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Metabolic Dysfunction in Pediatric HIV-Positive Patients

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Metabolic Dysfunction in Pediatric HIV-Positive Patients
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DAJUSTE, MARIE ESTHER ( Author, Primary )
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2008

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Blood ( jstor )
Fasting ( jstor )
Fats ( jstor )
Insulin ( jstor )
Insulin resistance ( jstor )
Obesity ( jstor )
Pediatrics ( jstor )
Percentiles ( jstor )
Plasmas ( jstor )
Triglycerides ( jstor )

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University of Florida
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University of Florida
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Copyright Marie Esther Dajuste. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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8/31/2008
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476764286 ( OCLC )

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METABOLIC DYSFUNCTION IN PEDIAT RIC HIV-POSITIVE PATIENTS By MARIE ESTHER DAJUSTE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Marie Esther Dajuste

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This document is dedicated to Daniel Rose mond Dajuste who had a passion for life.

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iv ACKNOWLEDGMENTS I would like to thank Dr. Peggy Borum fo r her guidance and dedication to this study. I gained much inspiration from her ex emplary devotion to he r work, patients, and students. I am also grateful to her for her continual support. I would like to express my gratitude to Dr. Bobbi Langkemp-Henken for her insight and direction in th e progress of this study. I would also like to thank Dr . Robert Lawrence for sharing his expertise in pediatric immunology at the University of Florida. His counsel was valuable to the success of this study. I would like to thank my lab members fo r their hard work in making this study possible. Special thanks go to Jennifer Rabbat, Gregory Young, Divya Juturu, and Benjamin Porras. I thank my fiancé, David Pierrelus, for be lieving in me and for his encouragement. Lastly, I thank my family for their con tinual love and support through all of my endeavors.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 Pathways Associated with the Metabolic Syndrome.............................................2 Obesity Links.................................................................................................2 Insulin Resistance and Dyslipidemia.............................................................7 Insulin Resistance and Endothelial Dysfunction............................................7 Insulin Resistance and Inflammation.............................................................8 The Metabolic Syndrome and HIV.......................................................................9 Gator Circle: A New, Noninvasive Met hod to Measure Visceral Adiposity.............11 Purpose.......................................................................................................................1 2 2 METHODS.................................................................................................................17 HIV-Associated Metabolic Dysfunction....................................................................17 Patient Data Tracking.................................................................................................24 Retrospective Analysis...............................................................................................26 Metabolic Modules.....................................................................................................27 Gator Circle................................................................................................................29 3 DEVELOPMENT OF THE METABOLIC MODULES...........................................36 Growth........................................................................................................................3 6 Body Composition and B ody Fat Distribution...........................................................37 Blood Lipids...............................................................................................................39 Blood Glucose and Insulin Status...............................................................................39 Blood Pressure............................................................................................................40 Mitochondrial Metabolism.........................................................................................40

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vi Renal Metabolism.......................................................................................................41 Bone Metabolism........................................................................................................41 Protein Nutrition Status..............................................................................................42 Anemia Status.............................................................................................................42 Dietary Intake.............................................................................................................43 4 DEVELOPMENT OF HIV-ASSOCI ATED METABOLIC DYSFUNCTION DEFINITION..............................................................................................................44 Obesity........................................................................................................................ 44 Fat Distribution...........................................................................................................47 Dyslipidemia...............................................................................................................49 Hypertension...............................................................................................................52 Insulin Resistance.......................................................................................................53 5 RESULTS...................................................................................................................59 6 SUMMARY................................................................................................................70 APPENDIX A DATABASE PARAMETERS....................................................................................73 B METABOLIC MODULES SUMMARY TABLE.....................................................76 C GROWTH MODULE.................................................................................................78 D BODY COMPOSITION AND BO DY FAT DISTRIBUTION MODULE...............84 E BLOOD LIPID ST ATUS MODULE.......................................................................106 F BLOOD GLUCOSE AND INSULIN STATUS MODULE....................................110 G BLOOD PRESSURE MODULE..............................................................................113 H MITOCHONDRIAL METABOLISM MODULE...................................................115 I RENAL METABOLISM MODULE.......................................................................120 J BONE METABOLISM MODULE..........................................................................124 K PROTEIN NUTRITION STATUS MODULE........................................................130 L ANEMIA MODULE................................................................................................132 M DIETARY INTAKE MODULE...............................................................................135 N GROWTH MEASUREMENTS CRF.......................................................................138

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vii O ANTHROPOMETRIC MEASUREMENTS CRF...................................................141 P DEXA CRF...............................................................................................................149 Q PHYSICAL ASSESSMENT CRF............................................................................150 R BODY IMAGE CRF................................................................................................153 S BLOOD LIPIDS CRF...............................................................................................157 T COMPREHENSIVE CHEMISTRY CRF................................................................158 U METABOLIC SPECIMEN TRACKING CRF........................................................162 V VITAL SIGNS CRF.................................................................................................170 W URINALYSIS CRF..................................................................................................172 X CREATININE CLEARANCE CRF.........................................................................174 Y MITOCHONDRIAL TOXICITY CRF....................................................................176 Z LACTATE CRF........................................................................................................182 AA COMPREHENSIVE HEMATOLOGY CRF...........................................................184 BB DIET CRF.................................................................................................................187 CC PEDIATRIC LAB REFERENCE RANGES............................................................192 DD DIVISION OF AIDS TABLE FOR GRADING THE SEVERITY OF ADULT AND PEDIATRIC ADV ERSE EVENTS................................................................238 LIST OF REFERENCES.................................................................................................244 BIOGRAPHICAL SKETCH...........................................................................................257

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viii LIST OF TABLES Table page 1-1 Current Methods of Eval uating Visceral Obesity....................................................14 2-1 Metabolic Syndrome Definition in Adults...............................................................18 2-2 Metabolic Syndrome Definition in Pediatrics..........................................................19 2-3 HIV-associated Metabolic Dysfunction Definition..................................................25 2-4 Questions addressed by the Metabolic Modules......................................................28 2-5 Gator Circle Equations.............................................................................................35 4-1 Obesity Cut-offs.......................................................................................................46 4-2 Growth Cut-offs.......................................................................................................49 4-3 Dyslipidemia Cut-offs..............................................................................................51 4-4 Hypertension Cut-offs..............................................................................................53 4-5 Glucose Cut-offs......................................................................................................55 4-6 Insulin Indices Most Conservative Cut-offs.............................................................55 4-7 HAMD Point System...............................................................................................57 5-1 Descriptive Characteris tics of Pediatric HIV+ Population and Prevalence of MS and HAMD...............................................................................................................59 5-2 Averages and Standard Devi ations of HAMD Risk Factors....................................61 5-3 Mean and S.D. for MSF and VC A for p1045 Control Population and HIV+ Population.................................................................................................................62 5-4 Gator Circle Phenotypes...........................................................................................62 5-5 Correlation of Gator Circle Va riables with Cumulative IRI....................................63 5-6 Prevalence on HAMD Risk Factors.........................................................................68

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ix LIST OF FIGURES Figure page 2-1 HAMD Parameters...................................................................................................24 2-2 Visceral Cavity Area................................................................................................30 2-3 Obtaining the Mean Skinfold...................................................................................31 4-1 Obesity Parameter....................................................................................................45 4-2 Growth Parameter....................................................................................................48 4-3 Dyslipidemia Parameter...........................................................................................51 4-4 Hypertension Parameter...........................................................................................52 4-5 Insulin Resistance Parameter...................................................................................54 4-6 HAMD Point Progression........................................................................................58

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science METABOLIC DYSFUNCTION IN PEDIAT RIC HIV-POSITIVE PATIENTS By Marie Esther Dajuste August 2006 Chair: Peggy Borum Major Department: Food Science and Human Nutrition The Metabolic Syndrome is a clustering of cardiovascular disease risk factors that include elevated blood pressu re, dyslipidemia, impaired glucose tolerance, insulin resistance and obesity. The Metabolic Syndrome has been defined by several organizations including the National Choles terol Education Program: Adult Treatment Panel III (NCEP:ATP III). Recent advances in the development of highly active antiretroviral therapy correlate with the emer gence of the metabolic syndrome profile in HIV positive patients. Variation in the estim ation of the prevalence of the metabolic syndrome in these patients occurs because of inconsistencies in diagnostic tools and clinical models. Diagnostic parameters called the Metabo lic Modules were provided as early detection tools of metabolic t oxicity. The Modules were orga nized in a menu format that allows health care providers to choose specifi c tests that are pertinent to an individual patient. They also included the Gator Ci rcle, a computation th at differentiates the visceral cavity area from the subcutaneous area. This inexpensive and noninvasive

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xi computation may prove to be especially us eful in the HIV positive population which is prone to experiencing body fat loss and fat accumulation concomitantly. The Modules were proposed to be employed within primary th erapy protocols, either internationally or domestically. The goal was to standardize tests in order to allow comparisons across clinics and protocols and to ensure that new drug therapies are assessed for these metabolic toxicities. An HIV-Associated Metabolic Dysfunc tion (HAMD) defini tion was developed with the intent of defining the metabolic disturbances that are specific to the HIV population and screening for early detection of metabolic abnormalities. The components were divided into two stages in which the fi rst stage consisted of parameter levels that were approaching abnormality and the second stage consisted of abnormal parameters. HAMD would allow for an at-risk group to be identified and preventive measures to be taken accordingly. The prevalence of the Metabolic Syndro me in a pediatric (126 years) HIV positive population (n=77) treated at Shands Pediatric Immunology Clinic during 2004 was determined based on a child-adjusted version of the ATP III definition. The prevalence of the Metabolic Syndrome within this population was 12.9%. The prevalence of HAMD was also determined in this population to be 31.2%, signifying that 31.2% of these patients had the equivalent of 3 or more abnormal metabolic parameters. Based on these results, HIV positive pediatric patients in Florida are at risk of developing metabolic abnormalities that lead to Metabolic Dysfunction.

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1 CHAPTER 1 INTRODUCTION Background The Metabolic Syndrome is a clustering of cardiovascular risk factors that include elevated blood pressure, dyslipidemia (high triglycerides, high LDL cholesterol and low HDL cholesterol), impaired glucose tolerance, insulin resistance and obesity (1). The syndrome is complex in that each component of the cluster of abnormalities is a risk factor for cardiovascular disease on it s own. Once termed Reaven Syndrome or Syndrome X, the exact cause of the Metabolic Syndrome is not known but its etiology is believed by researchers to be a combination of both genetic makeup and lifestyle choices (1,2). This syndrome is known by other name s including insulin resistance syndrome, metabolic cardiovascular syndrome, a nd dysmetabolic syndrome (3). The Metabolic Syndrome has been defined by several organizations including the World Health Organization (WHO), the Natio nal Cholesterol Education Program: Adult Treatment Panel III (NCEP:ATP III), the European Group for the Study of Insulin Resistance (EGIR), the American Association of Clinical Endocrinologists (AACE), and the International Diabetes Federation (IDF) (4 -8). Since there are currently no universal criteria for the diagnosis of the Metabolic Syndrome, its prevalence varies (9). According to the NCEP: ATP III criteria, 23.7 % of U.S. adults have the Metabolic Syndrome (10). Prevalence tends to increase with age and is the highest among Mexican Americans. Components of the Metabolic Syndrome are also present in children and adolescents (11). A study using the same a bove-mentioned criteria revealed a prevalence

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2 of 10% in U. S. adolescents, and 33.3% in overweight U.S. adolescents (12). This finding supports the idea that the developmenta l stages of the Metabolic Syndrome begin at an early age (13). Pathways Associated with the Metabolic Syndrome Randle was the first to describe the glucose-fatty acid cycle in which the physiological interactions of glucose and fat metabolism are linked. Normally an elevated glucose concentration stimulates the release of insulin which in turn suppresses nonesterified fatty acid (NEFA) release from adipose tissue storage (14). Exogenous stressors, namely excess energy intake can im pair this normal relationship of homeostasis between the nutrients (15). The links of obes ity and insulin resistance to other Metabolic Syndrome risk factors and their implicated roles in the developm ent of the Metabolic Syndrome are examined below. Obesity Links Adipose tissue can be found in two forms, white and brown . The white adipose tissue is more abundant and is involved in body fat st orage while the brown tissue is involved in thermogenesis, the generation of heat. Wh ite adipose tissue is innervated by the autonomic nervous system and is influenced by the sympathetic nervous system (SNS) (16). Stimulation of the SNS increases lipolysis. -1, 2 adrenergic receptors are also responsible for increasing lipolysis by init iating the activation of the lipolytic cascade (17). The activation of the cAMP dependent protein kinase A (PKA) results in the phosphorylation of perilipin and hormone se nsitive lipase (HSL) and consequently inducing lipolysis (17). Obesity can be considered as an energy st orage disorder where weight gain results from an energy imbalance (energy input exceeding output) (17). While an increase in

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3 total body fat has been linked to health complications, the accumulation of intraabdominal (visceral) fat is associated with low HDL-cholesterol, and increased triglycerides, small, dense LDL-cholestero l, hypertension, insulin resistance, type II diabetes and other morbidities (18). Elevated Nonesterified Fatty Acids Dietary fat is normally used by the muscle and liver for energy and the formation of lipid-derived molecules, respectively. Ex cess fat is stored as triglycerides in the adipose tissue. In times of energy shortage, stored triglycerides ar e lysed into glycerol and fatty acids. These nonesterified or fr ee, fatty acids (NEFAs) are released into circulation and are oxidized in the skeletal mu scle for energy (16). Acute elevations in NEFAs can lead to peripheral insulin resist ance (19). In normal individuals long-term elevations in NEFAs will be followed by in sulin secretion which compensates for the induced insulin resistance. In individuals with a family history of diabetes, the amount of insulin secreted is insufficient to compensate for the induced insulin resistance (20). Ectopic Fat Storage Another method in which obesity can lead to metabolic disturbances is referred to as the ectopic fat storage syndrome. In th is syndrome, energy intake exceeds energy expenditure, causing lipids to accumulate in other tissues of the body including muscle, liver, and pancreatic beta cells (21). According to RandleÂ’ s hypothesis, in creased fatty acid utilization could inhibit gl ucose oxidation in the muscle (14). It is proposed that increased intramyocellular lipids cause de fects in insulin sign aling, reduce insulinstimulated muscle glucose tr ansport activity, reduce glycog en synthesis, and impair suppression of hepatic glucose production (19). So me studies have found that decreased adipose tissue stores can also lead to insulin resistance (22,23).

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4 Obesity and Impaired Proliferation and Differentiation One of the proposed mechanisms to explain this syndrome is the impaired proliferation and differentiation of adipocytes . Adipocytes are deri ved from pleuripotent stem cell precursors: the mesenchymal stem ce lls (MSCs). MSCs can differentiate into an osteoblast, a chondroplast, myocyte and other cell types. Tr anscription factors (SREBP, PPAR), extracellular signals (cyt okines, prostaglandins, TNF), and hormones (corticosteroids, insulin) regulate cell differentiation in preadipocytes (24). Enlarged adipocytes correlate with insulin resistance better than other measures of adiposity (25). Also, they are more metabolica lly active and have highe r rate of lipolysis. Large adipocytes are thought to be due to a failure of fat cells to proliferate and/or differentiate (21). Obesity and Impaired Fat Oxidation The other manner in which these disturba nces are believed to occur is by the impairment of fat oxidation. This can either be due to a lack of mito chondria (the site of lipid oxidation), impairment in the signa ling to activate lipid oxidation, and/or a hindrance in fatty acid transport. PGC-1 is a transcriptional coactivator which is necessary for mitochondrial biogenesis and th e coordination of fibe r type switching. Reduced mitochondrial capacity could be i nduced by impairing PGC-1 (26). CPT-1 and AMPK regulate substrate utilization and defect s in these pathways will cause a decrease in fatty acid oxidation. CPT-1 catalyzes the ra te-limiting step in the transport of fatty acids into the mitochondria for their oxidation (27). AM PK has a pivotal role in maintaining energy homeostasis. AMPK is activated by AMP and inhibited by ATP therefore in times of high ATP production, the kinase is inhibited and Acetyl-Coenzyme

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5 A (CoA) Carboxylase (ACC) is dephosphorylated and fatty acid synthesis is stimulated while fatty acid oxidation is inhibited si multaneously (28). In times of low energy, inhibition of ACC enzyme via phosphorylati on causes a decrease in malonyl-CoA, which is an inhibitor of CPT-1 (28). Transcriptional and translational factors also play roles in regulating fatty acid oxidation. PPARand PPARregulate the transcription of the collection of genes involved in fatty acid oxidation. Furthermore, neural endocrine mechanisms may also regulate fatty acid oxidation while the SNS acting through the adrenoreceptors could decrease fatty acid oxidation (21,26). Obesity and Endothelial Dysfunction Endothelial dysfunction may also occur as a result of obesity. In the kidneys, damage to the vascular wall may be respons ible for hyperfiltration and the increase of albumin in the urine (29). Nitric oxide (NO) may have a protective effect against vascular inflammation and smooth muscle cell proliferation, whereas impaired NO action is proposed to reduce vasodila tion and lead to endothelial dysfunction. NEFAs impair endothelium-dependent vasodi lation and cause endothelial dysfunction. This may occur by the NEFAÂ’s acylation of CoA and thereby reducing the leve l of the free cytosolic CoA pool available for the activation NO synthase (30,86). Endothelial dysfunction may also lead to hypertension, which frequently occurs in obese subjects (31). Obesity and Adipokines Recently adipose tissue has been shown to be involved in more than just fat storage and mobilization. It may also have se cretory characteristics. Adipocytes secrete cytokines also known as adipokines. Some ad ipokines such as IL-6, resistin, adiponectin and leptin are secreted into th e blood stream and travel to othe r sites while others, such as

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6 TNFare secreted and act locally. Obesity seems to alter the levels of the secretion of these adipokines (32,33). Ad iponectin has been found to have both an insulin sensitizing and cardioprotective effect and obese subjec ts have lower levels of adiponectin. Hypoadiponectinemia may be somewhat responsi ble for the link betwee n visceral fat and insulin resistance (34). Another adipokine , leptin, regulates the size of the energy reserves in the body and fuel mobilization and utilization. Leptin is able to cross the blood-brain barrier and bind to its long-form r eceptor. Thus, a peripheral message of fat mass repletion is delivered to the central ne rvous system (CNS), which regulates energy intake and expenditure and limits fat deposition. Leptin secr etion is increased in obese subjects. In humans, IL-6 is secreted by subcutaneous fat and it stimulates lipolytic effects within the subcutaneous tissue (35). It also induces the e xpression of SOCS-3, a potential inhibitor of insu lin signaling (36). TNFlevels are increased in obesity and it induces lipolysis. It activates the mitogen act ivated protein kinases (MAPK) but inhibits auto-phosphorylation of tyrosine residues of the insulin receptor and suppresses the expression of the insulin receptor (37). In addition mechanisms involving inhibition of PPARhave been implicated as causing TNFÂ’s induction of insulin resistance (38). Resistin is also an adipokine that can induce insulin resistance (21). Obesity and Inflammation Obesity has been characterized as a proinflammatory state and excess adiposity is associated with the release of infla mmatory adipokines. These cytokines may contribute to an increase in IL-6 and C -reactive protein, a marker of inflammation (39,40). Increases in pro-inflam matory mediators also increase the risk of atherosclerotic complications (41). The increase in adi pokines may also induce an increase in pro-

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7 thrombotic factors (i.e. fibrinogen and pl asminogen activator inhibitor) (42). For example, leptin has a pro-aggregat ory effect on plat elets (41). As described above, obesity promotes th e development of in sulin resistance. Elevations in NEFAs can also lead to insulin resistance. Recent evidence suggests that reduced glucose transport may be due to NEFAs inhibiting proximal insulin signaling by inhibiting tyrosyl phosphorylation of th e insulin receptor substrates (19). Insulin Resistance and Dyslipidemia Insulin has been found to induce the expression of a key hepatic lipogenic transcription factor, SREBP-1c (43). This explains how hepa tic steatosis could be caused by hyperinsulinemia and hepatic glucose outpu t could be suppressed at the same time by insulin. Insulin can enhance lipogenesis a nd limit apoB secreti on (44). Decreased lipoprotein lipase activity can l ead to dyslipidemia in diabetic patients. The relationship between elevated VLDL triglycerides and lo w HDL levels is regulated by cholesterol ester transfer protein (CETP) which mediates th e transfer of triglycerides from VLDL to HDL. HDL is then degraded by hepatic lipa se, thus, HDL turnover increases in insulin resistant patients (45). Increased triglycerid e levels also cause th e production of small, dense LDL cholesterol particles which are eas ily oxidized and less re adily cleared (45). Insulin Resistance and Endothelial Dysfunction Insulin normally regulates vasodilation, ther efore, patients with insulin resistance are not sensitive to the normal actions of insulin, and vasodilation is impaired. Impairment in cellular cation transport can also occur as a result of insulin resistance and cause vasoconstriction (46). Hyperinsulinemia leads to increased sodium retention and increases SNS activity, which lead to hypert ension and insulin resistance, respectively (47,48).

