Novel Kidney Injury Biomarker Detected Subclinical Renal Injury in Severely Obese Adolescents with Normal Kidney Function

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2 Novel Kidney Injury Biomarker Detected Subclinical Renal Injury in Severely Obese Adolescents with Normal Kidney Function A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science in Clinical & Translational Research Department of Environmental Health Division of Epidemiology & Biostatistics College of Medicine by Nianzhou Xiao April, 2014 Division of Nephrology and Hypertension, Cincinnati Children s Hospital Medical Center, Cincinnati, Ohio, USA M.A. Biochemistry, State University of New York - Stony Brook, May 2005 M.D., Xi an Jiaotong University, School of Medicine, July 2000 Committee Chair: Erin Haynes, Ph.D.

3 ABSTRACT Background: Obesity is an independent risk factor for kidney injury. It is associated with albuminuria, hyperfiltration and progression of chronic kidney disease (CKD). Obesity-related kidney damage may occur long before detectable microalbuminuria or obvious change in glomerular filtration rate. Due to limitation of the current kidney function markers, detection of early kidney injury has been delayed in obese children. One sensitive and specific urinary biomarker, neutrophil gelatinase-associated lipocalin (NGAL) has been identified during the last decade for early risk prediction of kidney injury. We hypothesize that urinary NGAL is elevated in severely obese youth, representing existence of consistent kidney injuries. Objective: To investigate subclinical kidney injury in severely obese adolescents, using novel urinary biomarker, NGAL. Design/ Methods: This was a pilot, prospective study of severely obese adolescents who had no microalbuminuria or decreased estimated glomerular filtrating rate (egfr) at baseline, and after surgical weight loss. Urinary NGAL were measured in the obese cohort at baseline, 6 and 12 months post-operatively and in a local general pediatric population. Regression analysis was conducted to confirm the association between elevated NGAL levels and obesity status. T test and ANOVA was performed for comparison of variables. Results: The severely obese cohort (n=18) had a mean baseline BMI of kg/m 2. The controls (n=161) were younger (median age 5.1 years vs years, p < ) with more males (52% vs. 28%) in comparison to the obese cohort. Urine NGAL was significantly elevated in obese children compared to controls at baseline. Multivariate analysis showed that only obesity status was significantly associated with elevated urine NGAL levels. At 1 year postoperative follow-up, BMI in the bariatric participates decreased by 32% (p<0.001). The obese cohort had a further increase in urine NGAL at 6 months, followed by decline at 1 year. The overall change in NGAL levels through 1 year after bariatric surgery, however, was not significant. Conclusions: Our preliminary data indicates that severe obesity is associated with increased urinary excretion of NGAL in adolescents with normal kidney function, representing clinically silent but persistent structural and inflammatory kidney injury. Our findings suggest that aggressive weight controls should be considered in severely obese adolescents from kidney protection standpoint even without obvious kidney dysfunction. In addition, close long-term follow up of kidney status is warranted in this population. III

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5 TABLE OF CONTENTS List of Tables and Figures v Introduction.1-2 Methods Results 4-5 Discussion.5-7 Tables.8-10 Figures References Appendix V

6 LIST OF FIGURES AND TABLES Table 1: Characteristics of the obese cohort and general pediatric controls Table 2: Univariate correlations of urinary NGAL levels Table 3: Multivariable analysis: predictors of urine NGAL levels Figure 1: Study design Figure 2: Comparison of NGAL levels: Obese cohort at baseline vs. general pediatric controls Figure 3: Comparison of BMI in the obese cohort at baseline and, 6 and 12 months postoperative Figure 4: Comparison of NGAL levels: Obese cohort at baseline and, 6 and 12 months postoperative VI

7 Acknowledgements I would like to gratefully and sincerely thank my committee members for their guidance, understanding and patience for during my graduate studies at University of Cincinnati. I would also like to thank my fellowship program at the Nephrology and Hypertension Division in Cincinnati Children s for giving me the opportunity to study at UC graduate school. The Clinical and Translational Research Program has laid the foundation for my participation in scholastic activities in the long run. I would also like to thank my parents, Yanchao and Xiujuan, my dear husband Jianquan and our two sons, Renhan and Tianyi for their unwavering love and support. Any modest achievements I have made today won t have existed without my family. II

