty liver disease (NAFLD) is the most common chronic hepatopathy nowadays. [10] Despite the fact that it was shown to be associated to both overweight and obesity in children, it may be present in almost 50% of children with obesity. [11] In terms of laboratory parameters, the most constant finding that suggest liver inflammation due to obesity consists in increased levels of liver transaminases. [4] Nevertheless, these levels were found to fluctuate over time and they may be even normal in children with obesity and NAFLD or non-alcoholic steato-hepatitis (NASH) [32] suggesting that their assessment alone is not enough for clearly establishing the presence of liver inflammation and/or steatosis in patients with this nutritional status disorder. [33] Moreover, the study of Cho et al underlined that only ALT might be considered a reliable parameter of liver fibrosis assessment since it was significantly correlated with liver stiffness values measured on transient elastography. [34] Despite this fact, liver transaminases along with total direct bilirubin, fasting glucose, insulin, and lipid profile parameters remain extremely useful in patients with metabolic syndrome phenotype for diagnosing fatty liver disease. [32] The association between increased levels of TG and low HDL-chol values results in insulin resistance, [14] suggesting a clear association between obesity, dyslipidemia, liver fibrosis and metabolic syndrome. Similarly, our study revealed significantly higher levels of both liver transaminases, AST and ALT in children with obesity as compared to those with overweight. Moreover, we noticed a significant positive correlation between BMI and fasting blood glucose implying that the risk of developing insulin resistance and diabetes mellitus is directly related to the degree of obesity. As for lipid profile parameters, we found a negative correlation between BMI and HDL-chol levels indicating that atherosclerotic process might indeed occur during childhood in case of obese children sustaining the findings of Williams et al. [14] Taking into account that both increased levels of LDL-chol and low levels of HDL-chol contribute to the formation of atherosclerotic plaques, [16] our findings might hypothesize that the decrease of HDL-chol could be an early indicator of this process during childhood. Nevertheless, Table 5 The demographic analysis of the two groups according to the postpubertal period.

Postpubertal period (n = 19) Parameters
Obese group (n = 5) Mean ± SD (Median) Overweight group (n = 14) Mean ± SD (Median) P value  www.md-journal.com Table 6 Correlation between BMI and paraclinical and anthropometric parameters. according to the pubertal periods, our findings revealed a significant positive association between BMI and both AST and ALT during the pubertal period, and only between BMI and ALT during both prepubertal and pubertal periods, suggesting that these periods play an important role in the occurrence of obesity associated complications, such as liver inflammation or steatosis.