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8 Insulin Resistance and Inflammation Like obesity, insulin resistance is also view ed as a pro-inflammatory state. Insulin normally exerts an anti-inflammatory effect at the cellular and molecular level. This is shown by a low dose infusion of insulin reducing reactive o xygen species (ROS) generation by mononuclear cells (49). A co rrelation exists betw een fasting insulin concentrations and CRP concen trations in plasma, which i ndicates a relationship between insulin resistance and inflammatory processes (39). Insulin can promote lipid synthesis within arterial walls, and insulin has also been shown to have mitogenic effects, which promote vascular smooth muscle prolifera tion and migration (50,51). Thus, insulin appears to play a role in the de velopment of atherosclerosis. Although the Metabolic Syndrome has been defined by various groups, there is still no universally accepted definition of the Me tabolic Syndrome. The etiology of the syndrome is unknown although great strides have been taken to uncover some of the mechanisms that are responsible for the deve lopment of this syndrome. It is almost impossible to try to isolate the risk factor s in terms of proposing pathogenic pathways because they are very much interconnected. Both obesity and insulin resistance can be seen as pro-inflammatory states. In obe sity, NEFAs, ectopic fat accumulation, impaired fat oxidation, and the secretion of cytokines ar e the major pathways that lead to insulin resistance and the Metabolic Syndrome. Simila rly in the state of in sulin resistance, the regulation of lipogenic transcri ption factors, vasodilation, so dium retention, and secretion of proinflammatory cytokines aid in the progression of the Metabolic Syndrome. Whereas insulin resistance does not lead to obe sity, the reverse does occur. The resulting symptoms of insulin resistance are all in some way related to obesity. Obesity promotes

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9 insulin resistance and other fact ors, and insulin resistance in turn promotes factors that are related to obesity. The Metabolic Syndrome and HIV Prior to the initiation of highly active antiretroviral therapy (HAART), patients experienced wasting associated with the pr ogression of the HIV disease (52). A study published in 1985 found that th ese patients were underwei ght, their body fat contents were reduced and their intracellular water volumes were decreased, indicating a loss of lean body mass (53). These changes were proba bly the effects of altered metabolism and severe, progressive malnutriti on. Recent advances in the development of three different types of antiviral drugs, th e nucleotide and non-nucleotide analogues acting as reverse transcriptase inhibitors (NRT Is and NNRTIs) and the viral protease inhibitors (PI), and their introduction in the management of AI DS, have significantly improved the clinical course of the disease and pr olonged the life expectancy of patients with AIDS (54,55). However, prolonged HAART can lead to a ra nge of adverse effects, including the Metabolic Syndrome (dyslipidemia, insulin resistance, viscer al adiposity) and subcutaneous lipoatrophy, collectively called lipodystrophy syndrome (56,57). Other names for this syndrome include HIV-associated dyslipidemic lipodystrophy, and HIV associated lipodystrophy syndrome. Although these terms acknowledge the disturbances in the distribution of fat and lip id levels, they do not address the insulin resistance or the type of lipodystrophy pr esent (lipohypertrophy or lipoatrophy). The Metabolic Syndrome has become a subs tantial problem with a prevalence of 20-60% in HIV-infected adult patients re ceiving HAART (58). The variation in prevalence occurs because of in consistencies in diagnostic tools and clinical models (59). Since the metabolic disturbances may predispos e individuals to diab etes, heart disease,

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10 and vascular disease, early in tervention is crucial (60,61,2). HAA RT is also used to treat HIV-infected children and so these children sh ould be considered at risk for developing metabolic dysfunction. There is an urgent need to establish internationally acceptable criteria for the Metabolic Syndrome in children and adolescents (13,62). Recent scientific literature has questioned the existence of the Metabolic Syndrome as a diagnostic entity (63-65). The major incongruent points are whether diagnosing the Metabolic Syndrome has greater clinical bene fit than evaluating th e individual abnormal risk factors; and the risk factors that form the Metabolic Syndrome lack a common etiology. The lack of consensus on defini ng the Metabolic Syndrome and the debate on the existence of the Metabolic Syndrome may create a barrie r for efforts to screen and monitor metabolic dysfunction in general. Research has elucidated the potential for these factors to promote the development of heart dise ase, stroke, and vascul ar disease. It is fairly logical to expect that a clustering of several of these fa ctors will increase an individualÂ’s risk for these di seases. Moreover, several st udies have shown that mild abnormalities in cardiovascular disease risk fa ctors can lead to adverse events. For example, early stages of diabetes have an increased cardiovascular disease risk (66). Small increases in blood pressure can lead to clini cal events (66). These findings stress the importance of monitoring the progression of metabolic abnormalities in stages where multiple mild abnormalities could have powerful contributions. Other findings in current literature include the presence of nontraditional risk factors such as increased in flammatory markers and decrea sed adiponectin, as features that are often (but not always) observed in individuals with the Metabolic Syndrome.

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11 Since the observed effects of this syndro me are metabolic, affecting the physiology of glucose and fat, and are similar to th e metabolic dysfunction observed in the non HIVinfected population, referring to it as HIV-associated Metabolic Dysfunction (HAMD) seems appropriate. Major abnormalities present in HAMD include dyslipidemia, lipoatrophy and lipohypertrophy, insulin resistan ce, linear growth failure, hypertension, and obesity. Dyslipidemia refers to the elevati on in plasma triglycerides and cholesterol. Lipoatrophy refers to the loss of fat in pe ripheral areas and lipohype rtrophy is used to define the accumulation of fat in the visceral area (67). Insulin resi stance occurs when a greater amount of insulin is needed to e licit a normal metabolic response (68). Gator Circle: A New, Noninvasive Met hod to Measure Visceral Adiposity The prevalence and incidence of obesity in childhood has increa sed rapidly in both developed and developing countries (69,70). Research has exposed the unfavorable impact of obesity on physical a nd psychosocial health (3). While an increase in total body fat has been linked to health complicat ions, specifically the accumulation of intraabdominal (visceral) fat is a ssociated with low HDL-choleste rol, increased triglycerides and small, dense LDL-cholesterol, hypertension, insulin resistance, type II diabetes and other morbidities (18,69). These cardiovascul ar risk factors also make up the Metabolic Syndrome profile (71). A variety of anthropometric measuremen ts have been developed to measure visceral obesity of which, wa ist circumference has been c onsidered to be the best predictor of visceral fat ma ss and of the Metabolic Syndr ome profile (72,73,69). Despite the connection between visceral fat and th e Metabolic Syndrome, there is still no anthropometric measurement that can meas ure visceral fat mass directly (74,69). Magnetic Resonance Imaging (MRI) and Co mputed Tomography (CT) scans are the

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12 most accurate methods of measuring intra-abdomin al fat. In contrast, they are also the most costly and lengthy, and require personne l with advanced technical skills (73,69). Patients are required to remain still for an extended amount of time and in the case of pediatric patients, anesthesia and/or sedation may be necessary (82). Sedation of children for MRI and CT scanning is associated with risks of hypoxemia, inadequate sedation, and failed sedation (82). As shown in Table 1-1, current methods of measuring visceral abdominal fat are either inaccurate or expensive for use in rou tine clinical settings. Since the risk factors for cardiovascular disease are fairly stable and tend to track into adulthood, early identification is essential for the preven tion of cardiovascular disease in at-risk populations (75). Purpose The purpose of this investigation was fou r-fold. The first aim was to define the HIV-associated Metabolic Dysfunction in pediatric populations. The definition was determined with an effort to detect the va rious phenotypes that may be present in HAMD which diversified the parameters evaluated. A second aim was to continually devel op a population database to determine the prevalence of the HIV-associated Metabolic Dysfunction in patients at the Pediatric Immunology Clinic at Shands in Gainesville, Florida. Due to the dual stresses of both HIV infection and antiretrovi ral therapy, I hypothesized that this population was indeed at risk for HAMD. A third aim was to provide clinical tools that can be used to diagnose and monitor patients at-risk for developing the HIV-asso ciated Metabolic Dysfunction. These tools are referred to as the Metabolic Modules (M M). These Modules were designed with the

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13 intent that they would provide screens fo r metabolic toxicities that can be easily incorporated into primary therapy protocols (dom estic or international) to ensure that new drugs are assessed for these toxicities and to standardize the testing so that comparisons can be made across protocols. The final aim of this investigation was to propose the use of these Modules in the gathering of prospective data and recomm end needed changes to current diagnostic procedures.

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14 Table 1-1: Current Methods of Evaluating Visceral Obesity Method Advantages Disadvantages BMI • Easy to calculate • The correlation between BMI and body fat percentage is generally strong (78). • BMI increases may be due to increase in fat free mass rather than body fat compartments (78). • Given a certain WC, higher BMI values may not indicate an increased health risk (69) Waist Circumference (WC) • WC has been correlated with visceral fat and total fat mass (69) • WC in combination with BMI is a better predictor of abdominal fat than BMI is alone (76,77). • WC correctly identified >90% of children as being truly positive (high waist circumference and high trunk fat mass) or negative (low waist circumference and low fat trunk mass). (73). • Those with a WC above the 75th percentile had significantly higher odds of having higher blood pressure, high total cholesterol levels, high low-density lipoproteins cholesterol levels, and high triglyceride levels (75). • WC measurements correlate significantly with body fat mass in males and females, and correlate significantly with percentage body fat in females only. The associations with trunk fat were higher than were the associations with total body fat in both sexes (80). • WC reflects internal as well as subcutaneous adipose tissue and is influenced by variation in muscle and bone (69). • Standard definitions of the WC are not always followed, and some may vary between subjects in relation to bone landmarks, muscle, and adipose tissue. Some landmarks may be difficult to identify in obese subjects (69). • 14 different descriptions of the site for WC measurements have been identified (80). • When a person with an enlarged adipose tissue is standing, gravity pulls all the fat tissue downward, so waist circumference may not be accurate in assessing the intraabdominal fat, especially in those who are severely obese (69). • There are currently no guidelines for the classification of obesityrelated health risks among children and adolescents by using waist circumference (75)

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15 Table 1-1 Continued Method Advantages Disadvantages Waist-toHip Ratio (WHR) • WC and WHR are the most commonly used anthropometric measurements to quantify intraabdominal fat (79). • Together with WC, have good correlation with CT scan (79). • Studies have shown that WC alone is more useful and accurate than with WHR (73,69). Method Advantages Disadvantages Skin folds • Skinfold measurements are able to predict body fatness better than BMI because subcutaneous fat can be directly measured with a caliper (78). • Studies on the efficacy of anthropometric techniques for identifying children with high central adiposity are scarce (73). • Training and precision are required on the part of the person taking the measurement. Method Advantages Disadvantages Ultrasound • Stolk et al have reported that abdominal ultrasound, using strict protocol is a reliable and reproducible method to assess the amount of intra-abdominal adipose tissue compared to both CT and MRI (79). • It is not as costly in comparison to DXA, MRI, and CT scans (81). • No exposure to radiation (81). • Previously, the reproducibility of ultrasound has been questioned (79). • Ultrasonography is still more expensive than simple methods. Method Advantages Disadvantages DXA • Dual energy X ray absorptiometry (DXA) is a relatively simple technique for evaluating total and regional adiposity in children of all ages (73). • DXA cannot distinguish between intra-abdominal and subcutaneous fat (73).

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16 Table 1-1 Continued Method Advantages Disadvantages MRI singleslice scan • After CT, the most accurate anatomical method of measuring visceral obesity (69). • A single abdominal L4/L5 image correlates highly with visceral adipose tissue volume and also with metabolic variables. (69) • It is also less time consuming and costly than full scans. (69) • High cost, long duration, high technical skill requirement. (69) • Single slice MRI fat mass assessment does not appear to be able to predict the whole visceral fat mass due to regional intra-subject body variability. (69) Method Advantages Disadvantages CT scan • Gold standard for measuring visceral obesity (79). • High cost, long duration, high technical skill requirement. (69) • Exposure to radiation. (73,69) .

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17 CHAPTER 2 METHODS HIV-Associated Metabolic Dysfunction In order to define the HIV-associated Metabolic Dysfunction (HAMD), a literature search was performed in PubMed with key words, “Metabolic Syndrome definition” and “Metabolic Syndrome and adolescents” to dete rmine the existing definitions that define the Metabolic Syndrome. The inclusion criter ia were any pediatric study that defined the Metabolic Syndrome or used an established definition of the Metabolic Syndrome including those that utilized age-adjusted va lues. For adults, there are currently five formal definitions of the Metabolic Syndrome ( Table 2-1 ) (84,6-8). For the pediatric population, 10 studies were found ( Table 2-2 ) (84-90,1,62,12). Rodriguez-Moran created their own definition for the Metabolic Syndrom e and assigned points fo r the presence of abnormal risk factors (85). Cruz, Csabi, Chen, and Lambert also deviated from the established definitions (62,87,86,90)). Cruz assessed the prevalence of 0, 1, 2, 3 or more components of the Metabolic Syndrome (62). Chen did a factor analysis of the risk factors (86). Cook, De Ferranti, Duncan, and Weiss used adjusted versions of the ATP III definition to determine the preval ence of the Metabolic Syndrome (1,12,88,89). Goodman also used an adjusted version of the ATP III definition but contrasted the ATP III and WHO definitions (84).

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18 Table 2-1: Metabolic Syndrome Definition in Adults ADULT METABOLIC SYNDROME DEFINITIONS: World Health Organization (WHO-1999) Diabetes or Impaired Glucose Tolerance, Impaired Fasting Glucose or Insulin Resistance + 2 or more of these: Dyslipidemia: Triglycerides 150 mg/dl HDL < 35 mg/dl males, < 39 mg/dl females Hypertension: Blood pressure 140/ 90 mmHg (s,d) or on medication Obesity: BMI > 30 kg/m^2 and or WHR > 0.9 males, > 0.85 females Microalbuminuria: albumin > 20 mcg/min or albumin:creatinine > 30 mg/g European Group for the study of Insulin Resistance or Hyperinsulin emia (non-diabetic subjects) + 2 or more of these: Insulin Resistance (EGIR1999) Fasting Plasma Glucose 110 mg/dl Dyslipidemia: Triglycerides > 180mg/dl HDL < 40 mg/dl or treated for dyslipidemia Hypertension: Blood pressure >140/90 mmHg (s/d) Central Obesity: Waist circumference > 94cm males, > 80 cm females National Cholesterol Education Program 3 or more of these: Adult Treatment Panel III (NCEP:ATPIII-2001) Fasting Plasma Glucose 110mg/dl Hypertriglyceridemia: Triglycerides 150 mg/dl and/or Low HDL Cholesterol: < 40 (men), < 50 mg/dl (women) Hypertension: Blood pressure 130/ 85 mmHg (s,d) and/or medication Central obesity: Waist circumference > 102 cm males, > 88 cm females American Association of Clinical Endocrinologists Clinical judgement: (AACE_2003) Fasting plasma glucose between 110-126 mg/dl 2 hour post glucose challenge > 140mg/dl

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19 Table 2-1. Continued ADULT METABOLIC SYNDROME DEFINITIONS: Triglycerides 150 mg/dl Low HDL Cholesterol: < 40 mg/dl (men), < 50 mg/dl (women) Hypertension: 130/ 85 mmHg Overweight/Obesity: BMI 25 kg/m^2 Family history, age, sedentary, ethnic group International Diabetes Federation (2005) Combination of EGIR, ATP, and WHO Central obesity: Specific for ethnic group, In U.S. use ATP + any two of these: Triglycerides > 150mg/dl or treatment Low HDL Cholesterol: < 40 mg/dl (men), < 50 mg/dl (women) or treatment Raised Blood Pressure: 130/ 85 or treatment Fasting Plasma Glucose: 100 mg/dl or previous diabetes diagnosis, if >100mg/dl OGTT strongly recommended. Table 2-2: Metabolic Syndrom e Definition in Pediatrics PEDIATRIC METABOLIC SYNDROME DEFINITIONS: Research Group on Diabetes and Chronic Illnesses Step 1: Assign 1 point for one or more family phenotype of obesity (REGODCI-2004) Assign 1 poi nt for presence of low or high birth weight Assign 1 point for presence of obesity: BMI 90th percentile for age and gender Assign 1 point for presence of hypertension: BP 90th percentile for age and gender Need 2 or more points to go to step 2 Step 2: Fasting blood glucose: 110 mg/dl Fasting Triglyceride: 90th percentile for age and gender Fasting HDL: < 35 mg/dl

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20 Table 2-2. Continued. PEDIATRIC METABOLIC SYNDROME DEFINITIONS: Assign 1 point for each abnormal value 3 points = Metabolic Syndrome Chen et al. (1999) Fasting Plasma Insulin > 75th percentile for age, gender, ethnicity Hypertension: BP > 75th percentile for age & gender or on BP treatment Dyslipidemia: TG:HDL ratio > 75th ercentile for age, gender, & ethnicity Obesity: Ponderal Index (wt/ht3) 75th percentile for age, gender, & ethnicity Csabi et al. (2000) Simultaneous hyperinsu linemia, hypertension, impaired glucose tolerance, and dyslipidemia Hyperinsulinemia: Fasting Plasma Insulin > 18.7 µU/ml (>95th percentile) Hypertension: > 95th percentile for height & gender Impaired glucose tolerance: OGTT Dyslipidemia: TG < 97 and >130 and/or TC > 200mg/dl and/or HDL < 35mg/dl Cook et al. (2003) ATP III, age-adjusted values 3 or more of these: Fasting Plasma Glucose 110mg/dl Hypertriglyceridemia: Triglycerides 110 mg/dl and/or Low HDL Cholesterol: 40 mg/dl for both males and females Hypertension: Blood pressure 90th percentile for height, age, & gender Central obesity: Waist circumference 90th percentile for age, gender, & ethnicity Cruz et al. (2004) Components of the Metabolic Syndrome (0,1,2,3 or more) and insulin sensitivity

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21 Table 2-2. Continued. PEDIATRIC METABOLIC SYNDROME DEFINITIONS: Fasting Plasma Glucose 110mg/dl Impaired Glucose Tolerance: OGTT glucose b/t 140 mg/dl and 199 mg/dl Hypertension: Blood pressure 90th percentile for height, age, & gender Dyslipidemia: Triglycerides 90th percentile for age,gender & ethnicity, and/or HDL < 10th percentile for age & gender Central Obesity: waist circumference > 75th percentile for gender & ethnicity De Ferranti et al. (2004) Circulation ATP III age-adjusted values Fasting Plasma Glucose 110mg/dl Hypertension: Blood pressure 90th percentile for height, age, & gender Dyslipidemia: TG 100 mg/dl and/or HDL < 45 mg/dl for boys 1519 yrs, HDL < 50 mg/dl for other boys and girls The first step in defining HAMD was to ev aluate current parameters measured in the various definitions above. Abdominal obesity and fat distribution, dyslipidemia, hypertension, glucose intolerance and/or insulin resistance were consistently measured in the majority of the studies. Other factors such as microalbuminuria, and family history of disease were not consistently measured or documented. A trend observed within the literature was an attempt to simplify the defi nition by the use of fewer measurements to account for one parameter. In the HAMD de finition it was sought to incorporate all relevant factors that may lead to the Metabo lic Syndrome. For abdominal obesity or fat redistribution the literature supported the use of body mass index (BMI) as a marker of total body obesity and waist circumference as a measure of visceral adiposity or fat

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22 distribution (73,75-78). The cal culation of the Gator Circle was included for a clearer picture of the visceral cavity area since visceral obesity ha s been found to correlate the most with the risk for cardiovascular diseas e (18,69). Body fat percentage from BIA was also used to incorporate the effects of total body fat percentage on the progression of metabolic dysfunction (91-92). Triglycerides , HDL cholesterol, and LDL cholesterol were markers for the presence of dyslipidemia (93-95). High plasma LDL cholesterol has been shown to increase the risk of cardi ovascular disease. Measuring non-HDL gives a measure for LDL and other po tentially harmful c holesterol within th e body when direct measurement of LDL has not been made and calculation of LDL is not possible. NonHDL was included as a measure for dyslipidem ia because FriedewaldÂ’s equation for LDL calculation is only valid if tr iglycerides are less than 400 mg/dl (93). For hypertension, the National Task Force on Blood Pressure stre sses the importance of using percentiles adjusted for height, age, and gender for th e pediatric population to accurately compare the pediatric population to norms (95). Gl ucose intolerance was measured by fasting plasma glucose. Fasting plasma insulin wa s included because studies have shown that the development of abnormal fasting plasma insulin precedes an abnormal plasma glucose, which decreases the latterÂ’s potential as a screening agent (11,89). Indices are used to interpret the results of the fasting pl asma insulin. Insulin assays are not widely standardized (98). Insulin indi ces have been used to assess insulin resistance (99). Most indices require the use of fasting plasma glucose, fasting plasma insulin, and mathematical calculations (100). In this st udy, insulin values were not obtained and thus insulin resistance indices were not utilized. However, from a small study performed by another student in our laborator y, insulin values were obtained from a subset of this HIV-

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23 infected population and results from this study will be used to propose the need for insulin measurements. The diagnostic parameters within the HAMD definition refer to obesity, fat distribution, growth, hypertri glyceridemia, hypercholestero lemia, hypertension, glucose intolerance, and insulin resistance ( Figure 2-1 ). The definition consists of 11 parameters: BMI, waist circumference, body fat percen tage, Gator Circle, height z-score, triglycerides, LDL or nonHDL depending on triglyceride levels, HDL, resting blood pressure, fasting plasma glucose, and fasting plasma insulin ( Table 2-3 ). The definition was divided into two stages, Stage 1 and Stage 2, respectively. Stag e 1 pertained to the values for the parameters that were approach ing abnormal values and Stage 2 pertained to the values for the parameters that had reached abnormal values. Every abnormal value at the Stage 1 level was assigned 1 point each. Ab normal values at the Stage 2 level were assigned 2 points each. The two stages were exclusive in that a patient that received points for a parameter in Stage 1 could not receive points for that same parameter in Stage 2. It was possible to re ceive 0 points in Stage 1 and Stage 2 if the respective values obtained for a particular parameter were be low the specified cutoff ranges. Two groups of patients were identified; those that were at risk for HIV-associated Metabolic Dysfunction and those that had HIV-associat ed Metabolic Dysfunction. A patient was identified as being at-risk for HAMD if they had the equivalent of 2 metabolic abnormalities but less than the equivalent of 3 metabolic abnormalities. These patients would have from 4-5 points. The cut-off fo r having HAMD was set at an equivalent of having 3 or more metabolic abnormalities wh ich would amount to 6 or more points.

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24 These points could also be gained by having at-risk abnormalities th at amount to 6 or more points. Insulin Resistance Dyslipidemia Hypertension ObesityHAMD Parameters Growth Figure 2-1: HAMD Parameters Patient Data Tracking It is important for patien t progress to be monitored on a longitudinal basis to improve patient care. In order for this to occur, there must be a systematic manner of assessing patients. This is not always possible using indi vidual patient charts. The development of a database in which relevant questions regarding a patient or population can be addressed is crucial. A database that allowed for longitudinal monitoring of patients was developed in orde r to assess the risk of th e above-mentioned population for HIV-associated Metabolic Dysfunction. The data base consisted of 7 tables that contained 136 parameters ( Appendix A ). The tables consisted of th e Patient Demographics table, the Immunology table, the Growth and Body Composition table, the BIA table, the Food Intake table, the Chemistry table, and the Gator Circle table.