8 INTRODUCTION Severe obesity is increasing and now affects 4-6% of U.S. children and adolescents. [1] Obesity during adolescence is associated with a higher prevalence of chronic kidney disease (CKD) and other co-morbidities in adulthood, making obesity a huge public health burden. [2-4] It is critically important to detect kidney injury before there is clinical evidence of overt kidney functional decline (CKD development). Unfortunately, current non-invasive functional kidney markers: serum creatinine or serum cystatin C-based, estimated glomerular filtration rate (egfr) and proteinuria are insensitive for detection of early kidney injury in the severely obese patients, because: 1) Human beings have a large functional reserve. Kidney function/gfr can remain normal even in patients with up to 25-50% of kidney tissue loss; [5] 2) the obese population tends to have elevated GFR due to hyperfiltration; [6] 3) albuminuria is not prevalent among the majority of overweight and obese adolescents. [7, 8] Consequently, means to prevent or treat kidney injury are often delayed until overt, irreversible decline of kidney function has already occurred. Nevertheless, there have been major efforts to treat severe obesity in the pediatric population. One intervention is to conduct a bariatric procedure. The multi-center Teen- Longitudinal Assessment of Bariatric Surgery (Teen-LABS) study (NIH 5UM1DK072493) follows 242 severely obese adolescents who underwent bariatric procedures longitudinally. Long-term follow up of Teen-LABS participants will provide functional outcomes of the kidney in the target population. However, it will remain unknown whether bariatric procedures will blunt or even reverse obesity related kidney injuries in the severely obese teenagers because they are not detected by the currently available functional biomarkers. Novel, sensitive and specific biomarkers for the early detection of acute kidney injury (AKI) and prediction of CKD have been identified during the last decade [9-11]. Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kD ubiquitous iron-carrying protein normally expressed in small amounts in a variety of human tissues, including kidney, trachea, lungs, stomach, and colon. [9, 11] The biological role of early and rapid NGAL induction following kidney injury is to preserve kidney function, attenuate apoptosis, and enhance the proliferative response [12]. Multiple studies have shown that urine NGAL is increased after renal insults and the elevated levels predict acute kidney injury in both adults and children. [13] [14, 15] Those 1

9 findings led us to postulate that urinary NGAL is a strong candidate for early kidney injury detection and CKD prediction in severely obese youth. The object of our study was to investigate subclinical kidney injury in severely obese adolescents, using the novel urinary biomarker, NGAL. In the present pilot study, we hypothesized that subclinical obesity-related kidney injury exists in the absence of egfr decline or proteinuria in severely obese adolescents. In addition, we hypothesized that those subclinical kidney injuries might be reversible following significant weight loss. This study tests the hypothesis in two steps: 1) a cross-sectional comparison of urinary NGAL levels in severely obese adolescents and lean controls at baseline; 2) a prospective follow up of the trend of NGAL levels in the severely obese cohort after weight loss following a bariatric procedure. METHODS Study Population and Design Figure 1 showed the study design. For specific aim 1, we proposed to compare urinary NGAL levels in the severely obese cohort with those in a general pediatric cohort. For specific aim 2, we proposed to follow the severely obese cohorts for one year after the weight loss procedure. Biomarker levels were measured at baseline, 6 months and 1 year post operatively. Each obese participant was his/her self-control after bariatric procedure (prospective). The study population included severely obese participants who had already provided urine samples to the Cincinnati Children s Hospital Medical Center Pediatric Obesity Tissue Repository (CCHMC POTR) (IRB ) before and after undergoing bariatric surgery. We estimated that there would be urine samples from over 20 subjects available for analysis by the end of Inclusion criteria were: 1) 19 years of age; 2) cystatin C based egfr of > 60 ml/min/1.73m 2 (indicating normal kidney function) and no microalbuminuria (defined as urine albumin/ urine creatinine < 30 mg/gm); 3) no history of primary kidney disease (glomerular disease diagnosed by kidney biopsy, history of hematuria, kidney stone and abnormal kidney anatomy on image study). Patients who did not meet the inclusion criteria or were pregnant were excluded. General healthy pediatric control data were obtained from the CCHMC urinary biomarkers data set, which provided levels of urine NGAL of normal patients recruited from 2