BMI
In terms of lipid profile parameters and glycemia, our study revealed a significant positive correlation between BMI and glycemia, as well as TG only in children of prepubertal age. Moreover, according to the same age division, the significant negative correlation between BMI and HDL-chol was found also during the prepubertal period suggesting that obesity associated risks might have an earlier onset than expected. Despite their clear utility in assessing hepatic function, laboratory parameters are not enough for providing an accurate quantification of liver fibrosis, and they should be correlated with elastography parameters for a better assessment of fibrosis degree. [35] Elastography is an essential non-invasive method in assessing the evolution and prognosis of chronic liver conditions in children. [36] Nevertheless, concerns were raised regarding certain limitation of 2D-SWE method in terms of discrimination between low-grade fibrosis and normal liver tissue. [37] Moreover, it was also hypothesized that liver inflammation, necrosis, fatty infiltration or edema might represent possible confounders for liver stiffness measurements. [38] Thus, studies performed on adult patients underlined that liver inflammation results in higher stiffness values than those expected as a consequence of fibrosis alone. [39,40] A recent study performed on pediatric patients that aimed the same aspect concluded that liver stiffness values on transient elastography should be cautiously interpreted as an indicator of hepatic fibrosis in the setting of increased ALT. [38] On the contrary, the present study proved a significant positive correlation between BMI and elastography parameters assessed by 2D-SWE, underlining significant higher values of liver stiffness in children with obesity in comparison to those with overweight. Furthermore, this correlation persisted even after the age division of our sample during all 3 periods: prepubertal, pubertal and postpubertal, while the same positive significant correlation between BMI and ALT was noticed only during the prepubertal and pubertal ones.
Another fact worth mentioning is that multiple studies revealed important changes in liver function and morphology depending on maturation status alone underlining that liver tissue suffers important modifications in time such as increase in liver volume, asymmetric or an increase in connective tissue formation resulting in higher stiffness and viscosity properties. [41][42][43] The present study revealed higher values of E median in obese children as compared with overweight ones independently of the group age according to the pubertal status. Moreover, we found a positive significant correlation between BMI and elastography parameters independently of the pubertal period underlining that most-likely the changes in elastography parameters are due to excessive weight and not related to the child's age under pathological circumstances. Based on the above-mentioned findings, we feel entitled to underline the strong relationship between excessive fat accumulation and liver impairment even in children. Moreover, since both ALT and elastography parameters depend in a direct manner on BMI, overweight could be considered once more a real balance between self-awareness as a subsequent trigger for implementing weight-loss strategies, and the next stage of obesity. Thus, our findings might ruin the myth of overweight defined only as a stage for imminent obesity development.
This study has some limitations that are worth mentioning among which the relatively small sample size; the fact that we did not assess children below the age of 5 years taking into account the lack of compliance in small children that could have impaired the elastography assessment resulting in false measurements; the lack of liver biopsy for a more accurate diagnosis of liver fibrosis, but its indications are very limited in children due to the potential risks related to its invasiveness, presenting no indication in children with uncomplicated obesity, and also the fact that we were not able to assess precisely the dietary habits and diet quality in order to achieve a better perspective regarding their impact on the assessed parameters. Nevertheless, our study has multiple strengths: the complex assessment of overweight and obese children in terms of inflammatory status, liver impairment, lipid profile implying the assessment of multiple anthropometric, laboratory and elastography parameters; the pediatric age of the subjects; and the random selection of the children included in the study. Moreover, our findings were used for defining a national diagnostic protocol for obese children in order to achieve a better assessment of these patients regarding both short-and long-term complications, representing also a solid basis for proper and efficacious interventions meant to decrease the alarming incidence of this pathology in pediatric patients.
To the best of our knowledge, this is the first study that aimed to assess the differences between obese and overweight children indicating that overweight is an intermediary stage between normal weight and obesity, representing indeed an ideal opportunity for the prevention of further obesity development and its related complication since bad prognosis indicators like inflammatory and lipid profile markers, as well as liver impairment are strongly related to the increase in BMI. 2D-SWE = 2D-Shear Wave Elastography, ALT = alanine aminotransferase, AST = aspartate aminotransferase, BMI = body mass index, E = elasticity, ESR = erythrocyte sedimentation rate, HDL = high density lipoprotein, kPa = Kilo Pascal, LDL = low density lipoprotein, MUAC = medium upper arm circumference, n = number, NLR = neutrophils/lymphocytes rate, PLR = platelets/lymphocytes rate, TG = Triglycerides, TST = tricipital skin thickness.

Conclusions
Our study found significant differences in terms of inflammatory status markers expressed by significantly higher levels of leukocytes and lymphocytes in children with obesity in comparison to the overweight ones, and significant positive correlation between BMI and neutrophils, ESR and NLR. Moreover, liver steatosis due to obesity was proved by significantly higher values of liver transaminases, AST and ALT, and liver stiffness in the obese group, as well as a positive correlation between BMI and elastography parameters. Regarding lipid profile, we noticed a significant negative correlation between HDL-chol and BMI. All these findings underline that bad prognosis indicators are directly related to the increase in BMI emphasizing the real gap between overweight and obesity status in children. Nevertheless, further studies on bigger cohorts are required in order to benefit from a more complex approach of the differences between overweight and obesity in children.