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25Table 2-3: HIV-associated Me tabolic Dysfunction Definition DIAGNOSTIC PARAMETERSSTAGE 1 (at risk)*STAGE 2 (severe)† References OBESITYBMI 85-95 %ileBMI 95 %ileCDC Obesity Definition (36) Body Fat Percentile 85th %ile butBody Fat Percentile 95 th %ileKatzmarzyk (2004) (37) < 95 %ile adjusted for age, sex, adjusted for age, sex, ethnicity ethnicity FAT DISTRIBUTIONWC 75th %ile but < 90th %ile WC 90th %ile adjusted for MuellerProject Heartbeat (2004) (38) adjusted for age, gender, & ethnicityage, gender, & ethnicity VCA 75th %ile but < 90th or MSF VCA 90th %ile or MSF Taken from WCConsistent w/ WC 25 %ile but > 10th percentile adjusted 10 %ile adjusted for age for age GROWTHHeight z-score > -2 but <-1.5Height z-score < -2CDC Stunting Definition (36) HYPERTRIGLYCERIDEMIATG 75th %ile but < 90th %ile TG 90th percentile adjustedNCEP: Children & Adolescents (39) adjusted for age and genderfor age and gender HYPERCHOLESTEROLEMIALDL 75th percentile adjusted for age, genderLDL 90th %ile adjusted for age, genderNCEP: Children & Adolescents (39) or non HDL 75th %ile but < 90th or non HDL 90th %ile adjustednonHDL Srinivisan (Bogalusa HS) (40) %ile adjusted for age, gender for age, gender & ethnicity & ethnicity HDL >10th but < 25th adjustedHDL < 10th percentile adjustedNCEP: Children & Adolescents (39) for age & genderfor age, & genderCruz (2004), Lambert (2004) (9,14) HYPERTENSIONBP >90th but <95th %ile ad j ustedBP 95th percentile ad j ustedNational Hi g h Blood Pressure for hei g ht, a g e, and g enderfor hei g ht, a g e, and g enderEducation Pro g ram: 1997 Update GLUCOSE INTOLERANCEFasting Plasma Glucose Fasting Plasma Glucose American Diabetes 100-< 126 mg/dl> 126 mg/dlAssociation INSULIN RESISTANCE Fastin g Plasma Insulin: 1-2Fastin g Plasma Insulin: 3 orRespective Stud y CutOffs ( 33-35 ) abnormal indicesmore abnormal indices *1 pt. for each abnormal parameter. Maximu m amount of points for Stage 1 is 11 pts. † 2pts. for each abnormal parameter. Maximum amount of points fo r Stage 2 is 22 pts. Insulin indices include Fasting glucose to insulin ratio (FGIR), Homeostatic Model Assesssment of Insulin Resistance (HOMAIR), Quantitative In sulin-Sensitivity Check Index (QUICKI), Insulin Sensitivity Index (ISI McAuley), and Triglycerides/HDL cholesterol. An individual receives 0 points for normal values below Stage 1 level. Those with a total of 4-5 pts are considered at risk for HAMD. An individual with a total 6 or more points from Stage 1 and Stage 2 is considered to have the HAMD.

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26 Retrospective Analysis A retrospective analysis was conducted usi ng data collected from pediatric (1-26 years of age) HIV positive patients at Sh ands Pediatric Immunol ogy Clinic who attended clinic from January 2004 to December 2004. The protocol was reviewed and approved by the University of FloridaÂ’ s Institutional Review Board. Anthropometric and chemistry data from routine clinic visi ts were transferred from patient -specific Excel spreadsheets to a Microsoft Access database. Data were a udited for accuracy. The data were analyzed using the NCEP ATP III guidelines to determ ine if the population was at risk for the Metabolic Syndrome. The parameters used were the following: age, sex, race, weight, height, BMI, triglyceride concentration, to tal cholesterol, HDL and LDL cholesterol, body fat percentage, blood pressure, waist ci rcumference, fasting blood glucose, and umbilical, suprailiac, midback and tricep skinfolds. Reference populations were used for comparative data analysis. The control HI V negative population from the Pediatric AIDS Clinical Trials Group (pACTG) 1045 study was a reference population for a section (Gator Circle) of this study. Statistical testing was necessary to de termine if statisti cal significance was achieved in the analysis of the data. P earsonÂ’s correlation coefficient was used to determine if a linear relationship existed between the Gator Circle components and indices of insulin resistance. P-values were calculated using the Stat Tools program to determine if the Gator circle components of our population were si gnificantly different than those of the control group and to de termine the significance of the correlation coefficients.

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27 Metabolic Modules In an attempt to standardize the measurements collected across protocols to diagnose and monitor the Meta bolic Syndrome, the Metaboli c Modules were developed. A template was previously designed in our laboratory so that each module would answer a specific metabolic question ( Table 2-4 ). Below this question a list of measurements, the equipment required to do the measurements, a nd the amount of training that is required to obtain these measurements were included. Furthermore, a brief overview of steps involved in taking the measurements, and the case report form needed to record the measurements were also integrated. The individual Modules were separated into the following th ree categories: the frequently obtained parameters, the less frequently obtained parameters, and the parameters that pertain to di etary intake and anemia. The Modules included the following measurements: growth, body composition and body fat distribution, blood lipid levels, blood glucose and insulin status, blood pr essure, mitochondrial metabolism, renal metabolism, bone metabolism, protein nutrition status, anemia level, and dietary intake ( Appenidix B ). The actual measurements are cate gorized into level 1 and level 2 measurements. Level 1 measurements requ ire minimum training, site equipment, and cost whereas, level 2 measurements require advance training, equipment and cost. In testing that require blood samples, level 1 parameters allow draw ing initial non-fasting samples and then drawing fasting samples if the results are abnormal. Although nonfasting blood samples are not ideal, they we re included here for use in occasions where fasting samples are unattainable. Level 2 para meters required an initial fasting blood sample to be drawn.

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28 A standard operating procedure template was also designed to include step-bystep procedures for each measurement within the Modules. General instructions and specific cautions are also included in the standard operating procedures. Once templates for all measurements and standard operating procedures were made, a review of literature, educational websites, and persona l communication with h ealthcare providers provided the information needed to comple te these Modules. Case report forms were gathered from previous Pediatric Aids C linical Trials Groups (PACTG) protocols and revised for inclusion into the Metabolic Modules ( Appendices N-BB ). Visceral obesity has been shown to promote the development of the Metabolic Syndrome. Routine clinical a ssessment of the visceral cavity area is difficult due to the absence of simple, non-invasive methods. In these Modules, the measurement of body fat distribution includes a computation of th e visceral cavity area in relation to the subcutaneous area, which is referred to as the Gator Circle. Table 2-4: Questions addre ssed by the Metabolic Modules Metabolic Modules and Questions Answered Metabolic Module Question Growth Is the patien t growing normally? Body Composition and Body Fat Distribution Does the patient have an appropriate amount of lean body mass? Does the patient have lipoatrophy or lipohypertrophy? Blood Lipid Status Does the patient have dyslipidemia? Blood Glucose and Insulin Status Does the patient have insulin resistance and/or glucose intolerance? Blood Pressure Does the patient have hypertension? Mitochondrial Metabolism Does the patient have mitochondrial dysfunction?

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29 Table 2-4. Continued. Metabolic Modules and Questions Answered Metabolic Module Question Renal Metabolism Does the patient have renal dysfunction? Bone Metabolism Does the patient have bone disease? Protein Nutrition Status Does the pa tient have protein malnutrition? Anemia Does the patient have anemia? Dietary Intake Is th e nutrient intake meeting the nutrient requirements of the patient? Gator Circle The Gator Circle uses anthropometric meas urements to differentiate among visceral and subcutaneous adiposity. This is achieve d by examining the visceral cavity area (VCA), mean skinfolds (MSF), and wa ist circumference (WC) Z-score. Visceral Cavity Area The visceral cavity area was determined with the use of skinfolds and the umbilical waist circumference (UC) ( Figure 2-1 ). This representation is similar to that of a 1999 article by Kotler et al. where a cross-section of the abdomen was taken in HIV-infected and control subjects and visceral and subcut aneous fat were estimated by calculating their relative areas in the abdomen. The subc utaneous fat was measured via the upper abdominal (upper iliac) skinfold. Their resu lts suggested that an thropometric estimation of the subcutaneous tissue was more accurate than the estimation of visceral adipose tissue (96). In our study, the area of the subcutaneous fat was subtracted from the area of the total abdominal cavity. The equation for deriving the area of the visceral cavity originates from the equation used to calcu late the Arm Muscle Area (AMA). This

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30 equation is as follows: Arm Muscle Area = (C – ( x TSF))2/ 4 . Where C represents the mid – upper arm circumference and TSF repres ents the triceps skinfold thickness. The area of the subcutaneous fat, determined usi ng the Triceps Skin fold, is subtracted from the total arm area as determined by using the Upper Arm Circumference. It is assumed that the layer of subcutaneous fat around the arm is constant and that taking the Triceps skinfold of the arm is an accurate measure of the subcutaneous fat at any given point of the upper arm circumference. Figure 2-2: Visceral Cavity Area The same method was used for calculating the visceral cavity area; however the assumption regarding the uniformity of the s ubcutaneous fat layer around the arm could not be made with regards to the visceral cavity. Since the visceral cavity does not lie perfectly centered in the body, a mean of the 3 skinfolds: vertical umbilical skinfold Vertical Supra iliac Skinfold (VSISF) Vertical umbilical Skinfold ( VUSF ) Vertical Mid back Skinfold (VMBSF) Vertical Supra iliac Skinfold (VSISF) x 2 VCA: area of smaller shaded circle UC: circumference of larger unshaded circle UCA: area of larger unshaded circle

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31 (VUSF), vertical supra iliac skinfold (VSISF) and vertical mid back skinfold (VMBSF) was taken to represent the thickn ess of the subcutaneous layer ( Figures 2-2 and 2-3 ). The VSISF is routinely measured from the right si de. It was assumed that the left side was comparable to the right side and thus the valu e of the right side could be doubled. It is frequently difficult to obtain a mid back skinfold in the pediatric population due to limited subcutaneous adipose tissue. In these cas es, a constant of 5 is designated as the VMBSF for the patient. Mean skinfold (MSF) = vertical umbilical sk infold (VUSF) + 2 * vertical supra iliac skinfold (2VSISF) + vertical mid back skinfold (VMBSF)/4. Figure 2-3: Obtaining the Mean Skinfold When calculating the visceral cavity area, the umbilical waist circumference was measured at the umbilical level to the near est 1/10cm. This measure is typically about 2.3-3 cm below the waist. Traditionally, sk infolds are taken using fatfolds that are The subcutaneous layer of fat in the abdomen is not evenly distributed. By using the mean of the 4 skinfolds: vertical umbilical skinfold (VUSF), 2 * vertical supra iliac skinfold (2VSISF) and vertical mid back skinfold (VMBSF), we are able to look at the subcutaneous la y er

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32 obliquely oriented, or in line with the natu ral folds of the trunk as the patient bends. These oblique folds are not us eful in determining a repr esentative cross section of subcutaneous fat, therefore vertical skin folds were used instead. The vertical umbilicus skinfold was measured by pinching the abdomin al fat fold vertically about 2.5 cm to the right of the patientÂ’s umbilicus. The vert ical mid-back skinfold was measured by pinching the back fat fold about 2.5 cm to the ri ght of the spinal column at the level of the umbilicus. The vertical supra iliac skinfold was measured by pinching the side fatfolds vertically at the mid-axillary line and at the level of th e umbilicus (Fields-Gardner, personal communication). The visceral cavity area alone was not us eful for describing visceral adiposity because it did not effectively distinguish betw een the loss or gain of subcutaneous tissue and it did not account for the effects of age, gender or race on the visceral cavity. In addition, it was unclear if increa ses in the visceral cavity area were due to an increase in visceral fat or due to normal growth and development. There are currently no standards that exist for visceral cavity area. Howe ver, control data from p1045, a multicenter trial performed by the Pediatrics Aids Clin ical Trials Group (pACTG) evaluating the prevalence of morphologic and metabolic abno rmalities in HIV-inf ected and uninfected children and adolescents were used to provi de a norm to compare and evaluate the visceral cavity area and m ean skinfolds of our population. Waist Circumference Z score Waist circumference z-scores were used as measures of total abdominal obesity in respects to age, gender and ethnicity. The waist circumference Zscore alone could not distinguish between visceral or subcutaneous fat. Additionally, it could not differentiate

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33 between an increase in subcutaneous adipose tissu e, an increase in visceral adipose tissue or an increase in both. Yet, in combination with the visceral cavity area and the mean skinfolds, these factors are accounted for in the creation of the Gator Circle. The waist circumference Z scores were calculated by us ing waist circumference percentile tables from the Fernandez, Redden, Pietrobelli,and Allison, 2004 study evaluating waist circumference percentiles in nationally re presentative samples of African-American, European American, and Mexican-American Children and Adolescents (97). The 50th percentile from each table was designated as the mean for each age group and gender. It was assumed that a linear relationship exists between the waist circ umferences and their respective percentiles; the wais t circumference from the 68th percentile was extrapolated to calculate the standard deviation. The st andard deviation was calculated by taking the difference between the table value for waist circumference at the 50th percentile and the extrapolated value at the 68th percentile. This standard was used for children from 2 to 18 years of age. Waist percentiles for 19 year old patients were extrap olated using the slope and y intercept of each individua l percentile line. Extrapolat ions could not be made for patients greater than or equal to 20 years old because it could not be assumed that the waist continues to increase infinitely as ag e increases as there is a time within normal development when the waist normalizes. Fo r patients twenty years old or older the NHANES III waist percentile tables for the Unit ed States (1988-1994) by race-ethnicity and age were used. Studies have shown that patients whose waist circumference exceed the 75th percentile have significantly higher odds of having high bl ood pressure, high low density

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34 lipoprotein cholesterol levels and high triglyceride levels (75) . This cluster of symptoms is also found in patients with high de posits of visceral adipose tissue. Gator Circle The Gator Circle is the relationship am ong the waist circumference z-score, the visceral tissue by means of the visceral cav ity area and the subcutan eous tissue by way of the mean skinfolds. By comparing th e VCA and MSF of our population to the p1045 control population, the effects of age and ge nder on the size of these components were taken into account. The Gato r Circle equations are provided in the table below ( Table 25 ).

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35 Table 2-5: Gator Circle Equations Equations Visceral Cavity Area (VCA) ((UC -(3.14*((VUSF /10)+ (2* VSISF/10) +(VMBSF/10))/4))^2) /(4*3.14) Umbilical circumference (UC) Vertical Umbilical Skinfold (VUSF) Vertical Suprailiac Skinfold (VSISF) Vertical Mid Back Skinfold (VMBSF)* Mean Skinfolds (MSF) ((VUSF+2*VSISF+VMBSF)/4) Waist circumference (WC) Z-score ((Waist measured –Waist mean table)/Waist SD table) *VMBSF = “5” when unmeasurable

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36 CHAPTER 3 DEVELOPMENT OF THE METABOLIC MODULES The Metabolic Modules were developed w ithin our laboratory and consisted of a template of parameters indicating metabolic disturbances that are observed in pediatric HIV positive patients. The purpose of these Modules was to provide screening tools for metabolic abnormalities that are easy to use across various protocols and that provide thorough procedures for metabolic measuremen ts. The abnormal features that were described in the Modules include growt h, body composition, body fat distribution, blood lipids, blood glucose and insulin, blood pressure, mitochondrial metabolism, renal metabolism, bone metabolism, protein nut rition, anemia, and dietary intake ( Appendices B-M ). Measurements and laboratory tests we re chosen to assess each abnormal feature or parameter by the use of available literatur e on that parameter. Reference ranges for laboratory tests were provided ( Appendix CC ). The Pediatric Aids Clinical Trials Group (PACTG) is a worldwide organization invol ved in evaluating the treatments for HIVinfected children and adolescents, and for de veloping new approaches for the interruption of mother-to-infant transmission of the viru s. The PACTG was the first organization to utilize the Modules and as a result, components of the Modules were revised to be familiar to this group. Growth Prior to the initiation of highly active anti-retr oviral therapy (HAART), the loss of weight due to decreases in muscle mass or “wasting”, and decreased height were dominant issues in pediatric patients w ith HIV (52,107-108). After HAART’s initiation,

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37 the weight dilemma pendulum seemed to sw ing to the opposite side where increasing fat deposits in particular sites caused weight to increase (109). In or der to assess growth, parameters of age, gender, height/le ngth and weight are included according to recommendations by the National Center for H ealth Statistics (NCHS) for determining and comparing body mass index (BMI), percent iles and z-scores (110). The Standard Operating Procedure (SOP) and Case Report Forms (CRFs) for the Growth measurements were excerpted from the Pedi atric Aids Clinical Trial GroupÂ’s (PACTG) P1045 study which was a multi-center trial that examined the prevalence of morphologic and metabolic abnormalities in vertically HI V-infected and uninfected children and youth. Supporting information for the length m easurement SOP for infants including the formal definition of Frankfurt horizontal plane and proper subject positioning was excerpted from the Maternal and Child Health BureauÂ’s training module website (111). Body Composition and Body Fat Distribution Body composition, or the amount of fat mass, muscle mass, and bone mass within the body can greatly affect the homeostasis of metabolic functions. Increases in adipose tissue have been shown to alter the normal metabolic state and cause obesity, insulin resistance, hypertension, gluc ose intolerance, and dylipid emia (112). Research has revealed changing body composition of pa tients on HAART. Intra-abdominal fat accumulates within the visceral cavity (109,113,59). Fat clusters in the back of the neck are referred to as the buffalo hump and ad ipose tissue has also been observed to accumulate in the buttocks and breasts (59,67,113-114). To determine body fat percentage, the bioelectrical impedance anal ysis (BIA) was used. The BIA has been supported by several studies as a beneficial noninvasive method of measuring the percent of fat within the body (67,115,92). The BIA me asurement was included as a Level II

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38 parameter because protocols with limited fina ncial resources may not have access to this instrument. The SOP and CRF for the BIA measurements were taken from the PACTG procedures for BIA measurements. Anthropometric measurements, such as circumferences and skinfolds, have been ac knowledged as reliable estimators of body fat distribution (116,118,69). Circumfe rences at the mid-arm, mi d-thigh, umbilicus and hip sites were included as measurements to esti mate body fat and muscle mass. Skinfolds at the triceps, subscapular, and mid-thigh s ites were used as estimates of body fat percentage, muscle mass, and subcutaneous fa t. Skinfolds at the umbilicus, supra iliac and mid-back sites were used for the same reasoning above but also to calculate the visceral cavity area (VCA). The anthropom etric SOP and CRF were excerpted from the PACTG P1045 procedures for anthropometric measurements. The Gator Circle, as previously described, is a calcu lation of the VCA with regards to the space that the VCA incorporates within the umbilical cavity and with the effects of age, normal growth, and gender integrated. The Gator Circle was included as a noninvasive method of determining the proportion of th e umbilical cavity area to the total umbilical cavity. However, methods of greater precision a nd expense, such as the dual energy x-ray absorptiometry (DEXA) and Magnetic Resonance Imaging (MRI), do exist and are used for diagnosis of abnormal body composition and body fat distribution (117,73). The SOP for the Gator Circle has been proposed in this thesis. Th e DEXA was included as a Level II parameter for body composition. The SOP and CRF for the DEXA were excerpted from the PACTG P1045 procedures for DEXA an alysis. Visible evid ence of lipoatrophy and lipohypertrophy were used and their sever ity was stratified by th e National Institute of Allergy and Infectious DiseasesÂ’ (NIA ID) Division of Acquired Immunodeficiency

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39 SyndromeÂ’s (DAIDS) Severity grading table ( Appendix DD ). The CRFs for evidence of lipoatrophy were excerpted from the PACT G body image questionnaire and physical assessment form. The SOP and CRF for the DEXA were excerpted from the pACTG procedures for DEXA analysis. Diagra ms for growth, body composition and body fat distribution were included. Blood Lipids Historically, blood laboratory values have been used as a means of assessing the functionality of several human body system s. The blood lipid values have been implicated as risk factors for cardiovascular disease and therefore, should be closely monitored (119,120). Fasting lipids were preferred to nonfasti ng lipids because of th e effect of food on parts of the lipid profile (121-122). If fas ting blood samples were not available and/or were difficult to obtain, blood lipids should be analyzed for a nonfasting sample. If elevated concentrations were obtained from the initial nonfasting sample, then a fasting sample should be obtained. Triglyceride, HDL, calculated LDL, and total cholesterol labs were used to assess cardi ovascular disease risk. Since the calculation for LDL is only valid with triglycerides le ss than 400 mg/dl, a fasting LDL sample was included as a Level II parameter. The SOP for the blood lipid profiles was excerpted from the PACTG P1045 lipid profile procedures. The CRF was taken from the PACTG P1030 study CRFs. FriedewaldÂ’s equation for calculating LD L was taken from a 1972 article (123). Blood Glucose and Insulin Status Insulin resistance is a major component of the Metabolic Syndrome which can lead to diabetes mellitus and cardiovascular disease (124). Fasting plasma glucose, fasting blood insulin and the two hour glucose toleranc e test were used to determine insulin

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40 resistance, glucose intolerance and diabetes risk. Fasting plasma glucose was preferred but if unavailable, a nonfasting sample was evaluated. For insulin and the two hour glucose tolerance test only fasting labs we re assessed because of the limitations on the validity of the results if ta ken nonfasting. Since fasting insulin and the two hour glucose tolerance test are not routinely measured, they are included as Level II parameters. The SOPs and CRFs for the blood glucose and insu lin status parameters were excerpted from the PACTGÂ’s P1045 glucose and insulin status procedures. Blood Pressure Hypertension is another major risk factor for the Metabolic Syndrome, cardiovascular disease, and stroke (61). Blood pressure was used to measure hypertension. The most common problem that leads to invalid blood pressure measurement is the use of an inappropri ate cuff size. The National Kidney Foundation provides procedures and figures to promote proper cuff size se lection (125). The SOP for blood pressure was taken from the encyclopedi a of healthscout.com. The CRF for blood pressure was taken from the PA CTGÂ’s pediatric vital sign CRF. Mitochondrial Metabolism Mitochondrial toxicity is an emerging effect of anti-retroviral therapy use by HIV-infected patients (126-127). This toxici ty can be observed in terms of metabolic acidosis, liver or pancreatic disorders. The anion gap was used to determine if acidosis was present and if so, to be able to differentia te between the causes of metabolic acidosis. The liver function tests (LFTs) were used to determine if the liver was functioning optimally or if liver disease was present. Levels of amylase and lipase were used to assess if pancreatic disease was present. Lactic acid was included to determine if lactic acidosis was present. Mitochondrial DNA was used to assess mitochondrial dysfunction.