10 primary care clinics. Those subjects represent the general local pediatric population (control for aim 1). Subjects with known kidney diseases, abnormal kidney anatomy or egfr less than 60 ml/min/1.73m 2 or proteinuria on urinalysis were excluded. Demographic parameters, including age, gender, egfr, BMI and ethnicity (only available in the obese group) were obtained at the same time that urine and serum specimens were collected. Laboratory Analysis NGAL assays were carried out at CCHMC Nephrology Biomarker laboratory. Laboratory investigators were blinded to sample source and clinical outcomes. The assays were performed at the time of initial thawing to prevent degradation. Urine NGAL was measured using commercially available ELISA development kits (BioPorTo Diagnostics A/S) per manufacturer s instructions. This assay has been extensively validated in our Biomarker laboratory, and exhibits a co-efficient of variation of <5%. Biomarker results were expressed following log transformation. In addition, urinary microalbumin/creatinine ratio (<30 mg/gm is considered normal) was measured using a standardized automated clinical laboratory platform. egfr was estimated from serum cystatin C levels (also measured using a standardized automated clinical laboratory platform) using the Larsson s equation: egfr(ml/min) = 77.24(cyc C) for the obese cohort. [16] In the general pediatric controls, egfr was estimated from serum creatinine levels using the modified Schwartz equation: egfr (ml/min) = 0.413*(height/serum creatinine).[17] Statistical Analysis All statistical analyses were performed using SAS statistical software (version 9.3). Descriptive statistics for continuous variables were reported as median and interquartile range as most variables were not normally distributed. Univariate analyses were performed to explore the relationship between the dependent variable (urine NGAL levels) with demographic parameters. Pearson s correlation coefficients were calculated for all variables along with their associated p values to determine their statistically significant association with urine NGAL levels. NGAL values were log transformed in order to approximate the assumption of normal distribution of residuals. See Appendix AI for a frequency histogram of urine NGAL values, in which the 3

11 skewness of the dependent variable is graphically displayed. Appendix AII shows the frequent histogram of NGAL after log transformation. Multivariable regression was performed using all variables with a p value of < 0.05 in univariate analyses. Age and obesity status were included in the initial multivariable analyses. Variables with a p-value of < 0.05 were considered statistically significant and included in a final regression model, selected using both backward elimination and forward inclusion to ensure no eliminated variables subsequently became significant. See Appendix B for R 2, partial F test and p values as variables were removed from or added to the model. Regression diagnostics were performed on the final multivariable model with Jackknife residually > 1.96 and Cook s distance > 1 for plausibility. The assumption of normality was confirmed by examination of a normal probability plot of Jackknife residuals (Appendix C) in addition to examination of the histogram of log transformed NGAL (Appendix AII). T test was performed to compare means of the dependent variable in the obese cohort at baseline and the general pediatric controls. One way ANOVA was conducted for comparison of BMI and urine NGAL levels in the obese cohort at baseline, 6 month and 12 months post bariatric procedure. Institutional Review Board (IRB) Approval The original IRB protocol was approved by CCHMC on 9/22/2012. Reliance review for University of Cincinnati was requested through CCHMC IRB. RESULTS Cohort characteristics A total of 23 severely obese subjects was identified however, 5 of them had microalbuminuria and were excluded. Baseline characteristics of the enrolled 18 participants were shown in Table 1. Of them, 72% were female and 83% were Caucasian. Median age at surgery was 16.2 years while the median BMI was 47.8 kg/m 2. The median egfr values in the obese and control cohorts were not significantly different, median = ml/min/1.73m 2 and ml/min/1.73m 2 respectively (p = 0.089). 4

12 Elevated urinary NGAL levels: obese cohort at baseline vs. general pediatric population In univariate analysis (Table 2), urine NGAL levels differed significantly by age and obesity status (p < ), while no relationship was shown between gender and urine NGAL (p = ). Both age and obesity status were statistically significant at the p=0.05 significance level in univariate analysis and were therefore included in the original multivariate model. Age, however, was not found to be a significant predictor (p = ) thus was not included in the final analysis (Table 3). According to the final model, obesity increases the mean urine NGAL level by 709%. Figure 2 showed the significantly higher levels of NGAL in the obese cohort in comparison to the general pediatric controls. Urinary NGAL levels trends: before and after bariatric procedure Figure 3 showed that the obese cohort had significant weight loss through the first year after bariatric procedures (p < ). At 6 months, urine NGAL levels increased slightly from baseline before they trended down at 1 year. The overall change in biomarker levels through the first year after bariatric surgery, however, was not significant (F = 0.069). DISCUSSION Multiple mechanisms have been postulated to contribute to obesity related chronic kidney injury: hemodynamic changes of a hyperfiltering kidney, inflammation, oxidative stress, metabolic disorder (reduced insulin sensitivity) and other comorbidities, especially cardiovascular disease. [18-20] Kidney function, however, remains normal in the majority of these severely obese young patients (unpublished data from Teen Longitudinal Assessment of Bariatric Surgery, NIH UM1 DK072493). To develop severe obesity as a child or teenager and carry that obesity burden forward into adulthood results in kidney damage that is potentially greater than those seen with adult-onset obesity. [4]Therefore, absence of kidney injuries should not be assumed even with apparently normal kidney function. This prospective pilot study utilized a sensitive kidney injury marker, NGAL, to study the subclinical kidney injuries in severely obese youth. NGAL has been highlighted as a reliable biomarker of kidney injury in a variety of patient populations; for instance, children post cardiopulmonary bypass, renal allograft recipients (adult and children) and critically ill children with septic shock. [21-23] Our findings of significantly elevated urine NGAL levels strongly 5