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41 Lastly, acylcarnitine profiling was included as a means of determining if a fatty acid disorder was present. General instru ctions for lab tests were taken from labtestsonline.org. Except fo r the anion gap and the LFTs, which are routinely measured, all other mitochondrial metabolism tests were considered Level II parameters. The SOP for the anion gap, LFTs, amylase, lipase, and mitochondrial DNA were excerpted from the PACTGÂ’s P1045 study procedures. The SOP for acylcarnitine profiling was taken from the medical genetics website of D uke University (128). The CRF for the mitochondrial metabolism test were excerpt ed from the PACTGÂ’s P1045 study CRFs. Renal Metabolism Renal dysfunction is seen as an effect of HIV infection and as an effect of the Metabolic Syndrome (129-130). Serum BUN and creatinine levels were used to determine kidney function. Serum HCO3-, potassium, and phosphate were used to determine if renal tubular dysfunction was presen t. Urinalysis was used as an indicator of renal dysfunction. The urine protein: creat inine ratio was also used as a measure of renal dysfunction but as a Level II parameter. Creatinine clearance was used to assess the efficiency and rate of kidney filtration and included as a Level II parameter. General instructions for lab tests were taken from labtestsonline.org and webmd.com. The SOPs and CRFs for renal metabolism tests were excerpted from the PACTGÂ’s P1045 renal metabolism procedures. Bone Metabolism Bone disease has been observed as a longterm effect of HIV infection, HAART, or both (131,114). Radiographically proven fract ures from routine care x-rays were included as a level I parameter of bone fracture. The measurement of alkaline phosphatase was also included as a level I parameter for bone disease. The DEXA was

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42 included as a level II parameter to measur e bone density. Bone specific alkaline phosphatase (BSAP) was included as a more specific test for monitoring bone formation in patients with established bone disease. BSAP was included as level II parameter. Since, bone formation alone cannot efficiently depict bone turnover, the urinary crosslinked N-telopeptide (Ntx) was used as an indicator of bone resorption. The urinary cross-linked Ntx was also included as a leve l II parameter. The SOP for the alkaline phosphatase, BSAP, and Ntx were excerpted from the PACTGÂ’s P1045 study and the aruplab.com website. Thorough x-ray procedures are available from the National Health and Nutrition Examination Survey IIIÂ’s lab manual (132). The CRFs for the bone metabolism tests were excerpted fr om the PACTGÂ’s P1045 study CRFs. Protein Nutrition Status Protein energy malnutriti on is a common complicati on of HIV infection and HAART has the potential to cause malnourishmen t due to its side effects which include vomiting and diarrhea (133). When HIV infection and HAART are coupled in countries that already consist of malnourished indivi duals, complications associated with the disease may increase. Albumin was used to measure long-term protei n nutrition status. Prealbumin was used to measure short-term protein malnutrition status. The SOPs and CRFs for albumin and prealbumin were ex cerpted from the PACTGÂ’s P1045 nutrition status procedures and CRFs. Anemia Status Whereas anemia may not be a major abnor mality in patients with HIV-infection within the United States; in other countries it is an inherent abnormality in malnourished patients without HIV infection and is a ggravated by HIV infection (134-136). Hemoglobin, hematocrit, red blood cell count, and mean corpuscu lar volume were

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43 included as part of a complete blood count to assess anemia. The SOPs and CRFs for the anemia status tests were excerpted from th e PACTGÂ’s P1045 anemia status procedures and CRFs. Dietary Intake Similar to anemia and protein nutrition, the complications of HIV infection and HAART therapy can be intensified by poor nutrition (137,133). The 24 hour diet recall, the 3 day diet diary and diet ary assessment tool were included as means of assessing whether dietary intake is mee ting nutrient requirements. Th e 3 day diet diary requires that the subject is knowledgeable about food portions and was included as a level II parameter. The dietary assessment tool requires a food composition database and specific computer software which may be unfeas ible in some countries and consequently it was included as a level II parameter. Th e SOPs for dietary intake parameters were taken from our lab handbook of dietary in take evaluation procedures and Food and Agricultural Organization of the United Nations website (138). The CRFs for the dietary intake were excerpted from the PACTGÂ’s P1045 study CRFs.

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44 CHAPTER 4 DEVELOPMENT OF HIV-ASSOCI ATED METABOLIC DYSFUNCTION DEFINITION The HIV-Associated Metabolic Dysfunc tion (HAMD) defini tion was developed with the intent of depicting the types of metabolic abnorma lities that are common to the pediatric HIV positive population. The c oncept of the Metabolic Syndrome was incorporated to assess the conglomeration of metabolic abnormalitie s. Another goal of this definition was to be able to identify groups that are at-risk for developing multiple metabolic abnormalities and therefore at an increa sed risk for heart disease. In order to identify these groups the definition containe d two stages, Stage 1 and Stage 2. Stage 1 contained lower at-risk values for all com ponents and Stage 2 contained higher and more severe values for the components. Five f actors that were examined in the definition included obesity, linear growth, dyslipidemia, hypertension, and insulin resistance. The parameters that compose the HAMD defin ition were obesity, fat distribution, linear growth, hypertriglyceridemia, hypercholesterolemia, hyperten sion, glucose intolerance, and insulin resistance ( Table 2-3 ). Each parameter consisted of measurements that were obtained to assess the severity (or lack of) of the parameter. Obesity The prevalence of obesity has dramatical ly increased over the last three decades in industrialized countries (3). Although obe sity did not play a huge role in the HIV positive population at the emergence of this disease, it has evolved to play a significant role within this population because of the highl y active antiretroviral therapy that is used

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45 to treat the disease and th e increasing prevalence of obe sity itself (53,9,114,59). The obesity risk factor was divide d into two separate parameters ; one that pertained to total body obesity and body composition and the other to fat distribution within the body (Figure 4-1) . BMI has been used in epidemiological studies and has been suggested to be a good predictor of total body obesity (75,71). BMI was used as a measurement of total body obesity. The Stage 1 cut-off for BMI was set as greater than or equal to the 85th but less than the 95th percentile adjusted for age and gender according to definition of “overweight” by the Center for Disease Contro l (CDC). The Stage 2 cut-off was set at greater than or equal to the 95th percentile according the CDC’s “obesity” definition. Overall BMI Body Fat Percentage Fat Distribution WaistCircumference Gator Circle OBESITY Figure 4-1: Obesity Parameter

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46 Table 4-1: Obesity Cut-offs ObesityKatzmarzyk (2004) Waist Circumference 90th %ile (age, gender, ethnicity) Waist Circumference 75-<90th %ile (age, gender, ethnicity) CDC Obesity Definition BMI 95th %ile BMI 85-<95th %ile Obesity Reference Stage 2 (severe) Stage 1 (at risk) DIAGNOSTIC PARAMETER Body fat percentage percentiles determined from Bioelectrical Impedance Analysis (BIA) measurements were used as a measur e of body composition. Studies that compare the BIA to Dual X-ray Absorptiometry (DEXA) show that there is a high correlation between the results of the two methods although systematic differences do occur (92,115). There are several estimates of body fa t percentage, therefore it is important to use an estimate consistently throughout a popul ation to avoid inter-e stimate variations. The Arpadi method was used as re ported in an article by Horlic k et al. (139). The Stage 1 cut-off for percent body fat percentiles wa s set at greater than or equal to the 85th percentile but less than the 95th percentile adjusted for age, gender and ethnicity based on a study by Mueller et al. where the 85th percentile was considered as defining excessive body fat (104). Stage 2 was set at greater than or equal to the 95th percentile.

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47 Fat Distribution Another means of assessing obesity is to examine body fat distribution. Waist circumference was included as an indicator of abdominal obesity and predictor of visceral obesity (75,71,69,73). The Stage 1 cut-off for waist circumference was greater than or equal to the 75th percentile but less than the 90th percentile adjusted for age, gender, and ethnicity. This cut-off was set because a st udy by Savva et al. reported that patients whose waist circumference exceed the 75th percentile have significantly higher odds of having high blood pressure, high low density lipoprotein cholesterol levels and high triglyceride levels (140). Th e Stage 2 cut-off was set at gr eater than or equal to the 90th percentile because a study by Maffeis report ed that prepubertal children with waist circumferences greater than or equal to the 90th percentile had a greater chance of having multiple heart disease risk factors (141). The presence of intra-abdominal fat has been implicated in promoting an atherogenic profil e (142). The Gator Circle is a computation of intra-abdominal fat with relations to subcutaneous fat and normal growth. Comparative control data for the Gator Ci rcle components (VCA, VC%, WC Z-score) were obtained from the pACTG p1045 study. St age 1 cut-offs for VCA was greater than or equal to the 75th percentile but less than the 90th percentile adjusted for age whereas the Stage 2 cut-off was set at greater than or equal to the 90th percentile. The Mean Skinfold was found to be highly correlated (r=-0.93, p<.001) with the VC% and were used instead of the VC% to limit calculation variations. The Mean Skinfold Stage 1 cutoff was less than or equal to the 25th percentile but greater than the 10th percentile adjusted for age and the Stage 2 cut-off was 10th percentile.

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48 Growth Several studies have shown that HIV-inf ected children and adolescents grow less rapidly than their normal count erparts (143-144). This lack of linear growth, or stunting, has been observed to correlate with the subjec tÂ’s viral load, inferring that the virus itself is interfering with the normal metabolism and growth of the subjects (143-145). Height z-scores were used according to CDC growth charts to determine stunting in this population ( Figure 4-2 ). The Stage 1 cut-off was set at a z-score greater than or equal to 1.5 but less than -2. The Stage 2 cut-off was set at -2 z-score according to the stunting definition by the CDC (106). LINEAR GROWTH Height Z-score Figure 4-2: Growth Parameter

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49 Table 4-2: Growth Cut-offs Linear Growth Reference Stage 2 (severe) Stage 1 (at risk) DIAGNOSTIC PARAMETERCDC Stunting Definition Height z-score -2 Height z-score (-1.5)> (-2) Linear Growth Dyslipidemia Hypertriglyceridemia, hypercholesterolemi a, and low HDL concentrations have been cited as risk factors for heart disease (146) ( Figure 4-3 ). The cut-offs for the lipid profile were taken from the National C holesterol Education ProgramÂ’s Report for Children and Adolescents and also from th e National Cholesterol Education ProgramÂ’s Adult Treatment Panel III for patients older than 19 years of age. For children and adolescents percentiles were used when avai lable to incorporate the effects of normal growth on the measurements. The Stage 1 cutoff for triglycerides was greater than or equal to the 75th percentile but less than the 90th percentile adjusted for age and gender for children and adolescents and greater than or equal to 120mg/dl but less than 150mg/dl

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50 for older patients. The Stage 2 cut-off for tr iglycerides was greater than or equal to the 90th percentile and greater than or equal to 150mg/dl for children and adolescents, and older patients, respectively. Th e Stage 1 cut-off for LDL was greater than or equal to the 75th percentile but less than the 90th percentile adjusted for age and gender for children and adolescents and greater than or equal to 100mg/dl but less than 130mg/dl for older patients. The Stage 2 cut-off for LDL was greater than or equal to the 90th percentile and greater than or equal to 130mg/dl for ch ildren and adolescents, and older patients, respectively. The Stage 1 cut-off for HDL was greater than the 10th percentile but less than the 25th percentile adjusted for age and gender for children and adolescents. For adults, the Stage 1 cut-off was less than 60 mg/dl for women and less than 50 mg/dl for men. The Stage 2 cut-off for HDL was set at less than the 10th percentile for children and adolescents and less than 50 mg/dl and 40mg/dl for women and men, respectively. LDL is calculated using FriedewaldÂ’s equation. Th e limitation of this e quation begins to occur at triglyceride levels greater than 200 mg/dl a nd is invalid at trigly ceride concentrations greater than 400 mg/dl (147). In order to incorporate the effects of LDL in a population where hypertriglyceridemia is common due to therapy, non-HDL was used (105,148). The Stage 1 cut-off for non-HDL was greater than or equal to the 75th percentile but less than the 90th percentile adjusted for age and gender for children and adolescents and greater than or equal to 130m g/dl but less than 160mg/dl fo r older patients. The Stage 2 cut-off for non-HDL was greater than or equal to the 90th percentile and greater than or equal to 160mg/dl for children and adolescen ts, and older patien ts, respectively ( Table 43 ). The NCEP Adult cut-offs were used for all lipid measurements of subjects 19 years of age.

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51 DYSLIPIDEMIA Triglycerides HDL LDL or non HDL Figure 4-3: Dyslipidemia Parameter Table 4-3: Dyslipidemia Cut-offs Dyslipidemia Reference Stage 2 (severe) Stage 1 (at risk) DIAGNOSTIC PARAMETERNCEP: Children & Adolescents HDL < 10th%ile (age & gender) HDL 10-<25th%ile (age & gender) NCEP: Children & Adolescents nonHDLSrinivisan LDL 130 mg/dl or nonHDL 90th%ile (age, gender, ethnicity) LDL 110-< 130 mg/dl or nonHDL 75-<90th%ile (age, gender, ethnicity)HypercholesterolemiaNCEP: Children & Adolescents TG 90th%ile (age & gender) TG 75-<90 th%ile (age & gender)Hypertriglyceridemia

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52 Hypertension Obesity is linked to increasing hypertension in children and adolescents (3, 148). A recent study cited that the use of highly act ive anti-retroviral therapy increased the prevalence of hypertension in HIV-infected pa tients (149). Blood pr essure percentiles adjusted for height, age, and gender we re used based on recommendations by the National High Blood Pressure Education Prog ram: 1997 Update (95). The Stage 1 cutoff for blood pressure was great er than or equal to the 90th percentile but less than the 95th percentile ( Figure 4-4 ). The Stage 2 cut-off for blood pr essure was greater than or equal to 95th percentile ( Table 4-4 ). HYPERTENSION Blood Pressure Figure 4-4: Hypertension Parameter

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53 Table 4-4: Hypertension Cut-offs Hypertension Reference Stage 2 (severe) Stage 1 (at risk) DIAGNOSTIC PARAMETERNational High Blood Pressure Education Program: 1997 Update BP 95th%ile (height, age, gender) BP 90-95th%ile (height, age, gender) Hypertension Insulin Resistance The use of highly active anti -retroviral therapy has also been shown to lead to metabolic abnormalities that include insulin resistance and diabetes (60,67,150). Fasting plasma glucose was used as a measurement of glucose intolerance and diabetes according to the American Diabetes Association (109) ( Figure 4-5 ). The Stage 1 cut-off for fasting plasma glucose was greater than or equa l to 100mg/dl but less than 126 mg/dl as a measure of “prediabetes”. The Stage 2 cut-o ff was greater than or equal to 126 mg/dl as an indication of “diabetes” ( Table 4-5 ). Studies have shown that that the development of abnormal fasting plasma insulin precedes an abnormal plasma glucose, which decreases fasting plasma glucose’s potenti al as a screening agent. Fa sting plasma insulin was used to screen for insulin resistance. Insulin assays are not widely standardized (98). Therefore, insulin indices were used to interp ret the results of the fasting plasma insulin.

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54 The use of insulin indices requires fasting pl asma glucose concentration, fasting plasma insulin concentration, HDL con centration, triglycerides concentration and mathematical calculations. Various indices were used to interpret these results ( Table 4-6 ). Since one single cut-off was not defined for each index, the most conservative cut-offs were used where the least amount of individuals would be identified. The Stage 1 cut-off for insulin indices was the presence of two or less abnor mal indices according to their respective indices cut-offs. The Stage 2 cut-off for insulin indices was 3 or more abnormal indices. INSULIN RESISTANCE Glucose Intolerance Fasting Plasma Glucose Hyperinsulinemia Fasting Plasma Insulin (Insulin Indices) Figure 4-5: Insulin Resistance Parameter

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55 Table 4-5: Glucose Cut-offs Glucose Intolerance Reference Stage 2 (severe) Stage 1 (at risk) DIAGNOSTIC PARAMETERAmerican Diabetes Association FPG 126 mg/dl FPG 100<126 mg/dl Fasting Plasma Glucose Table 4-6: Insulin Indices Mo st Conservative Cut-offs Insulin Cut Off Values 3.5 (102)TG/HDL 5.8 (101)ISI 0.33 (153)QUICKI > 4 (151)HOMA < 7 (152)FGIR Cut off Value Parameter

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56 Points were assigned to the stages to assess whether a patient was at risk for multiple metabolic abnormalities and heart di sease and or whether they had multiple metabolic abnormalities ( Table 4-7 ). Every abnormal value at the Stage 1 level was assigned 1 point each. Abnormal values at the Stage 2 level were assi gned 2 points each. The two stages were exclusive in that a pa tient that received points for a parameter in Stage 1 could not receive points for that same parameter in Stage 2. It was possible to receive 0 points in Stage 1 and Stage 2 if th e respective values obtained for a particular parameter were below the specified cut-off range s. A subject was identified as being atrisk for HAMD if they had the equivalent of 2 metabolic abnormalities but less than the equivalent of 3 metabolic abnormalities. Th ese patients would have from 4-5 points. The cut-off for having HAMD was set at an equivalent of having 3 or more metabolic abnormalities which would amount to 6 or more points ( Figure 4-6 ). These points could also be gained by having at-risk abnormalities that amount to 6 or more points.

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57 Table 4-7: HAMD Point System DIAGNOSTIC PARAMETERSSTAGE 1 (at risk)*STAGE 2 (severe)† OBESITYBMI 85-95 %ile0BMI 95%ile0 Body Fat Percentile 85th %ile butBody Fat Percentile 95 th %ile0 < 95 %ile adjusted for age, sex, 0adjusted for age, sex, ethnicity ethnicity FAT DISTRIBUTIONWC 75th %ile but < 90th %ile 0WC 90th %ile adjusted for 0 adjusted for age, gender, & ethnicityage, gender, & ethnicity VCA 75th %ile but < 90th or MSF 0VCA 90th %ile or MSF 0 25 %ile but > 10th percentile adjusted 10 %ile adjusted for age for age GROWTHHeight z-score > -2 but <-1.51Height z-score < -20 HYPERTRIGLYCERIDEMIATG 75th %ile but < 90th %ile 0TG 90th percentile adjusted2 adjusted for age and genderfor age and gender HYPERCHOLESTEROLEMIALDL 75th percentile adjusted for age, gender0LDL 90th %ile adjusted for age, gender2 or non HDL 75th %ile but < 90th or non HDL 90th %ile adjusted %ile adjusted for age, gender for age, gender & ethnicity & ethnicity HDL >10th but < 25th adjusted0HDL < 10th percentile adjusted2 for age & genderfor age, & gender HYPERTENSIONBP >90th but <95th %ile adjusted0BP 95th percentile adjusted2 for height, age, and gende r for height, age, and gende r GLUCOSE INTOLERANCEFasting Plasma Glucose 1Fasting Plasma Glucose 0 100-< 126 mg/dl> 126 mg/dl INSULIN RESISTANCE Fasting Plasma Insulin: 1-21Fasting Plasma Insulin: 3 or0 abnormal indicesmore abnormal indices Stage 1 Subtotal3Stage 2 Subtotal8 Total 11

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58 Stage 2 Stage 1 6 points4-6 points 0-3 pointsHAMDNormal Figure 4-6: HAMD Point Progression Similar to anemia and protein nutrition, the complications of HIV infection and HAART therapy can be intensified by poor nutrition (137,133). The 24 hour diet recall, the 3 day diet diary and diet ary assessment tool were included as means of assessing whether dietary intake is mee ting nutrient requirements. Th e 3 day diet diary requires that the subject is knowledgeable about food portions and was included as a level II parameter. The dietary assessment tool requires a food composition database and specific computer software which may be unfeas ible in some countries and consequently it was included as a level II parameter. Th e SOPs for dietary intake parameters were taken from our lab handbook of dietary in take evaluation procedures and Food and Agricultural Organization of the United Nations website (138). The CRFs for the dietary intake were excerpted fro m the PACTGÂ’s P1045 study CRFs

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59 CHAPTER 5 RESULTS A descriptive summary of subj ect demographic characterist ics is provided in Table 5-1 ( Table 5-1 ). Seventy-seven patients were seen at Shands Pediatric Immunology clinic in 2004. The ages of the patients range d from 1-26 years, with an average age of 14.9 +/6.61 years. Forty percent of this population were male (n=31) while 60% were female (n=46). Twenty five percent of the patients were European American (n=19). Sixty one percent were African-American (n = 47). Eight percent were Hispanic American (n=10) and 4% were Biracial (n=3). In order to look at differences in the prevalence of risk factors that may be aff ected by age the population was divided into 3 groups; 1-12 year olds (n=33) , 13-19 year olds (n=21), and greater than 19 years old (n=23). Table 5-1: Descriptive Characteristics of Pediatric HIV+ Population and Prevalence of MS and HAMD N (%) Patients with MS Patients with HAMD Gender Male 31 (40) 1 (10) 9 (37.5) Female 46 (60) 9 (90) 15 (62.5) Total 77 (100) 10 (12.9) 24 (31.2) Race European-American 19 (25) 2 (20) 6 (25) African-American 47 (61) 7 ( 70 ) 16 (67) Hispanic-American 8 (10) 0 ( 0 ) 2 (8) Biracial 3 (4) 1 (10) 0 Age 1-12 y 33 (43) 1 (10 ) 6 (25) 13-19y 21 (27) 4 (40) 8 (33) 20-26y 23 (30) 5 (50) 10 (42)

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60 The prevalence of the Metabolic Syndro me was assessed using Cook et al.Â’s pediatric definition ( Table 2-2 ). The number of subjects th at met the criteria mentioned in chapter 2 was 77 patients. The preval ence of the Metabolic Syndrome according to Cook et al.Â’s definition was 12.9% ( Table 5-1). Glucose was abnormal in 2.6% of this population. Triglycerides were abnormal in 52.8% of the population. High-density lipoprotein concentration was abnormal in 16.9% of the subjects. Combined, systolic or diastolic pressure was abnormal in 51.4% of the subjects. Systolic blood pressure was abnormal in 50% of the subject s while diastolic blood pressu re was abnormal in 15.3% of the population. Waist circumference was abnormal in 13.2% of the subjects. The HAMD definition described in the prev ious chapter was applied to evaluate metabolic abnormalities in this population. Th e prevalence of HIV-associated Metabolic Dysfunction within this population was 31.2% ( Table 5-1 ). BMI percentiles were calculated for 77 patients based on available da ta. Obesity was prevalent in 16% of the patients, while the percentage of overweight individuals was 14%. The mean BMI was 22.01 +/6.77. Mean and standard deviations for HAMD risk factors are provided in Table 5-2 ( Table 5-2 ). Thirty seven patients had body fat percentage data. The percentage of patients with body fa t percentiles greater than the 90th percentiles was 5%, while that of patients with body fa t percentiles great er than the 75th percentile but less than the 90th percentile was 3%. The average bo dy fat percentage was 19.3 +/12.3. Waist percentiles were calculated in 68 pa tients. Thirteen percent of the patients had a waist circumference grea ter than or equal to the 90th percentile and 18% had waist percentiles greater than the 75th percentile but less than the 90th percentile. The mean waist circumference was 74.09 +/19.07 cm. In examining the Gator Circle, the average

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61 VCA was 388.24 +/160.46. The average mean skinfold was 16.87 +/10.96mm. Using control data obtained from the pACTG P1045 study, VCA and MSF cut-offs were determined. WC z-score was 1.5 but < 2 in 8.8% of the population and was 2 in 22% of the population. VCA z-score was 0.93 but < 1.25 in 7.8% of the population and 1.25 in 9% of the patients. MSF Z-score was -0.45 but greater than -1 in 11.5% of patients and was -1 in 15% of patients. Table 5-2: Averages and Standard Deviations of HAMD Risk Factors N Mean S.D. Age 77 14.9 6.61 Risk Factors: BMI 76 22.01 6.77 BMI %ile 76 59.2 31.28 PBF 37 19.3 12.3 PBF %ile 37 46.83 27.03 Waist (cm) 68 74.09 19.07 Waist %ile 68 57.4 27.23 VCA 64 388.28 160.46 MSF (mm) 61 16.9 11.0 Gator Circle 63 57.61 164.22 Height z-score 76 -0.50 1.15 TG (mg/dl) 61 141.92 98.81 TG %ile 61 79.45 20.72 LDL (mg/dl) 69 95.11 29.4 HDL (mg/dl) 70 53.46 16.17 nonHDL* (mg/dl) 7 151.56 44.95 Systolic BP 72 118.12 8.64 Systolic %ile 72 88 10.46 Diastolic BP 72 66.2 9.5 Diastolic %ile 72 67 18.03 Glucose 75 84.98 9.79 PBF= Percent Body Fat VCA= Visceral Cavity Area MSF= Mean Skinfold *nonHDL only necessary when TG 250 mg/dl The VCA, MSF, and WC z-score could be either small, normal, or large with a total of 27 possible phenotypes ( Table 5-3 ). Phenotypes that were c onsidered detrimental were those that portrayed lipoatrophy, visceral obesity, subcutaneous obesity or any combination of these three phenotypes. Any large phenotype was considered unfavorable

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62 due to increasing abdominal obesity. There are several phenotypes that were unlikely. For example, it is improbable that a patient with a small VCA and small MSF would have a large WC z-score. Within the HIV-infected population, small MSFs were of concern because they suggested lipoatrophy. Table 5-4 depicts the correlation of the Gator Circle components with cumulative insulin resistance indices assessment ( Table 5-4 ). Table 5-3: Mean and S.D. for MSF a nd VCA for p1045 Control Population and HIV+ Population Control HIV+ MSF Age group Mean SD Median 10%ile 90%ile Mean SD <=8 8.1 6.3 6.4 3.6 12.8 6.6 1.2 >8-10 14 8.8 11 4.7 23.5 13.1 4.7 >10-12 16.1 9.8 13.9 6.2 28 13.8 9.4 >12-14 14 9.3 10.5 6 28.4 17.0 14.2 >14-16 20.5 10.1 20.2 7.1 32.4 21.5 11.8 >16-18 16 5.8 15.8 8.8 22.6 20.9 8.6 >18 19.8 5.5 19.9 12.4 25.4 22.2 13.0 Control HIV+ VCA Age group Mean SD Median 10%ile 90%ile Mean SD <=8 259.33 63.41 230.37 201 366.04 200.24 53.85 >8-10 292.44 88.07 272. 45 228.14 401.26 292.42 33.82 >10-12 384.55 138.42 329.91 275.73 630.06 323.08 89.59 >12-14 411.59 184.26 351.66 289.41 522.55 408.47 231.88 >14-16 480.24 142.54 423.39 343.42 692.45 448.12 112.78 >16-18 412.3 65.08 421.24 308.38 495.65 460.46 75.01 >18 515.48 162.12 440.56 382.82 754.14 498.52 169.73 Table 5-4: Gator Circle Phenotypes Small WC Zscore MSF small MSF normal MSF large VCA small Small Small VCA normal Unlikely Unlikely VCA large Unlikely Unlikely Unlikely Normal WC Zscore MSF small MSF normal MSF large VCA small Unlikely Small/Normal VCA normal Normal VCA large Unlikely Unlikely

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63 Table 5-4. Continued. Large WC Zscore MSF small MSF normal MSF large VCA small Unlikely Unlikely VCA normal Unlikely Unlikely VCA large lipoatrophy subcutaneous obesity visceral obesity lipoatrophy and subcutaneous obesity visceral and subcutaneous obesity lipoatrophy and visceral obesity Table 5-5: Correlation of Gator Circ le Variables with Cumulative IRI Variable Correlation with r p Value WC z-score IRI 0.671 <.01 VC% IRI -0.255 >.05 VCA IRI 0.734 <.01 MSF IRI 0.544 <.05 *IRI= Insulin Resistance Indices GC= Gator Circle Height z-scores were calculated for this population. Nine percent of the population experienced stunting while 5% ha d height z-scores less than -1.5 but greater than -2. The mean height z-score was -0.5 +/1. Triglyceride labs were available for 61 pa tients. Forty-one pe rcent of the patients had triglyceride concentrations greater than the 90th percentile and 30% had triglyceride concentrations greater than or equal the 75th percentile but less than the 90th percentile. The average triglyceride concentration wa s 141.92 +/98.81 mg/dl. Sixty nine patients had LDL (or non-HDL) values of which 14% had LDL (or non-HDL) percentiles greater than the 90th percentile and 20% had LDL pe rcentile greater than the 75th percentile but less than the 90th percentile. The average LDL c oncentration was 95.11 +/29.4 mg/dl. Non-HDL was assessed to replace LDL for patients with triglyceride concentrations greater than or equal to 2 50 mg/dl. The average nonHDL concentration was 151.56 +/-

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64 44.95 mg/dl. Seventy patients had HDL lab valu es. Patients with HDL concentrations greater than the 90th percentile totaled 24%; while those with HDL concentrations greater than or equal to the 75th percentile but less than the 90th percentile was 19%. The mean HDL concentration was 53.46 +/16 mg/dl. Blood pressure was measured in 72 patients. Thirty-five percen t of the population had systolic blood pressure s greater than the 95th percentile, while 14% had systolic blood pressures greater than the 90th percentile but less than the 95th percentile. Ov erall, 36% of patients had blood pressures greater than the 95th percentile, while 14 % had blood pressures greater than the 90th percentile but less than the 95th percentile. The mean systolic blood pressure was 118.12 +/8.64 mmH g and the mean diastolic blood pressure was 66.2 +/9.5 mmHg. Glucose labs were available for 75 patients. No patients had glucose concentrations greater than or equal to 126 mg/dl. Four patients (5%) had glucose concentrations greater than 100 mg/dl but less than 126 mg/dl. Discussion The prevalence of the Metabolic Syndro me in this population (12.9%) using Cook et al.Â’s definition is more than three times that observed within normal U.S. adolescents (4.2%) as seen in a study by Cook et al. us ing the Third National Health and Nutrition Examination Survey. The statistical significance of this relationship should be tested in future studies. The prevalence of the Metabolic Syndrome amongst overweight (9.1%) and obese (41.7%) subjects were also hi gher than observed in CookÂ’s study (6.8%, 28.7%, respectively). Differences in prevalen ce may be due to differences between the populations in relation to the presence of risk factors (age, etc.).

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65 Using the HAMD definition, it was determined that 14% of patients were at risk of becoming obese and would requ ire intervention. Waist circ umference was approaching an abnormal level in 18% of the patients. Visceral cavity area was approaching an abnormal level in 7.8 % and abnormal in 9.4% of the patients. In comparison to the control group, the pediatric HI V+ populationÂ’s VCA was not significantly different in 5 age groups with a p-value > .05 (8-<10yr s, 10-<12yrs, 14-<16yrs, 16-<18yrs, 18yrs) and significantly lower in 2 age groups w ith a p-value <.05 (<8yr s, 12-14yrs). Mean skinfolds were approaching abnormal high le vels in 11.5% of patients and abnormal low levels in 19.7 % of patients. In comparis on to the control group, this populationÂ’s MSF was not significantly different with a P>.05 for all age groups except the < 8 group which had a p-value <.05. The average Coefficients of variances (CV) were determined for intra and inter-patient VCA measurements. The CV for the VCA was smaller for intrapatient measurements than for the inte r-patient, (5.24% and 42%, respectively) suggesting that more than one population was re presented in our Gator Circle data. More studies are needed to examine the prevalence of the Gator Circle phenotypes within the HIV+ population. The number of patients with body fat percentage measurements limits the accuracy of assessing its results within this population. The mean height z-score was -0.5 +/1. The 95% CI was 0.262. Negative height z-scores were observed in 65.8% of the patients suggesting a high prevalence of insufficient linear growth in this population. However, the trend of decreasing height zscores tended to attenuate with age, sugges ting that younger patients (1-12 years) are at an increased risk for linear growth failure than the older patients . Another important issue would be whether the pati ent acquired the HIV virus vert ically or horizontally and

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66 the timeframe when this occurred. For exampl e, if the older patients tended to acquire the virus vertically in the late r stages of their life when lin ear growth was complete than linear growth failure would not be identified here. Abnormal triglycerides (48%) and LDL (70 %) concentrations were more prevalent in the youngest group of 0-12 year olds ( Table 5-5 ). It is important to note that as a whole, the mean triglyceride was 141.92 +/ 98.81. In contrast, HDL (64%) was more prevalent in the older gro up of 20-26 year olds. Abnormal blood pressure prevalence increased with age. Abnormal systolic blood pressure (34.7%) was more prevalent than abnormal diastolic blood pressure (9.7%) suggesting greater vascular wall damage. This prevalence may be influenced by the use of improper cuff sizes for children and adolescents, although, the use of HAART has been associated with increas es in blood pressure (149). The results from the glucose analysis s uggest that this population does not need close monitoring of glucose intolerance and insulin resistance. However, scientific literature and a separate small study performe d in this population s uggest otherwise. Thus, it can be deduced that fasting plasma glucose is not a sensitive screening for glucose intolerance and insulin resistance. As a whole, some parameter components increased with age while others either decreased with age or fluctuated. BMI, WC percentile, and VCA fluctuated, with BMI and WC decreasing from the first age group (1 -12 years) to the second age group (13-19 years) and then increasing from the second age group to the third (20-26 years). VCA increased from the first age group to the sec ond and then decreased from the second age group to the third. Mean skinfo ld, body fat percentage, height z-scores, triglycerides, and

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67 LDL decreased as age increas ed. HDL, and systolic and diastolic blood pressure increased as the age increased.

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68 Table 5-6: Prevalence on HAMD Risk Factors

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69 Table 5-6 Continued BMI: Body Mass Index WC: Waist Circumference VCA: Visceral Cavity Area MSF: Mean Skinfold TG: Triglycerides nonHDL calculated when TG 250 mg/dl

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70 CHAPTER 6 SUMMARY The aim of this study was to define HIV-associated Metabolic Dysfunction (HAMD), compare it to the Metabolic Syndrom e and determine its pr evalence within a pediatric HIV-infected population. In defi ning HAMD, metabolic parameters that were of concern in pediatric HIV-infected populat ion were incorporated. These parameters included a computation of th e Visceral Cavity Area (VCA) and its relation to the subcutaneous area thereby examining vi sceral obesity and lipoatrophy, which are common occurrences within this population (1-5). In an atte mpt to unify and standardize the evaluation of metabolic abnormalities within the normal population and the HIVinfected population, screening and monitoring tools referred to as Metabolic Modules were provided that contain metabolic parame ters, their standard operating procedures, and case report forms. The prevalence of the Metabolic Syndrome according to an ATP age-adjusted definition was 12.9%. The prev alence of HAMD, a metabolic dysfunction definition more specific to the HIV-infect ed population was 31.2%, suggesting that the HIV-infected population is at risk of acquiring metabolic disturbances. Recommendations The HAMD definition should be corrobor ated by a longitudinal study where the outcomes from the development of these parame ters can be assessed. In this way, it can be observed whether the patients that do indeed have HAMD develop diabetes and cardiovascular disease. Refinements c ould be made to the HAMD definition as necessary. Furthermore, a longitudinal study wo uld allow for researchers to be able to

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71 evaluate whether the outcome s of HAMD are due to the severity of the abnormal components, the duration of the abnormal compon ents or both. This will enable health care providers to make valuable decisions on when and how therapy should be administered. Based on the results of this study severa l recommendations are given for future monitoring of metabolic disturbances in patien ts. Due to the limitations of a retrospective study, data were not available for all subjec ts and parameters. For example, body fat percentage could not be fully assessed in th is study because of limited data. Another limitation of a retrospective study is that it can not be assured that labs were taken in a fasting state. Therefore, a pr ospective study is proposed to ensu re that collected data is as complete as possible. Moreover, the results of this study support th e concept that fasting plasma glucose is not a sensitive screening indi cator of insulin resistance. Fasting plasma insulin is not routinely measured in patients, thus a prospective study will allow for the controlled evaluation of fasting plasma insulin as an indicator of insulin resistance. Since insulin assays are less standardized than gluc ose and lipid assays, it is not advised that a cut-off value using the fasting plasma insuli n value be utilized (6). Rather, insulin resistance indices should be used to evaluate whether a patient is insulin resistant. Being able to differ between visceral and subcutan eous fat in patients is very important, especially in the HIV-infected population wh ere both fat loss and fat accumulation could be occurring at the same time and within the same body region. The Gator Circle consisted of the relations hip of the Visceral Cavity Area (VCA), the mean skinfolds (MSF) which respresent the subcutaneous area and the overall waist circumference z-score. The Gator Circle components had a moderate correlation with

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72 indices of insulin resistance and aids in iden tifying individuals that have an increasing visceral cavity area or decreasing subcutane ous area. By doing so, it also helps in the identification of individuals that have the disturbances promoted by lipoatrophy and lipohypertrophy. However, the Gator Circle co mponents still remain to be validated by accurate measurements of both visceral and subcutaneous fat which can be performed by magnetic resonance imaging. The validation of the Gator Circle components is proposed as a recommendation so that it can be us ed for future monitoring of metabolic disturbances in patients. The assessment of LDL concentration usi ng FriedewaldÂ’s equation is limited by the value of the triglyceride c oncentration (7). Non-HDL is proposed to be used as a measurement of LDL and other atherogenic part icles when triglycerides concentration is too high to maintain accuracy and a fasting LDL concentration is not feasible. In pediatric populations, it is essential to have correct blood pressure cuff sizes to ensure accurate measurements of blood pre ssure. Larger-than-appr opriate cuff sizes can lead to false low measurements and smaller-t han-appropriate cuff size s can lead to false high measurements (8-9). In this study, it could not be substantia ted that the results obtained in regards to blood pressure were not affected by the use of incorrect blood pressure cuff sizes. The use of correct bl ood pressure cuff sizes as recommended by the National High Blood Pressure Education Progra m will help to ascerta in the accuracy of hypertension risk prevalence within this population.

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APPENDIX A DATABASE PARAMETERS

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74 Immunology Growth and Body Composition BIA Measure ments Chemistry Patient Demographics Gator Circle Food Intake Access ID Gator ID Date of Collection CDC CD 20% CD 20AB CD3% CD3AB CD4% CD4AB CD8% CD8AB CD45RA CD45RO T4/T8 VRL LD split Access ID Gator ID Date of Collection Height* Weight* Ht% Wt% Wt/Ht% Ht Z-score Wt Z-score Wt/Ht Z-score BMI BMI Z Arpadi FFM Goran FFM Arpadi TBW Schaefer FFM Gator Circle % Body fat Intake % Calories Access ID Gator ID Date of Collection Z* R* P* X* Access ID Gator ID Birthdate Age Gender Race Zip Code First Clinic Date Sibling Access ID Gator ID Date of Collection Waist Hip W/H ratio USF SISF MBSF VC% VCA Gator Circle Waist % Waist Zscore Access ID Gator ID Date of Collection % Calories from fat % SatÂ’d Fat % Mono Fat % Poly Fat

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75 Immunology Growth and Body Composition BIA Measure ments Chemistry Patient Demographics Gator Circle Food Intake Intake % Proteins Intake % w/ Supplementary calories Intake % w/Supplementary proteins UAC* TSF* TSF% Sub-scap* Sub-scap% AMC UAA UAA % AMA AMA% AFA AFA% USF* USF% Waist/Hip Ratio Blood Pressure-Systolic Blood Pressure –Diastolic Fasting AlkPhos AST ALT BiliDirect Lipase Amylase RBC Hgb Hct Platelet Cnt MCV MCHgb MCHgb Conc RBC Width WBC ReticCorr ReticUncorr Insulin

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76 APPENDIX B METABOLIC MODULES SUMMARY TABLE Summary of Metabolic Modules Pr ocedures and Required Resources Parameter Level I Resources Required Level II Resources Required Parameters Frequently Obtained that Pertain to HIV Associated Metabolic Syndrome Growth Age Gender Length/Height Weight Body Composition and Body Fat Distribution Circumferences at the Mid Upper Arm, Umbilicus, Hip, and Mid Thigh sites Skinfolds at the Triceps, Suprailiac, Subscapular, Umbilicus, Mid Thigh, and Mid Back sites Visible evidence of lipoatrophy or lipohypertrophy. Gator Circle BIA DEXA for Body Composition and Body Fat Distribution Blood Lipid Status Initial Non fasting then Fasting Triglyceride, HDL , LDL and Total Cholesterol if abnormal Initial Fasting Triglyceride, HDL and Total Cholesterol Fasting LDL Blood Glucose and Insulin Status Initial Non fasting then Fasting Blood Glucose if abnormal Initial Fasting Blood Glucose Fasting Blood Insulin 2 Hour Glucose Tolerance Test with both Glucose and Insulin Measurements Blood Pressure Resting Blood Pressure

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77 Summary of Metabolic Modules Pr ocedures and Required Resources Parameter Level I Resources Required Level II Resources Required Parameters Infrequently Obtained that Pertain to HIV Associated Metabolic Syndrome Mitochondrial Metabolism Anion Gap followed by Lactic Acid test if abnormal ALT, AST, Albumin, Alkaline phosphatase Non fasting Lactic Acid Mitochondrial DNA Acylcarnitine Profiling (fasting) Lipase, Amylase Renal Metabolism Serum BUN Serum Creatinine, Creatinine Clearance Serum HCO3-, Potassium, Phosphate Urinalysis Urine Protein:Creatinin e ratio Bone Metabolism Radiographically Proven Fractures Alkaline phosphatase DEXA for Bone Density Bone Specific Alkaline Phosphatase Urinary CrossLinked Ntelopeptide Parameters that Pertain to Dietar y Intake and Physical Activity Protein Nutrition Status Plasma Albumin Plasma Prealbumin Anemia Hemoglobin Hematocrit Red Blood Cell Count Mean Corpuscular Volume Dietary Intake 24 hour dietary recall 3 Day Diet Diary Dietary Assessment

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78 APPENDIX C GROWTH MODULE Growth Module Question: Is the patient growing normally? Measurement: Age Level 1 Equipment: Case Report Form (CRF) Training: Minimal Overview Of Steps Involved: Ask patient or caregiv er date of birth. Record data on CRF. Information Obtained: Age is obtained to use in comp arison to reference populations. CRF Template: Measurement: Gender Level 1 Equipment: CRF Training: Minimal Overview Of Steps Involved: Brief physical exam. Record data on CRF. Information Obtained: Gender is obtained to use in co mparison to reference populations. CRF Template: Measurement: Length /Height Level 1 Equipment: Length board for children less than 2 years of age. Stadiometer for children over 2 years of age. Training: pACTG short course Overview Of Steps Involved: Measure length for children less th an 2 years of age using Metabolic Module SOP. Measure height for children over 2 years of age using Metabolic Module SOP. Record data on CRF. Information Obtained: Length or height for age percentile or z-score provides a comparison of the linear growth to that of a re ference population of the same age and gender. Body Mass Index (BMI) per centile or z-score provides a comparison of

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79 the relationship of weight to height to that of a reference population of the same age and gender. Note that both height and weight data are required to determine BMI. CRF Template: Taken from EVW0078 Measurement: Weight Level 1 Equipment: Digital infant scale for children less than 2 years of age. Digital scale for children 2 years of age and older. Training: pACTG short course Overview Of Steps Involved: Measure weight using Metabolic Module SOP. Record data on CRF. Information Obtained: Weight for age percentile or zscore provides a comparison of the ponderal growth to that of a refere nce population of the same age and gender. Body Mass Index (BMI) per centile or z-score provides a comparison of the relationship of weight to height to that of a reference population of the same age and gender. Note that both height and weight data are required to determine BMI. CRF Template: Taken from EVW0078

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80 Growth SOP Question : Is the patient growing normally? Measurement: Length/Height Level 1 General Instructions: Length or height should be meas ured at each clinic visit. Length: SOP: 1. Length should be measured using a length board and in the recumbent position for infants younger than 24 months of age or children aged 24 to 36 months who cannot stand unassisted . 2. The length board should have a fixed hea dpiece with a moveable footpiece which is perpendicular to the surface of the table that the length board is on. 3. Length measurements should be obtained while the child is dressed in light underclothing or a diaper. The child's shoes must be removed. Hair ornaments should be removed from the top of the head. 4. The child should be placed on his back in th e center of the length board so that the child is lying straight and his shoulders and buttocks are flat against the measuring surface. The child's eyes s hould be looking straight up. Both legs should be fully extended and the toes s hould be pointing upward with feet flat against the footpiece. 5. Accurate length measurements require two measurers. One measurer holds the infantÂ’s head, with the infant looking ve rtically upward and the crown of the head in contact with the headpiece in the Frankfort Horizontal Plane. The head of the infant is firmly but gently held in pos ition. The measurer gently cups the infantÂ’s ears while holding the head. Make sure the infant's chin is not tucked in against his chest or stretc hed too far back. 6. While the second measurer holds the in fants head in the proper position, the measurer aligns the infantÂ’s trunk and legs, extends both legs, and brings the footpiece firmly against the heels. The measurer places one hand on the infantÂ’s knees to maintain full extension of the le gs. The infantÂ’s toes are pointing upward. 1 1 The formal definition of the Frankfort horizont al plane is a line extending from the most inferior point of the orbital ma rgin to the left tragion. The tr agion is the deepest point in the notch superior to the tragus of the auricle. When the h ead is positione d correctly, the Frankfort horizontal plane is pa rallel to the fixed headpiece. For length measures, the Frankfort plane is aligned perp endicular to the plane of th e measuring table and parallel to the headpiece.

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81 7. It is important that both legs be fully extended for an accurate and reproducible length measurement. If only one of the infa nt’s legs is extended during the length measurement, the measurement may be unreliable and inaccurate. Correctly positioning the infant for a length measurement generally cannot be accomplished without two measurers . Height: SOP: 1. For subjects 24 months of age or older, height should be measured using a calibrated, wall-mounted stadiometer. 2. For best results, the subject is measured w earing a gown that allows the measurer to visualize the subject's body position. 3. The subject stands with bare feet close together, body and legs straight, arms at sides, relaxed shoulders, and head, back, bu ttocks, and heels agai nst the wall or shaft of the stadiometer (Diagrams 1a-1d). 4. Instruct the subject to lo ok straight ahead and stand tall, keeping heels on the ground. 5. Bring the headboard down to the top of the subject's head while at eye-to-eye level with the subject and record the height. Measurement: Weight Level 1 General Instructions: Weight should be measured at each clinic visit. SOP: 1. For subjects less than 24 mont hs of age, use an electron ic or beam balance with nondetachable weights. The scale should have a maximum weight of 20 kilograms (40lbs) and weigh in 0.01 kg ( 10 g) or ½ ounce increments. Use a calibrated scale. Zero the scale prior to each measure. There should not be any length devices attached to the infant scale. 2. For subjects 24 months of age or older use an electronic or beam scale with nondetachable weights. Zero th e scale prior to each measure. Use a calibrated scale. 3. Instruct the subject to sta nd with both feet centered on th e scale with arms at the sides. The subject should not move or hold onto anything during the measurement (Diagrams 2a-2c). 4. Allow the scale to stabilize a nd record the weight in th e units provided by the scale (lbs. or kg).