13 suggested that subtle kidney injuries have occurred in the severely obese adolescents even though their traditional kidney functional markers (egfr and urine protein) were normal. Our findings need to be reconciled with NGAL s reported involvement in low-grade chronic inflammation accompanying obesity. Catalán et al reported higher NGAL protein expression in the visceral fat depot of obese patients compared to lean subjects. [24] Their data demonstrated a significant positive association between NGAL gene expression levels and inflammatory markers (P < 0.01). Our findings are also consistent with the concept that obesity is associated with a mild chronic inflammatory response. [25] However, it should be noted that any contribution of NGAL due to visceral fat deposits alone would be expected to elevate circulating NGAL levels, but NOT urinary NGAL levels. It is well known that any circulating NGAL protein that is filtered through the kidney glomerulus is avidly and completely reabsorbed by the proximal tubules of the normal kidney. [26] In the absence of kidney injury, very little NGAL is detectable in the urine. Thus, our findings of dramatically increased urinary NGAL are consistent with the hypothesis of obesity-induced early kidney damage. NGAL levels were robustly higher in the obese cohort in comparison to the controls but a small portion of the subjects from the two cohorts had NGAL levels that overlapped. However, the exact BMI data was not available for the controls. Since this cohort represents the general pediatric population (recruited from CCHMC clinics), and the current prevalence of severe obesity in U.S. children and adolescents is 4-6% [27, 28], it is reasonable to suspect that several severely obese subjects were included in the control group. This is a likely explanation for the overlapping NGAL levels in a few subjects from both groups. Substantial effort has been made to discover and validate novel urinary biomarkers to detect acute kidney injury over the past decade. In addition to NGAL, other promising urine biomarkers, such as interleukin-18 (IL-18) and kidney injury molecule 1 (KIM-1) might also be plausible candidates to study subclinical kidney injury for obese patients. In future studies, combining these three may provide complementary information, with NGAL and IL-18 reflecting on-going inflammatory events in real time and KIM-1 indicating the more chronic fibrotic changes [29-32]. Our study followed NGAL changes post bariatric procedure, prospectively. Despite the significant weight loss after the procedures, urine NGAL remained elevated at 1 year. This was an unexpected finding that might represent persistent sub-clinical kidney injury, non-reversible 6

14 kidney damages or both. Participants in our study group were less obese but yet still obese by the end of the first year post bariatric procedure. A large adult study has shown that maximum weight losses after bariatric procedure were observed after 1 to 2 years.[33] It is possible that patients in our cohort will continue to lose weight during the second year post-surgery. Therefore, a future follow study of our cohort at 2 years post-surgery is warranted as the next step. This pilot study has several limitations. First, the control cohort was much younger than the obese cohort and had more males. However, univariate and multivariate analysis showed that age and gender were not statistically important risk factors for urinary NGAL elevation. Obesity status was the only independent variable that was included in the final model. Second, we did not have access to all characteristics for the subjects, such as years of being obese/ lean, hydration status, ethnics and BMI in the control subjects. All those factors may theoretically affect their urinary NGAL levels. Future studies should recruit controls with extensive demographic information available to validate our findings. The third limitation is that kidney function was estimated by different means for the controls and the obese subjects so one may argue that both groups may have different kidney function status. There are multiple ways to estimate GFR. Serum creatinine and serum Cystatin C are the most widely used clinical means to calculate GFR. They both have limitations but overall, creatinine based egfr is more muscle mass dependent than cystatin C egfr, hence rendering the latter as a more accurate measure of true GFR. Cystatin C measurements were not available in the normal control population.. Last, our obese cohort is small with only 18 patients. Future, larger study will be needed to increase the power. In summary, our pilot study showed a strong association of obesity and kidney injuries. To our best knowledge, this is the first study using urinary biomarker to reveal subclinical kidney damages in the obese adolescents. Physicians should counsel those young patients for means to control weight before kidneys develop functional loss, and continue to follow their kidney outcomes. 7

15 Table 1: Characteristics of the obese cohort and general pediatric controls Characteristic Obese cohort (n = 18) Controls (n = 161) p values Age (years) 16.2 (15.1, 17.9) 5.1 (3.8, 9.8) < Gender Male 5 (28%) 84 (52%) Female 13 (72%) 77 (48%) Race Caucasian 15 (83%) NA African-American 3 (17%) egfr* (98.52, ) (87.55, ) BMI (43.28, 51.68) NA Continuous variables presented as median (interquartile range) Categorical variables presented as percentages egfr - estimated glomerular filtration rate; BMI, body mass index. Used cystatin C based for the obese cohort and serum creatinine based calculation for the controls. n = 81 missing 8

16 Table 2: Univariate correlations of urinary NGAL levels Variable Pearson correlation coefficient P value Age <.0001 Gender Obesity status <.0001 Pearson correlation coefficients of simple linear regression of urine NGAL levels with each independent variable are presented. All urine NGAL levels were log transformed. Obesity status: obese = 1, general = 0 9

17 Table 3: Multivariable analysis: predictors of urine NGAL levels Variable Parameter estimate Standard Error T Value P value R-Square Intercept <.0001 Age Obesity status < Multivariable regression analysis of urine NGAL levels with each independent variable is presented. All urine NGAL levels were log transformed. Obesity status: obese = 1, general = 0 10

18 Figure 1: Study design Prospective (Aim 2) Bariatric Procedure Cross sectional (Aim 1) Morbidly obese adolescents 6 months 1 year Lean controls 11

19 Figure 2: Comparison of urine NGAL: Obese cohort at baseline vs. general pediatric controls Obesity status: obese = 1, general = 0 NGAL levels were log transformed. 12

20 Figure 3: Comparison of BMI in the obese cohort at baseline, 6 and 12 months post bariatric procedure (one way ANOVA) Time: baseline=0, 6 month = 6, and 12 month = 12 BMI - body mass index 13

21 Figure 4: Comparison of urine NGAL: Obese cohort at baseline, 6 and 12 months post bariatric procedure (one way ANOVA) Time: baseline=0, 6 month = 6, and 12 month = 12 NGAL levels were log transformed. 14

22 REFERENCES 1. Kelly, A.S., et al., Severe obesity in children and adolescents: identification, associated health risks, and treatment approaches: a scientific statement from the American Heart Association. Circulation, (15): p Serra, A., et al., Renal injury in the extremely obese patients with normal renal function. Kidney Int, (8): p Vivante, A., et al., Body mass index in 1.2 million adolescents and risk for end-stage renal disease. Arch Intern Med, (21): p Inge, T.H., et al., The effect of obesity in adolescence on adult health status. Pediatrics, (6): p Ibrahim, H.N., et al., Long-term consequences of kidney donation. N Engl J Med, (5): p Chagnac, A., et al., Glomerular hemodynamics in severe obesity. Am J Physiol Renal Physiol, (5): p. F Nguyen, S., et al., Being overweight modifies the association between cardiovascular risk factors and microalbuminuria in adolescents. Pediatrics, (1): p Mueller, P.W. and S.P. Caudill, Urinary albumin excretion in children: factors related to elevated excretion in the United States population. Ren Fail, (3-4): p Devarajan, P., The use of targeted biomarkers for chronic kidney disease. Adv Chronic Kidney Dis, (6): p Chaudhary, K., et al., The emerging role of biomarkers in diabetic and hypertensive chronic kidney disease. Curr Diab Rep, (1): p Fassett, R.G., et al., Biomarkers in chronic kidney disease: a review. Kidney Int, (8): p Mishra, J., et al., Amelioration of ischemic acute renal injury by neutrophil gelatinaseassociated lipocalin. J Am Soc Nephrol, (12): p Clerico, A., et al., Neutrophil gelatinase-associated lipocalin (NGAL) as biomarker of acute kidney injury: a review of the laboratory characteristics and clinical evidences. Clin Chem Lab Med, (9): p Cruz, D.N., et al., Plasma neutrophil gelatinase-associated lipocalin is an early biomarker for acute kidney injury in an adult ICU population. Intensive Care Med, (3): p Dent, C.L., et al., Plasma neutrophil gelatinase-associated lipocalin predicts acute kidney injury, morbidity and mortality after pediatric cardiac surgery: a prospective uncontrolled cohort study. Crit Care, (6): p. R Larsson, A., et al., Calculation of glomerular filtration rate expressed in ml/min from plasma cystatin C values in mg/l. Scand J Clin Lab Invest, (1): p Schwartz, G.J., et al., New equations to estimate GFR in children with CKD. J Am Soc Nephrol, (3): p Savino, A., et al., Obesity-related renal injury in childhood. Horm Res Paediatr, (5): p Eknoyan, G., Obesity and chronic kidney disease. Nefrologia, (4): p Laville, M., [Renal consequences of obesity]. Nephrol Ther, (2): p