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82 HEIGHT MEASUREMENT DIAGRAMS: 1a. Correct Stance 1b. Correct Position 1d. Incorrect Stance 1c. Incorrect Position

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83 WEIGHT MEASUREMENT DIAGRAMS: 2a. Correct Stance 2c. Incorrect Stance 2b. Correct Position

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84 APPENDIX D BODY COMPOSITION AND BODY FAT DISTRIBUTION MODULE Body Composition and Body Fat Distribution Module Questions: Does the patient have an appropriate amount of lean body mass? Does the patient have lipoatrophy or lipohypertrophy? Measurement: Bioelectrical Impedance Analysis (BIA) Level 2 Equipment: BIA Instrument Training: pACTG short course Overview Of Steps Involved: Patient lies still on mat and is connected to BIA instrument. Measure the resistance of the flow of electric current through fat tissue using BIA Instrument and Metabolic Module SOP. Record measurement on CRF. Information Obtained: Estimate Body Composition (Percent Body Fat vs. Lean Body Mass). CRF Template: Taken from EVW0078 Measurement: Body Composition/ Fat Distribution Level 2 1 Equipment: DEXA Scan Training: Certified DEXA technician Overview Of Steps Involved: Patient lies down for 10-20 minut es beneath a whole body scanner. Body composition is measured using Metabolic Module SOP. Record measurements on CRF. Information Obtained: Body composition: lean body mass, fat mass, bone mass, and localization of fat for arms, trunk, legs and whole body. Body fat percentage and localization. CRF Template: Taken from DGW0030 Measurement: Circumferences at the mid arm and mid thigh sites Level 1 Equipment: Insertion tape, Grease marking penc il or washable felt-tip marker Training: pACTG short course Overview Of Steps Involved: Measure upper arm and thigh midpoi nt using the insertion tape according to Metabolic Module SOP. Measure circumference of mid arm and mid thigh using the insertion tape according to Metabolic Module SOP. 1 1DEXA scan: Same technique for Body Composition and Fat Distribution

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85 Record measurements on CRF. Information Obtained: Estimation of body fat and muscle mass. CRF Template: Taken from EVW0078 Measurement: Circumferences at the umbilicus and hip sites Level 1 Equipment: Flexible, non-stretchable measur ing tape (fiberglass or paper) specific to 1 mm. Training: pACTG short course Overview Of Steps Involved: Measure umbilicus and hip circumference using non-stretchable measuring tape according to Metabolic Module SOP. Record measurements on CRF. Information Obtained: Estimation of body fat and muscle mass. CRF Template: Taken from EVW0078 Measurement: Skinfolds at the triceps, subscapular, and mid thigh sites Level 1 Equipment: Lange caliper Training: pACTG short course Overview Of Steps Involved: Vertical skinfold pinch is taken at triceps, subscapular and mid thigh sites using Metabolic Module SOP. Record measurement on CRF. Information Obtained: Estimation of body fat percentage, muscle mass, and subcutaneous fat. Subtracting skinfold thickness from arm circumference can be used to approximate arm muscle circumference. CRF Template: Taken from EVW0078 Measurement: Skinfolds at the umbilicus, suprailiac, and mi d back sites Level 1 Equipment: Lange caliper Training: pACTG short course Overview Of Steps Involved: Vertical skinfold is taken at umbilicus, suprailiac, and mid back sites using Metabolic Module SOP. Record measurement of CRF. Information Obtained: Estimation of body fat percentage, muscle mass, and subcutaneous fat. Estimate visceral cavity area and its relationship to the abdominal subcutaneous area. CRF Template: Taken from EVW0078 Measurement: Visible evidence of lipoatrophy or lipohypertrophy Level 1 Equipment: Training: pACTG short course Overview Of Steps Involved:

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86 Examine patient for signs of fat accumulation (abdomen, neck, buttocks, and breasts) and fat loss in the peripheral areas (face, arms, legs). Record on CRF Information Obtained: Presence of lipoatr ophy or lipohypertrophy CRF Template: Taken from EVW0116, QLW0069 Measurement: Gator Circle Level 1 Equipment: Training: pACTG short course Overview Of Steps Involved: Calculating the Gator Circle involve s using the measurements obtained above for umbilical circumference, USF, SISF, MBSF, to determine the visceral cavity area. The wais t circumference Z-score obtained by using waist standardization tables is used to calculate the Gator Circle. The Gator Circle is the product of the VC% and the WC Z-score. Record on CRF Information Obtained: Computation of the visceral cavit y area which is used to assess abdominal fat distribution. CRF Template: Added to EVW0078

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87 Body Composition and Body Fat Distribution SOP Questions: Does the patient have an appropriate amount of lean body mass? Does the patient have lipoatrophy or lipohypertrophy? Measurement: Bioelectrical Impedance Analysis (BIA) Level 2 General Instructions: BIA should be performed at each clinic visits. Subjects should be 24 months of age. SOP: 1. Prior to performing measurements the BIA instrument should be calibrated using the manufacturerÂ’s instru ctions and the result from this calibration should be written down on the case report form. 2. Subject should be positioned on a non-c onducting surface and atleast 50 cm from any electrical devices. 3. Subject should lay down on their back with arms not touching their trunk. Their thighs should not touch and their ankles s hould be atleast 20 cm. apart. Their head should be level. 4. Subject should remove their shoes and socks and any metallic objects. Other clothing may be worn. 5. SubjectÂ’s skin on top of the hand and wrist and top of the foot and ankle should be cleaned with alcohol. 6. Four electrodes should be attached by m eans of tape to the hand, wrist, foot, and ankle (Diagram 3). 7. Record the resistance and/or re actance provided by the instrument. Measurement: Body Composition and Fat Distribution Level 2 General Instructions: A DEXA scan should be performed The following is a summary of pr ocedures for whole-body DEXA scans performed for body composition measurements and regional (hip and spine) DEXA scans. Please follow the manufact urerÂ’s instructions for operating your instrument. Whole-body and hip and spine scans should be done using standard operating procedures defined by the manuf acturer. The subject should be asked about and examined for metal that could be in the scan path. Typical things to look for are earrings, eyeglasses, wristwat ches, coins, rings, buttons, buckles, zippers, and support braces. The subject should remove shoes, and it may be necessary to remove skirts, slacks, etc. If in doubt, it is best to remove the object in question.

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88 1 Whole body Protocol SOP: 1. When prompted by the program, ask the s ubject to lie down on the scan table. 2. The centerline on the table pad should divi de the subject's body in half. Use the lines on the table pad to ensure the subject is lying straight on the table. The subject's head should be di rectly below (about 1") the horizontal line located along the top of the table pad. 3. Have the subject tilt his/her head back slightly during the scan. This makes placement of the head region cut line easier to place. 4. A foam wedge (or pillow) should not be us ed under the subject's head as it will affect the body composition results. 5. Have the subject place hands palms down alongside the body with fingers together. Do not overlap hands and legs. The subject's entire body sh ould fit within the scan lines on the table pad. 6. If the subject is too wide to fit with in the boundary lines, the technician should proceed to the Hemiscan Protocol section below. For tall s ubjects, please proceed to the Tall Subjects section below. 7. The subject's feet should be held together using a Velcro strap, and the subject is asked not to move until directed to do so. 8. Continuing the scan will cause the x-ray t ube to ramp up to th e appropriate current and voltage. The operator should check to make sure the orange "X-Ray On" light is lit, and remain in the room to check the progress of the scan acquisition as it appears on the screen. 9. The subject's head should appear with a few blank scan lines above it. As the scan proceeds, the total-body image should be in a straight line vertically on the screen. If these conditions are not met, the scan should be stopped. The scan arm will move to the original start position and the local izer light will come on. The subject should be repositioned as needed. 10. When the detector goes past the subject's feet, the auto stop fe ature then interrupts the scan and closes the shutter. A me ssage appears on the screen, allowing the operator to continue the scan or shut down the system. After the scan ends the shutter closes, the voltage and current ramp down, and a messages appears for the operator to wait. The scan arm moves to the home position a nd a screen message appears to inform the operator that the scan is over and to remove the subject from the table. The technician should get to the Total-body Scan Options screen, then save the scan file. 11. The operator may now exit the Total-body porti on of the program to do another type of scan. Hemiscan Protocol for Obese Subjects SOP: 1 DEXA Scan: Same technique for Body Composition and Fat Distribution.

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89 1. The left side of the body is aligned within the left margin of th e scanning table, and the subject is scanned as prev iously described. This allows the patient’s left side to b e completely within the cut lines, so that all the “overflow” occu rs on the right side. This overflow should be ignored. 2. During the scan analysis at the Central Reading Site, the midline of the body is selected by the technician to demarcate th e left half of the body as the region of interest, and a scan report is produced. 3. The regional scan data are multiplied by 2 to give the whole-body estimate of body composition. Tall Subjects Protocol General Instructions: If a person is too tall to fit within the lines, prioritize what part to “cut off” using the following list. Go down the list from top to bottom, and cut off as many parts as needed to fit the person on the table. Make note of the parts that were cut off for future reference. Top half of the head (to the tops of the ears) Lower foot (toes) Bottom half of the head (to the jaw) SOP: 1. Position against the board, relaxed. 2. Do not use support under the knees for size, only for comfort when absolutely necessary. 3. If necessary, use a head pillow carefully only after the scanner has passed over the head region. When inserting a pillow unde r the head, please make sure that the subject does not move. Hip and Spine Protocol General Instructions: For hip and spine scans, the subject -positioning techniques and the scan acquisition procedures should be done using the standa rd operating procedures defined by the instrument manufacturer. Measurement: Circumferences at the mid arm and mid thigh sites Level 1 General Instructions: Circumference measurement should be performed at each clinic visit. Subjects should be 24 months of age. Circumference measurements are made once before repeating them a second time in the same sequence by the same observer. Document measurement conditions

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90 (i.e., type of scale used, whether length or height was measured, subject's behavior during procedure, etc.). Always take two measurements in each category. A third measurement will be needed when the second measurement differs from the first one by > 0.5 cm. All measurements are taken on the right si de of the subject being measured. If measurements need to be taken on the left side due to abnormalities, be sure to record this on the case report form. Whenever possible, measurements should be taken by a team of two measurers. One measurer takes the measurements wh ile the other measurer records. The measurer taking the measurements calls out the results to the recorder. The recorder repeats the results and then calls out the name of the next measurement. The measurer keeps the measuring instru ment in place until the recorder repeats the number. The recorder checks the ex amineeÂ’s position during the procedure. The subject's cooperation is extremely important for obtaining accurate measurements. Always take two measurements in each category. Circumferences should be recorded with the zero end of the tape held by the left hand above the remaining part of the tape held by the right hand. The plane of the tape around the body part should be perpe ndicular to the long axis of the body part being measured. Care should be taken to ensure that the tape is touching the surface of the skin, but is not al tering contours or compressing tissue . Maintenance of a perpendicular plane w ith the tape touching but not altering contours of the body can be challenging wh en measuring the obese individual and requires extra care on the part of the examiner. Mid arm circumference: SOP: 1. To locate the midpoint, the s ubjectÂ’s elbow is flexed to 90 degrees with the palm facing superiorly (Diagram 4a). 2. The measurer stands above or behind the subject and locates the lateral tip of the acromion by palpating laterally along the s uperior surface of the spinous process of the scapula. 3. The tape is placed from the acromion pr ocess to the tip of the olecranon and the midpoint is marked (Diagram 4b-4c). 4. The arm is now repositioned to hang loosel y at the side with the palm facing the thigh. 5. The tape is passed around the arm from left to right, and the free and fixed ends are transferred. 6. Ensuring the tape is at the same leve l as the mid-upper-arm mark, the measurer tightens the tape so that it touches the sk in all around the circumference but does not compress the tissue or alter the contour of the arm (Diagram 4d). Because the arm in cross-section is not an ex act circle but rather oval, so me difficulty may be met in ensuring that the tape actually touches the sk in on the medial side of the arm. If necessary, the middle finger of the left hand can be used to gently press the tape to the skin.

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91 7. The circumference is then read and reco rded twice in centimeters to the nearest millimeter. Mid thigh circumference: SOP: 1. The subject lies down with legs extended. 2. The thigh circumference is measured at the midpoint, located with the subjectÂ’s upper leg flexed to 90 degrees in the supine position (Diagram 11a). 3. The measurer stands alongside the subject and locates the inguinal crease in the midline of the leg. (The midline is also identifiable from the anterior superior iliac spine.) (Diagram 11b). 4. The subject relaxes the leg being measured with knee flexed and foot flat on surface on which the subject is lying. 5. The tape is placed from this point to th e top of the patella, and the midpoint is marked (Diagram 11c). 6. The tape is passed around the thigh at mid-th igh level. Ensuring that the tape is at the same level as the mid-thigh mark all the way around the leg, the measurer tightens the tape so that it divides the skin all around the circumference but does not compress the tissue or alter the cont our of the leg (Diagram 11d). 7. The circumference is then read. The meas urement is made to the nearest 0.1 cm. Measurement: Circumferences at the umbilicus and hip sites Level 1 Umbilicus circumference SOP: 1. Subject should stand during this measurem ent with feet hip-distance apart. 2. Ask the subject not to try to hold in th e stomach during the measurements. All measurements should be made af ter the subject has exhaled. 3. To ensure reproducibility, locate the leve l of the umbilicus (belly button) and measure the waist circumference at this level. The tape measure should be perpendicular to the long axis of the body (Diagram 7a). 4. Perform this measurement twice, recordi ng results in centimeters to the nearest millimeter. Hip circumference: SOP: 1. Subject should stand during this measurem ent with feet hip-distance apart. 2. Ask the subject not to try to hold in the stomach during the measurements. 3. Viewing the subject from the side, visually identify the widest width of the hip. The widest point is generally where there is maximal protuberance of the buttocks (Diagram 7b). 4. Measure the circumference at that point, making sure the measuring tape is exactly perpendicular to the long axis of the body.

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92 5. Perform this measurement twice, recordi ng results in centimeters to the nearest millimeter. Measurement: Skinfolds at the triceps, subscapular, and mid thigh sites Level 1 General Instructions: Skinfold measurements should only be performed on subjects greater than 24 months of age. Skinfold measurements are made once before repeating them a second time in the same sequence by the same observer. Document measurement conditions (i.e., type of scale used, whether length or height was measured, subject's behavior during procedure, etc.). Always take tw o measurements in each category. A third measurement will be needed when the second measurement differs from the first one by the following: Skinfold thickness, > 2 mm for every 10 mm in the first measurement, i.e., 2 mm tolerance for 0up to 10-mm first m easurements; 4-mm tolerance for 10up to 20-mm first measurements; 6-mm tolerance for 20up to 30-mm first measurements; 8-mm for 30up to 40-mm first measurements; and 10-mm tolerance for first measurements > 40 mm. All measurements are taken on the right si de of the subject being measured. If measurements need to be taken on the left side due to abnormalities, be sure to record this on the case report form. Lange calipers should be calib rated with standard metal blocks on the day of each exam. Calipers require calibrating to less than 1.5 mm (i.e., 1.0 mm or less) at each of the 10-, 20-, 30-, 40and 50-mm te st distances (Diagram 5a-5c) . Calipers that do not meet the standard s must be removed from service and repaired. The fold of skin should be firmly gras ped between the left thumb and forefinger (for right-handed observers) and then raise d. The fold may be pinched and raised several times to make certain that no muscul ature is grasped. The skinfold is held firmly with the thumb and index finger , and the calipers are placed below the thumb and finger. The grip on the caliper is released completely, allowing the spring to compress the fold. With the fo ld held, the reading should be taken 3 seconds after caliper jaw pressure is released. A firm grip on the skinfold, not exceedi ng the pain threshold, eliminates or at least substantially reduces the variations in the apparent thickness of skinfold that would result from wide differences in the pulling force of the fingers. It should be noted that the fold is held with the thum b and index finger of the left hand and is not released until the caliper is removed.

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93 The width of the skin that is enclosed between the fingers cannot be standardized, in its absolute size, for all the sites of the body. With a larger subcutaneous layer, for example, on the thigh, a wider segment of the skin must be “pinched” in order to form a fold compared with areas wh ere the adipose tissue is poorly developed, as it is on the dorsum of the hand. For a given site, the width of the skin should be minimal, still yielding a well-defined fold. The depth of the skinfold at which the cal ipers are placed on the fold also requires comment. The two sides of the fold are not likely to be parallel when the skin is lifted by one hand, being narrower near the crest and larger toward the base. When the calipers are placed at the base, the resulting measurement is too large. The correct distance from the crest of a tr ue fold is obtained when the surfaces are approximately parallel to each other and to the contact surfaces of the calipers. It is extremely important to measure skin folds accurately. Even after extensive practice, it is possible to make errors due to slight misplacement of the calipers or misreading of the dial. To avoid su ch errors, the follo wing procedure is recommended: Skinfolds should be lifted two or three times to determine the fold to be measured before placing the calipers. Avoid becoming overly anxious to put the calipers in place before determining what is really to be measured. The calipers are placed below the thumb and index finger, and the dial is read. The calipers must be removed and the skinfold released between each measurement. Triceps skinfold: SOP: 1. The level for the triceps skinfold is the sa me as that for the midarm circumference, as marked with the felt pen. It is mi dway between the acromion and the olecranon when the arm is bent at a right angle, a nd measurement is made at the marked point (Diagrams 4c-4d). 2. With the subject's arm dropped and hanging l oosely, the skinfold is raised from the underlying muscle fascia at this point with a sweeping motion of the fingers to the point at which the observer is holding the fold between the index finger and thumb. The skinfold calipers are then applied to this vertical fold (Diagrams 6a-6b). 3. Perform this measurement twice, recordi ng results in centimeters to the nearest millimeter. Subscapular skinfold: SOP:

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94 1. The point of measurement is located imme diately below the inferior angle of the scapula (Diagram 10b). 2. The subject stands with his/her back to the observer (Diagram 10a), with shoulders relaxed and arms hanging loosel y at the sides (Diagram 10c). This posture is most important to prevent movement of the scapulae. 3. The skinfold is picked up, as for the tr iceps skinfold, by a sweeping motion of the finger and thumb, and the calipers are applie d at a slight angle following the natural cleavage of the skin (Diagram 10c). Perf orm this measurement twice, recording results in centimeters to the nearest millimeter. Mid-thigh skinfold: SOP: 1. The point of measurement is located on th e superior surface of the thigh, in the midline at the level of the mid-thigh circumference measurement. 2. The subject lies down with legs positioned as for the mid-thigh circumference measurement. 3. The skinfold is picked up, as for the triceps skinfold measurement, by a sweeping motion of the finger and thumb, and the cal ipers are applied following the natural cleavage of the skin (Diagram 12). 4. Perform this measurement twice, recordi ng results in centimeters to the nearest millimeter. Measurement: Skinfolds at the umbilicus, s upailiac and midback sites Level 1 Umbilicus skinfold: SOP: 1. The point of measurement is located about 2 cm to the personÂ’s right of his/her umbilicus (belly button) (Diagram 8a). 2. Subject should stand during this measuremen t with feet hip-distance apart and arms relaxed at the side. 3. Ask the subject not to try to hold in the stomach during the measurements. 4. The skinfold is picked up, as for the triceps skinfold, by a sweeping motion of the finger and thumb, and the calipers are applied vertical to the Mid Waist Circumference (Diagrams 8b). All measurem ents should be made after the subject has exhaled. 5. Perform this measurement twice, recordi ng results in centimeters to the nearest millimeter. Suprailiac skinfold: SOP:

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95 1. The point of measurement is located at th e mid-axillary line and at the umbilical level. 2. Subject should stand during this measuremen t with feet hip-distance apart and raise his/her right arm to the side. 3. Ask the subject not to try to hold in th e stomach during the measurements. The skinfold is picked up, as for the triceps skinfold, by a sweeping motion of the finger and thumb, and the calipers are applied ve rtical to the Mid Waist Circumference (Diagram 9). All measurements should be made after the subject has exhaled. 4. Perform this measurement twice, recordi ng results in centimeters to the nearest millimeter. Mid back skinfold: SOP: 1. The point of measurement is located about 2 cm to the personÂ’s right of his/her spinal column at the umbilical level (Diagram 8c). 2. Subject should stand during this measuremen t with feet hip-distance apart and arms relaxed at the side. 3. Ask the subject not to try to hold in the stomach during the measurements. 4. The skinfold is picked up, as for the tr iceps skinfold, by a sweeping motion of the finger and thumb, and the calipers are applied vertical to the Mid Waist Circumference (Diagram 8d). 5. All measurements should be made after the subject has exhaled. Perform this measurement twice, recording results in centimeters to the nearest millimeter. 6. Note that for some slim children, subcutane ous fat at this site may be too small to obtain a skinfold. Such a situation shoul d be noted on the physical assessment form. Measurement: Visible evidence of lipoatrophy or lipohypertrophy Level 1 SOP: 1. During physical examination, examiner should note any fat accumulation in the abdomen, neck, buttock and breasts or fa t atrophy in the arms, legs or face. 2. Severity grading should be reco rded using pACTG Severity Table.

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96 BIA DIAGRAM: 3.

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97 ARM CIRCUMFERENCE DIAGRAMS: 4a. Elbow flexed 90 degrees 4b. Acromion process to olecrenon 4c. Midpoint mark 4d. Tape tightened

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98 PROPER HANDLING AND CALIBRATION OF LANGE CALIPER DIAGRAMS: 5a. Proper handling 5b. Example of 10 test distance 5c. Example of 30 test distance

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99 TRICEP SKINFOLD DIAGRAMS: 6a. Hold fold between index and thumb 6b. Calipers applied to vertical fold

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100 UMBILICAL AND HIP CIRCU MFERENCE DIAGRAMS: 7a. Waist Circumference at the umbilical level 7b. Circumference at widest width of hip

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101 UMBILICAL AND MID-BACK SKINFOLD DIAGRAMS: 8a. Measure at 2cm to right of umbilicus 8b. Calipers applied at 2cm right mark 8c. Measure at 2cm to right of spinal column 8d. Calipers applied at 2cm right mark

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102 SUPRAILIAC SKINFOLD DIAGRAM: 9. Calipers applied at mid-axilla ry line at umbilicus level

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103 SUBSCAPULAR SKINFOLD DIAGRAMS: 10a. Subject stands with back facing observer 10b. Measure below inferior angle of scapula 10c. Calipers applied at slight angle following natural skin cleavage

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104 MID THIGH CIRCUMFERENCE DIAGRAMS: 11a. Upper leg flexed 90° to locate midpoint 11b. Locate inguinal crease at midline of leg 11c. Midpoint is marked 11d. Circumference at mid-thigh mark

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105 MID-THIGH SKINFOLD DIAGRAM: 12. Caliper applied at mid-thigh mark following skinÂ’s natural cleavage

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106 APPENDIX E BLOOD LIPID STATUS MODULE Blood Lipid Status Module Question: Does the patient have dyslipidemia? Measurement: Fasting (or non fasting) plasma triglycerid e concentration Level 2 (1) Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting (or non fasting) venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Triglyceride concentration is compared to reference standards. Used with other blood lipid measur ements to assess cardiovascular disease risk. CRF Template: Taken from LBW0011 Measurement: Fasting (or non fasting) high density lipoprotein (HDL) concentration Level 2 (1) Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting (or non fasting) venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: HDL concentration is compared to reference standards. Used with other blood lipid measur ements to assess cardiovascular disease risk. CRF Template: Taken from LBW0011 Measurement: Fasting (or non fasting) total cholesterol conc entration Level 2 (1) Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Total cholesterol concentration is compared to reference standards.

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107 Used with other blood lipid measur ements to assess cardiovascular disease risk. CRF Template: Taken from LBW0011 Measurement: Fasting (or non fasting) Low Density Lipoprotein (LDL) Concentration Level 2 (1) Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting venous blood using Metabolic Module SOP. Send samples to lab for analysis. Use FriedewaldÂ’s equation to derive LDL concentration. Record data on CRF. Information Obtained: LDL concentration is compared to reference standards. Used with other blood lipid measur ements to assess cardiovascular disease risk. CRF Template: Taken from LBW0011

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108 Blood Lipid SOP Question: Does the patient have dyslipidemia? Measurement: Fasting (or non fasting) plasma triglycerid e concentration Level 2 (1) General Instructions: For level 1: A fasting sample is preferred but if not possible, an initial nonfasting sample should be taken being sure to record the time that the subject last consumed food. If the result is abnormal then, a fasting ( 8 hrs) sample should follow. For level 2: An initial fasting ( 8 hrs) sample should be taken. SOP: 1. Collect one (4 mL) Serum Separator Tube (SST). 2. Let blood clot for 30 minutes in a verti cal position. 3. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 4. Separate serum from cells and ali quot into 4-5 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label aliquots with subject ID, site ID, date and time of collection, and specimen type. Measurement: Fasting (or non fasting) high dens ity lipoprotein (HDL) concentration General Instructions: For level 1: A fasting sample is preferred but if not possible, an initial nonfasting sample should be taken being sure to record the time that the subject last consumed food. If the result is abnormal then, a fasting ( 8 hrs) sample should follow. For level 2: An initial fasting ( 8 hrs) sample should be taken. SOP: 1. Same as SOP for fasting plasma triglyceride above. Measurement: Fasting (or non fasting) total cholesterol conc entration Level 2 (1) General Instructions: For level 1: A fasting sample is preferred but if not possible, an initial nonfasting sample should be taken being sure to record the time that the subject last consumed food. If the result is abnormal then, a fasting ( 8 hrs) sample should follow. For level 2: An initial fasting ( 8 hrs) sample should be taken. SOP: 1. Same as SOP for fasting plasma triglyceride above.

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109 Measurement: Fasting (or non fasting) Low Density Lipoprotein (LDL) Concentration General Instructions: For level 1: A fasting sample is preferred but if not possible, an initial nonfasting sample should be taken being sure to record the time that the subject last consumed. If the result is abnormal then, a fasting ( 8 hrs) sample should follow. For level 2: An initial fasting ( 8 hrs) sample should be taken. SOP: 1. Same as SOP for fasting plasma triglyceride above. LDL is calculated using Friedewald’s formula: LDL=Total cholesterol – Triglyceride/5 – HDL. 2. If triglyceride concentration is 400 mg/dl then, Friedewald’s formula cannot be used to calculate LDL. Therefore, it must be measured directly by collecting fasting plasma.

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110 APPENDIX F BLOOD GLUCOSE AND INSULIN STATUS MODULE Blood Glucose and Insulin Status Module Question: Does the patient have insulin resis tance and/or glucose intolerance? Measurement: Fasting (or non fasting) blood glucose Level 2 (1) Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting (or non fasting) venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Blood glucose level is compared to reference standards in order to determine insulin resistance, glucos e intolerance and diabetes risk. CRF Template: Taken from PE4811 Measurement: Fasting blood insulin Level 2 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting (or non fasting) venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Blood insulin level is used with other blood glucose measurements to determine insulin resistance, glucose to lerance and diabetes risk. CRF Template: Taken from SPW0309 Measurement: 2 hour glucose tolerance test Level 2 Equipment: Needle and tube, standard glucose beverage Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting venous blood for init ial fasting glucose and insulin measurement using Metabolic Module SOP. Patient drinks pre-measured amount of glucose drink. Two hour post-dose glucose measurem ent using Metabolic Module SOP. Information Obtained: Confirm insulin resistance, glucose intolerance, and diabetes risk. CRF Template: Taken from SPW0309

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111 Blood Glucose and Insulin Status SOP Question: Does the patient have insulin resis tance and/or glucose intolerance? Measurement: Fasting (or non fasting) blood glucose Level 2 (1) General Instructions: For level 1: A fasting sample is preferre d but if not possible an initial nonfasting sample should be taken being sure to record the time that the subject last consumed food. If the result is abnormal then, a fasting ( 8 hrs) sample should follow. For level 2: An initial fasting ( 8 hrs) sample should be taken. SOP: 1. Collect one chilled (3 mL) gray-top Sodium Fluoride/Potassium Oxalate anticoagulated tube and label it with s ubject ID, site ID, date and time of collection, and specimen type. 2. Place the specimen immediately on ice, and complete processing steps 3 & 4 within 30 minutes of collection . 3. Centrifuge gray-top tube at 800 to 1,000 x g for 10 minutes. 4. Remove plasma and prepare replicate 0.5 mL plasma aliquots and freeze at minus 70oC. 5. Label aliquots with subject ID, site ID, date and time of collection, and specimen type . Measurement: Fasting blood insulin Level 2 General Instructions: Subjects MUST be in a fasting state. Subjects should consume no food or beverage (other than normal amounts of plain water) for at least 8 hours before collection of entry specimens. Subjects s hould be asked whether they have fasted for this minimum length of time prior to co llection of entry specimens. If fasting requirements have not been met, subjects should return to th e clinic on another day in a fasting state. Although subjects must fast prior to collec tion of entry specimens, they should be specifically instructed to take all requir ed medications according to their regular schedules and with the usual amounts of wa ter, to the extent that they can be tolerated in a fasting condition. SOP: 1. Collect one (4 mL) Serum Separator Tube ( SST). Label tube with subject ID, site ID, date and time of collection, and specimen type. 2. Let blood clot for 30 minutes in a verti cal position. 3. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g.

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112 4. Separate serum from cells and ali quot into four 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label aliquots with subject ID, site ID, date and time of collection, and specimen type . Measurement: 2 hour glucose tolerance test Level 2 General Instructions: For 3 days before the test, the patient shoul d be asked to eat a balanced diet that includes at least 150 to 200 grams of carbohydrates per day. SOP: 1. Collect a baseline sample for fasting ( 8 hrs) glucose and fasting ( 8 hrs) insulin assays using the procedure described above. 2. After collection of baseline specimens , the subject will immediately consume 75g of chilled oral dextrose in solutio n accompanied by up to 200mL of water. The dextrose solution should be consumed within 5 minutes. T=0 is the time that the consumption of the dextrose solution is completed. The subject will wait for 120 minutes after consumption of dextrose solution. Caution: The subject should not eat, drink, or smoke during the test interval. 3. Collect 2 hour post glucose ingestion blood samples for glucose and insulin assays using the procedure described above.

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113 APPENDIX G BLOOD PRESSURE MODULE Blood Pressure Module Question: Does the patient have hypertension? Measurement: Resting Blood Pressure Level 1 Equipment: Blood pressure cuff Training: Appropriate center training Overview Of Steps Involved: Blood pressure cuff is positioned around patientÂ’s arm and blood pressure is measured using Metabolic Module SOP. Record measurement on CRF. Information Obtained: Systolic and Diastolic pressure whic h is used with age, gender, total cholesterol, HDL and LDL to dete rmine risk for heart disease and stroke. CRF Template: Taken from PE0032

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114 Blood Pressure SOP Question: Does the patient have hypertension? Measurement: Resting Blood Pressure Level 1 General Instructions: A blood pressure reading should be taken at every clinic visit. Strenuous exercise should be avoided 1-2 hrs prior to measurement. Blood pressure cuffs should be ageand size-specific.* The cuff bladder width should be approximately 40% of the circum ference of the arm measured at a point midway between the olecranon and acromion. Values should be interprete d according to normal values adjusted for age, gender, and height percentile. (See National Hi gh Blood Pressure Education ProgramÂ’s 1996 Update on Task Force Report). SOP: 1. Patient should be seated for at least 5 minutes with arm resting on a table. The arm should be slightly bent so that it is at the same level as the heart. The upper arm should be bare, with sleeve rolled up, but not tight or uncomfortable. 2. Wrap the blood pressure cuff snugly around the upper arm, positioning it so that the lower edge of the cuff is 1 inch above the bend of the elbow. 3. Locate the large artery on the inside of the elbow by feeling for the pulse and place the head of the stethoscope (or mi crophone) over this artery, below the cuff. It should not rub the cuff or any clothing because these noises may block out the pulse sounds. Correct positioning of the stethoscope is important to get an accurate recording. 4. Close the valve on the rubber inflating bul b and then squeeze it rapidly to inflate the cuff until the dial or column of mercury reads 30 mmHg (millimeters of mercury) higher than the usual systolic pre ssure. If the usual sy stolic pressure is unknown, the cuff should be inflated to 210 mmHg. 5. Open the valve slightly, allowing the pre ssure to fall gradually (2 to 3 mmHg per second). As the pressure falls, the level on the dial or mercury tube at which the pulsing is first heard is recorded. This is the systolic pressure. As the air continues to be let out, the sounds will disappear. The point at which the sound disappears is recorded. This is the diastolic pressure. *See National Kidney Foundation DOQI guidelines at http://www.kidney.org/professionals /kdoqi/guidelines_bp/guide_13.htm.

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115 APPENDIX H MITOCHONDRIAL METABOLISM MODULE Mitochondrial Metabolism Module Question: Does the patient have mitochondrial dysfunction? Measurement: Anion Gap Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting venous blood ( 8 hrs) using Metabo lic Module SOP. Send samples to lab for analysis. Use electrolyte panel to calculate anion gap: Na+ (Cl+ HCO3 -) Record data on CRF. Information Obtained: The concentration of all unmeasured an ions in the plasma which is used to determine the presence of acidos is and to differentiate between causes of a metabolic acidosis. CRF Template: Added to PE4811 Measurement: Liver Function Tests (ALT, AST, ALP, albumin) Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting venous blood ( 8 hrs) using Metabo lic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: High serum levels of ALT, AST, and ALP are indicative of liver disease. Albumin is synthesized by the liver a nd is a marker of the liverÂ’s ability to synthesize proteins. CRF Template: Taken from PE4811 Measurement: Lipase, Amylase Level 2 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw non fasting venous blood us ing Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Levels of lipase and amylase which ar e markers for pancreatic disease.

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116 CRF Template: Taken from PE4811 Measurement: Lactic Acid Level 2 Equipment: Needle and tube Training: Certified phlebotomist a nd ability to centrifuge Overview Of Steps Involved: Draw non fasting venous blood us ing Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Lactic Acid is used to determine presence of lactic acidosis. CRF Template: Taken from F0886 Measurement: Mitochondrial DNA Level 2 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Amount of mitochondrial DNA is used with other mitochondrial metabolism measurements to dete rmine if there is mitochondrial dysfunction. CRF Template: Taken from EVW0089 Measurement: Acylcarnitine profiling Level 2 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw fasting venous blood ( 8 hrs) using Metabo lic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Differentiation of fatty acids bound to car nitine is used to determine if a fatty acid oxidation disorder is present. CRF Template: Taken from EVW0089

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117 Mitochondrial Metabolism SOP Question: Does the patient have mitochondrial dysfunction? Measurement: Anion Gap Level 1 General Instructions: If this test is abnormal a nonfasting l actic acid test shoul d be performed. SOP: 1. Collect one (4 mL) Serum Separator Tube (SST). 2. Let blood clot for 30 minutes in a verti cal position. 3. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 4. Separate serum from cells and ali quot into 8-9 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label serum aliquots with subject ID, s ite ID, date and time of collection, and specimen type. Measurement: Liver Function Tests (ALT, AST, ALP, albumin) Level 1 General Instructions: SOP: 1. Collect one (4 mL) Serum Separator Tube (SST). 2. Let blood clot for 30 minutes in a verti cal position. 3. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 4. Separate serum from cells and ali quot into 8-9 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label serum aliquots with subject ID, s ite ID, date and time of collection, and specimen type. Measurement: Lipase, Amylase Level 2 General Instructions: SOP: 1. Collect one (4 mL) Serum Separator Tube (SST). 2. Let blood clot for 30 minutes in a verti cal position. 3. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 4. Separate serum from cells and ali quot into 8-9 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label serum aliquots with subject ID, s ite ID, date and time of collection, and specimen type.

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118 Measurement: Lactic Acid Level 2 General Instructions: Venous lactate levels are highly depe ndent on collection techniques. The procedures listed below s hould be followed precisely. Note that the specimen must be collected in a ch illed gray-top tube and processed within 30 minutes of collection. Lactate can be run on the same specimen collected for the baseline glucose for the OGTT if the specimen was coll ected without a tourniquet . The subject must have avoided strenuous exercise the day of the blood drawing and the day before the blood drawing. SOP: 1. In order to minimize subject discomfort , apply a topical anesthetic (e.g. Emla® Cream) prior to venipuncture. 2. Have subject sit, relaxed for 5 minutes prior to venipuncture. 3. Instruct subject to not clench the fist befo re or during the procedure and to relax the hand as much as possible. 4. Use of a tourniquet should be avoided, if at all possible. If a t ourniquet is necessary, then apply the tourniquet ligh tly and draw lactate first be fore the other samples with the tourniquet still in place. 5. Collect one chilled (3 mL) gray-top Sodium Fluoride/Potassium Oxalate anticoagulated tube. Label tube with subjec t ID, site ID, date and time of collection, and specimen type. 6. Place the specimen immediately on ice, and complete specimen processing Steps 7 & 8 within 30 minutes of specimen collection . 7. Centrifuge grey-top tube at 800 to 1,000 x g for 10 minutes. 8. Remove plasma and prepare replicate 0.5 mL plasma aliquots and freeze at minus 70oC. 9. Label aliquots with subject ID, site ID, date and time of collection, and specimen type. Measurement: Mitochondrial DNA Level 2 SOP: 1. Collect one (3 mL) purple-top EDTA anticoagulated blood tube. 2. Process EDTA blood tube according to the PACTG Virology Manual, available online at http://www.niaid.nih.gov/daids/vir_manual/ . 3. Freeze plasma in three 0.6 mL aliquots and store at minus 70oC. 4. Prepare twice-washed PBMCs according to the PACTG Virology Manual, available online at http://www.niaid.nih.gov/daids/vir_manual/ .

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119 5. Prepare replicate dry cell PB MC pellet aliquots at 1 x 106 cells per vial. 6. Freeze dry cell PBMC aliquots at minus 70oC. 7. Ship frozen vials on dry ice. Measurement: Acylcarnitine profiling Level 2 General Instructions: Subject should be in fasting ( 8 hrs) state. SOP: 1. Collect one (1 mL) sodium he parin (green-top) blood tube. 2. Separate plasma from cells as soon as possible by centrifugation at 1500 X g for 30 minutes. 3. Freeze plasma in two 0.50 mL aliquots and store at minus 70oC. 4. Ship vials frozen.

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120 APPENDIX I RENAL METABOLISM MODULE Renal Metabolism Module Question: Does the patient have renal dysfunction? Measurement: Serum BUN Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: The amount of nitrogen in the blood is used with serum creatinine to evaluate kidney function. CRF Template: Taken from PE4811 Measurement: Serum Creatinine Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Measures glomerular filtration rate (GFR). Used with BUN to evaluate level of kidney function. CRF Template: Taken from PE4811 Measurement: Serum HCO3-, Potassium, Phosphate Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Amount of CO2 in blood is used to de termine if there is renal tubular dysfunction. CRF Template: Taken from PE4811 Measurement: Urinalysis Level 1 Equipment: Dipstick

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121 Training: Short course, trained microscopist Overview Of Steps Involved: Use dipstick to semi-quantify the amount of protein. Perform microscopic exam to look for the number of WBC, RBC and waxy and hyaline casts. Information Obtained: Amount of protein in urine and presen ce of casts are indicators of renal dysfunction. CRF Template: Taken from F0868 Measurement: Urine protein:creatinine ratio Level 2 Equipment: Sterile urine container, dipstick Training: Short course Overview Of Steps Involved: Obtain spot (not 24 hr) urine sample. Send sample to lab for analysis. Record data on CRF. Information Obtained: Quantitative measurement of proteinuria is used with other renal metabolism measurements to assess kidney function. CRF Template: Taken from PE4811 Measurement: Creatinine clearance Level 2 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: The rate and efficiency of kidney filtration. CRF Template: Taken from LBW0045

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122 Renal Metabolism SOP Question : Does the patient have renal dysfunction? Measurement: Serum BUN Level 1 General Instructions: Subject should avoid eating food high in protein the day prior to this test. SOP: 7. Collect one (4 mL) Serum Separator Tube (SST). 8. Let blood clot for 30 minutes in a verti cal position. 9. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 10. Separate serum from cells and ali quot into 8-9 0.5 mL cryo vials. 11. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 12. Label serum aliquots with subject ID, s ite ID, date and time of collection, and specimen type. Measurement: Serum creatinine Level 1 General Instructions: Subject should avoid strenuous exerci se the day prior to this test. SOP: 5. Same as SOP for serum BUN above. Measurement: Serum HCO3-, potassium, phosphate Level 1 SOP: 1. Same as SOP for serum BUN above. Measurement: Urinalysis Level 1 SOP: 1. Collect random urine (10 mL) in a clean container. 2. Refrigerate urine in transit to the laboratory. 3. Aliquot urine into ten (1 mL) cryovials. 4. Freeze urine aliquots at minus 70oC within 8 hours of collection. 5. Label aliquots with subject ID, site ID, date and time of collection, and specimen type. 6. Observe sediment under low power microscope. Measurement: Urine protein: creatinine ratio Level 2 SOP: 1. Same as SOP for urinalysis above.

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123 Measurement: Creatinine Clearance Level 2 SOP: 1. Same as SOP for serum BUN above. 2. Calculate creatinine clearance using the Sc hwartz formula = (k x 1.73 x Height) / SCr (Where k is a constant, Height is in centimet ers, and SCr = serum creatinine in mg/dl.). If age is less than 1 year, k = 0.45. If age is greater than or equal to 1 year, k = 0.55.

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124 APPENDIX J BONE METABOLISM MODULE Bone Metabolism Module Question: Does the patient have bone disease? Measurement: Radiographically proven fractures Level 1 Equipment: X-ray machine Training: Certified x-ray technician Overview Of Steps Involved: Patient lies down and remains still on the x-ray table. X-ray pictures are take n at desired locations. Record measurements on CRF. Information Obtained: Presence of bone fractures. CRF Template: Taken from PE4850 Measurement: Alkaline Phosphatase (ALP) Level 1 Equipment: needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Level of ALP in the blood, which is used as an initial screen for bone disease. CRF Template: Taken from PE4811 Measurement: Bone density Level 2 Equipment: DEXA scan Training: Certified DEXA technician Overview Of Steps Involved: Patient lies down for 10-20 minut es beneath a whole body scanner. Bone density is measured using Metabolic Module SOP. Record measurements on CRF. Information Obtained: Bone density measurement is compared to reference values to determine fracture risk. CRF Template: Taken from DGW0030 Measurement: Serum bone specific alkaline phospha tase (BSAP) Level 2 Equipment: Needle and tube

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125 Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood using Metabolic Module SOP. Send samples to lab for analysis. Record data on CRF. Information Obtained: Used with other Bone metabolism parameters to determine fracture risk. CRF Template: Taken from PE4811 Measurement: Urinary Cross-linked N-Te lopeptide (Ntx ) Level 2 Equipment: Sterile urine container Training: Short Course Overview Of Steps Involved: Obtain a second-morning void urine sample. Send sample to lab for analysis. Record data on CRF. Information Obtained: Used with other Bone metabolism parameters to determine fracture risk. CRF Template:

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126 Bone Metabolism SOP Question: Does the patient have bone disease? Measurement: Radiographically proven fractures Level 1 General Instructions: X-rays would not be routinely done but if a subject has x-rays available from routine care it can be examined for fract ures. X-rays may be obtained from various locations. SOP: 1. Ask subject to lie down on x-ray table. 2. Depending on the desired location of the xray, position subject beneath the x-ray machine. 3. Gently pull x-ray machine down coming close to but not touching the desired location. 4. Ask subject to remain still and hold this position until picture is taken. 5. Proceed to control room and take x-ra y pictures following standard operating procedures for the x-ray instrument. 6. Be sure to take pictures from at least two different angles (three if necessary), repositioning subject as needed. Specific procedures are availa ble from NHANES III lab manual: http://www.cdc.gov/nchs/data/nhanes/ nhanes3/cdrom/NCHS/MANUALS/XRAY.PDF Measurement: Alkaline Phosphatase General Instructions: SOP: 1. Collect one (4 mL) Serum Separator Tube (SST). 2. Let blood clot for 30 minutes in a verti cal position. 3. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 4. Separate serum from cells and ali quot into 8-9 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label serum aliquots with subject ID, s ite ID, date and time of collection, and specimen type. Measurement: Bone Density Level 2 General Instructions: The following is a summary of pr ocedures for whole-body DEXA scans performed for body composition measurements and regional (hip and spine) DEXA scans. Please follow the manufact urerÂ’s instructions for operating your instrument. Whole-body and hip and spine scans should be done using standard

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127 operating procedures defined by the manuf acturer. The subject should be asked about and examined for metal that could be in the scan path. Typical things to look for are earrings, eyeglasses, wristwat ches, coins, rings, buttons, buckles, zippers, and support braces. The subject should remove shoes, and it may be necessary to remove skirts, slacks, etc. If in doubt, it is best to remove the object in question. Whole body Protocol SOP: 1. When prompted by the program, ask the s ubject to lie down on the scan table. 2. The centerline on the table pad should divi de the subject's body in half. Use the lines on the table pad to ensure the subject is lying straight on the table. The subject's head should be di rectly below (about 1") the horizontal line located along the top of the table pad. 3. Have the subject tilt his/her head back slightly during the scan. This makes placement of the head region cut line easier to place. 4. A foam wedge (or pillow) should not be us ed under the subject's head as it will affect the body composition results. 5. Have the subject place hands palms down alongside the body with fingers together. Do not overlap hands and legs. The subject's entire body sh ould fit within the scan lines on the table pad. 6. If the subject is too wide to fit with in the boundary lines, the technician should proceed to the Hemiscan Protocol section below. For tall s ubjects, please proceed to the Tall Subjects section below. 7. The subject's feet should be held together using a Velcro strap, and the subject is asked not to move until directed to do so. 8. Continuing the scan will cause the x-ray t ube to ramp up to th e appropriate current and voltage. The operator should check to make sure the orange "X-Ray On" light is lit, and remain in the room to check the progress of the scan acquisition as it appears on the screen. 9. The subject's head should appear with a few blank scan lines above it. As the scan proceeds, the total-body image should be in a straight line vertically on the screen. If these conditions are not met, the scan should be stopped. The scan arm will move to the original start position and the local izer light will come on. The subject should be repositioned as needed. 10. When the detector goes past the subject's feet, the auto stop fe ature then interrupts the scan and closes the shutter. A me ssage appears on the screen, allowing the operator to continue the scan or shut down the system. After the scan ends the shutter closes, the voltage and current ramp down, and a messages appears for the operator to wait. The scan arm moves to the home position a nd a screen message appears to inform the operator that the scan is over and to remove the subject from the table. The technician should get to the Total-body Scan Options screen, then save the scan file. 11. The operator may now exit the Total-body porti on of the program to do another type of scan.