23 21. Wheeler, D.S., et al., Serum neutrophil gelatinase-associated lipocalin (NGAL) as a marker of acute kidney injury in critically ill children with septic shock. Crit Care Med, (4): p Di Nardo, M., et al., Impact of severe sepsis on serum and urinary biomarkers of acute kidney injury in critically ill children: an observational study. Blood Purif, (1-3): p Heyne, N., et al., Urinary neutrophil gelatinase-associated lipocalin accurately detects acute allograft rejection among other causes of acute kidney injury in renal allograft recipients. Transplantation, (12): p Catalan, V., et al., Six-transmembrane epithelial antigen of prostate 4 and neutrophil gelatinase-associated lipocalin expression in visceral adipose tissue is related to iron status and inflammation in human obesity. Eur J Nutr, Alnaeeli, M., et al., Erythropoietin signaling: A novel regulator of white adipose tissue inflammation during diet-induced obesity. Diabetes, Devarajan, P., Neutrophil gelatinase-associated lipocalin: a promising biomarker for human acute kidney injury. Biomark Med, (2): p Claire Wang, Y., S.L. Gortmaker, and E.M. Taveras, Trends and racial/ethnic disparities in severe obesity among US children and adolescents, Int J Pediatr Obes, (1): p Koebnick, C., et al., Prevalence of extreme obesity in a multiethnic cohort of children and adolescents. J Pediatr, (1): p e Miyauchi, K., et al., Upregulated IL-18 expression in type 2 diabetic subjects with nephropathy: TGF-beta1 enhanced IL-18 expression in human renal proximal tubular epithelial cells. Diabetes Res Clin Pract, (2): p Liang, D., et al., Effects of interleukin 18 on injury and activation of human proximal tubular epithelial cells. Nephrology (Carlton), (1): p van Timmeren, M.M., et al., Tubular kidney injury molecule-1 (KIM-1) in human renal disease. J Pathol, (2): p Ko, G.J., et al., Transcriptional analysis of kidneys during repair from AKI reveals possible roles for NGAL and KIM-1 as biomarkers of AKI-to-CKD transition. Am J Physiol Renal Physiol, (6): p. F Sjostrom, L., et al., Effects of bariatric surgery on mortality in Swedish obese subjects. N Engl J Med, (8): p

24 Appendix A: Histogram of dependent variable: NGAL I. Histogram of urine NGAL levels (ng/ml) in all study subjects. II. Histogram of log transformed urine NGAL levels in all study subjects. 17

25 Appendix B: Strategy for selecting variables I. R 2, partial F test and p value as variables were removed using backward elimination. Variables included in the final model were obesity status. Step Variable Removed R2 Partial F test p value 1 Gender Age R 2 represents the R 2 value after the corresponding variable was removed from the model II. R 2, partial F test and p value as variables were removed using forward inclusion. Variables included in the final model were obesity status. Step Variable Removed R2 F Value p value 1 Obesity < R 2 represents the R 2 value after the corresponding variable was included in the model 18

26 Appendix C: Normal probability plot of jackknife residuals for final regression model. Variables included in the final model were obesity status. * +++* Normal Probability Plot ++++* * ******* ********* ******* ******* ++**+ ******** * +*+*+***** *