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128 Hemiscan Protocol for Obese Subjects SOP: 1. The left side of the body is aligned within the left margin of th e scanning table, and the subject is scanned as prev iously described. This allows the patient’s left side to be completely within the cut lines, so th at the entire “overflow” occurs on the right side. This overflow should be ignored. 2. During the scan analysis at the Central Reading Site, the midline of the body is selected by the technician to demarcate th e left half of the body as the region of interest, and a scan report is produced. 3. The regional scan data are multiplied by 2 to give the whole-body estimate of body composition. Tall Subjects Protocol General Instructions: If a person is too tall to fit within the lines, prioritize what part to “cut off” using the following list. Go down the list from top to bottom, and cut off as many parts as needed to fit the person on the table. Make note of the parts that were cut off for future reference. Top half of the head (to the tops of the ears) Lower foot (toes) Bottom half of the head (to the jaw) SOP: 1. Position against the board, relaxed. 2. Do not use support under the knees for size, only for comfort when absolutely necessary. 3. If necessary, use a head pillow carefully only after the scanner has passed over the head region. When inserting a pillow unde r the head, please make sure that the subject does not move. Hip and Spine Protocol General Instructions: For hip and spine scans, the subject-pos itioning techniques and the scan acquisition procedures should be done using the standard operati ng procedures defined by the instrument manufacturer. Measurement: Serum bone specific alkaline phos phatase (BSAP) Level 2 SOP: 1. Collect one (4 mL) Serum Separator Tube (SST).

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129 2. Let blood clot for 30 minut es in a vertical position. 3. Within 1 hour of collection, spin blood tube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 4. Separate serum from cells and aliquot into 8-9 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label serum aliquots with subject ID, site ID, date and time of collection, and specimen type. Measurement: Urinary Cross-linked N-Te lopeptide (Ntx) Level 2 SOP: 1. Collect random urine (10 mL) in a clean container. 2. Refrigerate urine in transit to the laboratory. 3. Aliquot urine into ten (1 mL) cryovials. 4. Freeze urine aliquots at minus 70oC within 8 hours of collection. 5. Label aliquots with subject ID, site ID, date and time of collection, and specimen type.

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130 APPENDIX K PROTEIN NUTRITION STATUS MODULE Protein Nutrition Status Module Question: Does the patient have protein malnutrition? Measurement: Albumin Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood sample from patientÂ’s arm using Metabolic Module SOP Send sample to lab for analysis. Report data on CRF. Information Obtained: Compare to reference values to determine patientÂ’s long-term protein nutrition status CRF Template: Taken from PE4811 Measurement: Prealbumin Level 2 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood sample from patientÂ’s arm using Metabolic Module SOP. Send sample to lab for analysis. Report data on CRF. Information Obtained: Compare to reference values to dete rmine patientÂ’s short-term protein nutrition status. CRF Template: Taken from PE4811

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131 Protein Nutrition Status SOP Question: Does the patient have protein malnutrition? Measurement: Albumin Level 1 SOP: 1. Collect one (8.5 mL) Serum Separator Tube (SST). 2. Let blood clot for 30 minutes in a verti cal position. 3. Within 1 hour of collection, spin blood t ube for 10 minutes in a swinging bucket centrifuge or for 15 minutes in a fixed angle centrifuge at 1100 to 1300 X g. 4. Separate serum from cells and ali quot into 8-9 0.5 mL cryo vials. 5. Freeze serum aliquot vials at minus 70oC within 8 hours of collection. 6. Label serum aliquots with subject ID, s ite ID, date and time of collection, and specimen type. Measurement: Prealbumin Level 2 SOP: 1. Same as SOP for albumin above.

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132 APPENDIX L ANEMIA MODULE Anemia Module Question: Does the patient have anemia? Measurement: Hemoglobin from Complete Blood Count (CBC) Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood sample from patientÂ’s arm using Metabolic Module SOP. Send sample to lab for analysis. Report data on CRF. Information Obtained: Measures amount of oxygen-ca rrying protein in the blood. Used with other Anemia parameters to determine if patient has anemia. CRF Template: Taken from PE4811 Measurement: Hematocrit from CBC Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood sample from patientÂ’s arm using Metabolic Module SOP. Send sample to lab for analysis. Report data on CRF. Information Obtained: Measures the percentage of space the red blood cells take up in the blood. Used with other Anemia Module parameters to determine if patient has anemia. CRF Template: Taken from PE4811 Measurement: Red Blood Cell Count from CBC Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood sample from patientÂ’s arm using Metabolic Module SOP. Send sample to lab for analysis. Report data on CRF. Information Obtained:

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133 Measures the number of red blood cells per volume of blood. Used with other Anemia Module parameters to determine if patient has anemia. CRF Template: Taken from PE4811 Measurement: Mean Corpuscular Volume (MCV) from CBC Level 1 Equipment: Needle and tube Training: Certified phlebotomist and ability to centrifuge Overview Of Steps Involved: Draw venous blood sample from patientÂ’s arm using Metabolic Module SOP. Send sample to lab for analysis. Report data on CRF. Information Obtained: Measures the average size of red blood cells. Used with other Anemia Module parameters to determine if patient has anemia. CRF Template: Taken from PE4811

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134 Anemia SOP Question: Does the patient anemia? Measurement: Hemoglobin from Complete Blood Count (CBC) Level 1 SOP: Caution: Subject should avoid smoking the da y of this exam as smoking can increase hemoglobin levels. 4. Collect one (7 mL) purple-top EDTA anticoagulated blood tube. 5. Process EDTA blood tube according to the PACTG Virology Manual, available online at http://www.niaid.nih.gov/daids/vir_manual/ . 6. Freeze plasma in 7-8 0.5 mL aliquots and store at minus 70oC. 4. Ship vials frozen. Measurement: Hematocrit Level 1 SOP: 1. Same as Hemoglobin SOP above. Measurement: Red Blood Cell Count Level 1 SOP: 1. Same as Hemoglobin SOP above. Measurement: Mean Corpuscular Volume Level 1 SOP: 1. Same as Hemoglobin SOP above.

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135 APPENDIX M DIETARY INTAKE MODULE Dietary Intake Module Question: Is the nutrient intake meeting the nu trient requirements of the patient? Measurement: 24 hour Dietary Recall Level 1 Equipment: CRF Training: Short course Overview Of Steps Involved: Ask patient to recall actual food and drink consumed on specified days, usually the immediate past 24 hours. Information Obtained: Amount of patient dietary intake during a 24 hour period. CRF Template: Taken from LGW0056 Measurement: 3 day diet diary Level 2 Equipment: CRF Training: Short course Overview Of Steps Involved: Ask patient to provide concise diet ary intakes for three 24 hour periods, preferably two weekdays and one weekend day. Information Obtained: Amount of patient dietary intake during a three 24 hour period. CRF Template: Taken from LGW0056 Measurement: Dietary Assessment Level 2 Equipment: Diet Analysis tool Training: Short course Overview Of Steps Involved: Input dietary intakes into a dietary analysis software. Analyze and graph dietary data acquired from all other dietary measurements. Graph triglycerides and total cholesterol periodically. Information Obtained: Percentage of nutrients consumed by patient during a 24 hour period or three 24 hour periods. Changes in diet and lipid profile over specific period. CRF Template: N/A

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136 Dietary Intake SOP Question: Is the nutrient intake meeting the nu trient requirements of the patient? Measurement: 24 hour Dietary Recall Level 1 General Instructions: The 24-hour dietary recall cons ists of a listing of foods and beverages consumed the previous day or the 24 hours prior to the recall interview. Foods and amounts are recalled from memory with the aid of an interviewer who has been trained in methods for soliciting diet ary information. The interv iew is usually conducted face to face, but may also be conducted by telephone. In some situations, the recall is self-administered by the subj ect, but this approach may not yield sufficiently reliable data. A brief activity history may be incorporated into the interview to facilitate probing for foods and beverages consumed. SOP: 1. Ask subject to provide concise diet ary intakes for preceding 24 hour period with portion sizes included. Use visu al aids and models to aid subject recollection. Include questions regarding physi cal activity, vitamin consumption, and gastrointestin al issues. Measurement: 3 day diet diary Level 2 General Instructions: A diet diary requires that the subject (or observer) re port all foods and beverages consumed for a specified pe riod (usually one to seven days). Two weekdays and one weekend day is preferred to incorpor ate the changes in diet that may occur within during the weekend. If nutrient intakes are to be calculated, the amounts consumed should be estimated as accurately as possible. Amounts may be determined by weighing or by estimating volumes. SOP: 1. Ask subject to keep a 3 day diary of all foods and beverages consumed, preferably two weekdays and one weekend day. Subject should accurately estimate amount of all foods and beverages consumed. Measurement: Dietary Assessment Level 2 General Instructions: Converting food consumption data to nutrient intakes re quires both a food composition database and ideally comput er programs for nutrient calculation. Available regional food composition data should be carefully evaluated for relevance of foods and food descriptions, as well as for accuracy and completeness of the food and nutrient data. Computer software should be used for nut rient calculations wh ich are tedious and prone to error if done manually. Quality c ontrol procedures for data entry should

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137 be carefully adhered to. So ftware should be carefully evaluated, both for program features, such as the ease of data entry, reporting capabilities and hardware requirements, as well as for the quality of the nutrient database. SOP: 1. Enter nutrient data into computer software. 2. Enter nutrient data into databa se for nutrient calculations.

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138 APPENDIX N GROWTH MEASUREMENTS CRF

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141 APPENDIX O ANTHROPOMETRIC MEASUREMENTS CRF

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149 APPENDIX P DEXA CRF

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157 APPENDIX S BLOOD LIPIDS CRF

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158 APPENDIX T COMPREHENSIVE CHEMISTRY CRF

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162 APPENDIX U METABOLIC SPECIMEN TRACKING CRF

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170 APPENDIX V VITAL SIGNS CRF

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172 APPENDIX W URINALYSIS CRF

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174 APPENDIX X CREATININE CLEARANCE CRF

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176 APPENDIX Y MITOCHONDRIAL TOXICITY CRF

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182 APPENDIX Z LACTATE CRF

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184 APPENDIX AA COMPREHENSIVE HEMATOLOGY CRF

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187 APPENDIX BB DIET CRF

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192 APPENDIX CC PEDIATRIC LAB REFERENCE RANGES HDL (plasma/serum) Male Female Test Age n mg/dL mmol/L n mg/dL mmol/L 1 1-9 y 50* 35-82 0.91-2.12 50 35-82 0.91-2.12 10-13 y 56* 36-84 0.93-2.17 56 36-84 0.93-2.17 14-19 y 60* 35-65 0.91-1.68 60 35-65 0.91-1.68 2 5 y ** 37-72 0.96-1.86 ** 34-69 0.88-1.78 10 y 31-70 0.80-1.81 34-69 0.88-1.78 15 y 29-61 0.75-1.58 34-69 0.88-1.78 20 y 29-58 0.75-1.50 36-70 0.93-1.68 3 0-20 mo 43*** 8-61mg 0.21-1.58 43*** 8-61mg 0.21-1.58 2-<7 y 95 23-70mg 0.60-1.81 71 12-64mg 0.31-1.66 7-<12 yrs 99 25-59mg 0.65-2.05 133 23-80mg 0.60-2.07 12-<16 yrs 199 19-76mg 0.49-1.97 181 25-83mg 0.65-2.15 16-<19 yrs 91 26-75mg 0.67-1.94 157 21-77mg 0.54-1.99 4 0-24 mo 12-60m 0.31-1.55 12-60m 0.31-1.55 2-< 7 yrs 26-68 0.67-1.76 16-62 0.41-1.61 7-<12 yrs 28-76 0.73-1.97 26-67 0.67-1.99 12-<16 yrs 22-73 0.57-1.89 28-79 0.73-2.05 16-<19 yrs 28-72 0.73-1.86 24-74 0.62-1.92

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193 Specimen Type (s) 1Serum 2Plasma 3, 4Plasma/serum Reference (s) 1Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 2Kottke BA, Moll PP, Michels VV, et al. Levels of lipids, lipoproteins and apolipoproteins in a defined population. Mayo Clin Proc 1991; 66:1198-1208. 3Murthy JN, Soldin SJ. Pediatric reference ranges for HDL-cholesterol on the Vitros 500 analyzer. Clin Chem 1999; 45: A23. (Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003; 331: 135-46. Method (s) 1,3Dextran-sulfate precipitation/choleste rol oxidase. Vitros 700 (1) and 500 (3) (Ortho-Clinical Diagnostics, Raritan, NJ). 2LDL and VLDL precipitated using polyethylene glycol 6000. Supernatant used to determine HDL-cholesterol by an enzymatic method. See reference. 4The HDL cholesterol assay is a homogenous method for directly measuring HDL levels without the need for off-line pretreatment or centrifugation steps. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1Healthy children. Results are 2.5-97.5th percentiles. *No significant differences were found for males and females. These ranges were therefore derived from combined data. 2Results are 5-95th percentiles read off graphs in reference. **See reference for numbers. 3***Male and female combined. The study used hospitalized patients and employed Chauvenet's criteria for removing outliers and a computerized approach from the Hoffman technique to obtain the 2.5-97.5th percentiles. The 2.5 and 97.5th percentiles are lower during the first 2 y of life. For children > 2 y, the 25th percentiles are lower than th ose previously described by Lockitch, et al. Clin Chem 1988;34: 1622-5. 4****Reference ranges were obtained by comparing results from previously published data and using regression equations.

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194 Total Cholesterol Male Female Test Age n mg/dL mmol/L n mg/dL mmol/L 1 0-1 mo 37 45-177 1.16-4.58 27 63-198 1.63-5.12 2-6 mo 354 60-197 1.55-5.09 243 66-218 1.71-5.64 7-12 mo 401 89-208 2.30-539 252 74-218 1.91-5.64 2 1-3 y 49* 44-181 1.15-4.70 49* 44-181 1.15-4.70 4-6 y 38* 108-187 2.80-4.80 38* 108-187 2.80-4.80 7-9 y 72* 112-247 2.90-6.40 72* 112-247 2.90-6.40 10-11 y 28 125-230 3.25-5.95 34 127-244 3.30-6.30 12-13 y 32 127-230 3.30-5.95 40 125-213 3.25-5.55 14-15 y 39 106-224 2.75-5.80 50 130-213 3.35-5.55 16-19 y 41 110-220 2.85-5.70 68 106-217 2.75-5.60 3 1-30 d 62 54-151 1.40-3.90 74 62-155 1.60-4.01 31-182 d 77 81-147 2.09-3.80 75 62-141 1.60-3.65 183-365 d 53 76-179 1.97-4.63 45 76-216 1.97-5.59 1-3 y 136 85-182 2.20-4.71 111 108-193 2.79-4.99 4-6 y 112 110-217 2.84-5.61 113 106-193 2.74-4.99 7-9 y 124 110-211 2.84-5.46 104 104-210 2.69-5.43 10-12 y 111 105-223 2.72-5.77 109 105-218 2.72-5.64 13-15 y 126 91-204 2.35-5.28 105 108-205 2.79-5.30 16-18 y 112 82-192 2.12-4.97 110 92-234 2.38-6.05 4 0-1 mo ** 38-174 0.98-4.50 ** 56-195 1.45-5.04 2-6 mo 53-194 1.37-5.02 59-216 1.53-5.59 7-12 mo 83-205 2.15-5.30 68-216 1.76-5.59 1-3 y 37-178 0.96-4.60 37-178 0.96-4.60 4-6 y 103-184 2.66-4.76 103-184 2.66-4.76 7-9 y 107-245 2.77-6.34 107-245 2.77-6.34 10-11 y 120-228 3.10-5.90 122-242 3.16-6.26 12-13 y 122-228 3.16-5.90 120-211 3.10-5.46 14-15 y 101-222 2.61-5.74 125-211 3.23-5.46 16-18 y 105-218 2.72-5.64 101-215 2.61-5.56

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195Specimen Type (s) 1-4sPlasma/serum Reference (s) 1Soldin SJ, Rakotoarisoa FTS. Pediatric reference ranges for amylase and cholesterol on Kodak Ektachem 500 in the first year of life. Clin Chem 1996; 42: S308. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Hicks JM, Bailey J, Beatey J, et al. Pediatric reference ranges for cholesterol. Clin Chem 1996;42: S307. (Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003; 331: 135-46. Method (s) 1,2Cholesterol oxidase method. Vitros 700 (Ortho-Clinical Diagnostics, Raritan, NJ). 3Boehringer Mannheim reagents on the Hitachi 747 analyzer (Boehringer Mannheim Diagnostics, Indianapolis, IN). 4Cholesterol esterase catalyzes the hydrolysis of cholesterol esters to produce free cholesterol, which along with preexisting free cholesterol, is oxidized in a reaction catalyzed by cholesterol oxidase. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1,3Values are 2.5-97.5th percentiles. Study used hospitalized patients and a computerized approach to removing outliers. 2The study population was healthy children. Non-parametric methods used to determine the reference values. The central 95% were used. *Males and females wre not studied separately; n refers to the total number of males and females studied in each age group. 4**Reference ranges were obtained by comparing results from previously published data and using regression equations.

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196 Triglycerides Male Female Test Age n mg/dL mmol/L n mg/dL mmol/L 1 0-7 d 149 21-182 0.24-2.06 142 28-166 0.32-1.88 8-30 d 283 30-184 0.34-2.08 172 30-165 0.34-1.86 31-90 d 247 40-175 0.45-1.98 171 35-282 0.40-3.19 91-180 d 132 45-291 0.51-3.29 126 50-355 0.57-4.01 181-365 d 286 45-501 0.51-5.66 261 36-431 0.41-4.87 2 1-3 y 49* 27-125 0.31-1.41 49* 27-125 0.31-1.41 4-6 y 38* 32-116 0.36-1.31 38* 32-116 0.36-1.31 7-9 y 72* 28-129 0.32-1.46 72* 28-129 0.32-1.46 10-11 y 28 24-137 0.27-1.55 34 39-140 0.44-1.58 12-13 y 32 24-145 0.27-1.64 40 37-130 0.42-1.47 14-15 y 39 34-165 0.38-1.86 50 38-135 0.43-1.52 16-19 y 41 34-140 0.38-1.58 68 37-140 0.42-1.58 3 Cord blood 397** 9-92mg 0.10-1.04 397** 9-92mg 0.10-1.04 serum arterial Cord blood serum venous 397** 12-86mg 0.13-0.97 397** 12-86mg 0.13-0.97 4 0-7 d *** 19-174 0.21-1.97 *** 26-159 0.29-1.80 8-30 d 37-279 0.42-3.15 33-270 0.37-3.05 31-90 d 42-279 0.47-3.15 34-340 0.38-3.84 1-3 y 25-119 0.28-1.34 25-119 0.28-1.34 4-6 y 30-110 0.34-1.24 30-110 0.34-1.24 7-9 y 26-123 0.29-1.39 26-123 0.29-1.39 10-11 y 22-131 0.25-1.48 37-134 0.42-1.51 12-13 y 22-138 0.25-1.56 35-124 0.40-1.40 14-15 y 32-158 0.36-1.78 36-129 0.41-1.46 16-19 y 32-134 0.36-1.51 35-134 0.40-1.51

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197Specimen Type (s) 1Serum 2-4sPlasma/serum Reference (s) 1Soldin SJ, Morse AS. Pediatric reference ranges for calcium and triglycerides in children < 1 year old using the Vitros 500 Analyzer. Clin Chem 1998;44:A16. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Perkins SL, Livesey JF, Belcher J. Reference intervals for 21 clinical chemistry analytes in arterial and venous umbilical cord blood. Clin Chem 1993;39:10411044 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003; 331: 135-46. Method (s) 1,2Glycerol phosphate oxidase on Vitros 700 analyzer (Ortho-Clinical Diagnostics, Raritan, NJ). 3Hitachi 737 with Boehringer Mannheim reagents (Boehringer Mannheim Canada, Montreal Canada). 4The sample is pre-incubated with lipase enzyme reagent, which converts triglycerides into free glycerol and fatty acids. The liberated glycerol is determined enzymatically using glycerol dehydrogenase and NAD. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1The study used plasma/serum obtained from hospitalized patients and employed Chauvenet's criteria to remove outliers and a computerized approach adapted from the Hoffman technique to obtain the 2.5-97.5th percentiles. Fasting samples were not obtained in these neonates and infants, which accounts for the somewhat elevated 97.5th percentiles. 2A healthy population of children 1-19 y was studied. Results are 2.5-97.5th percentiles. *No significant differences were found for males and females. These ranges were therefore derived from combined data. 3Results are 2.5-97.5th percentiles. **No significant differences were found for males and females. These ranges were therefore derived from combined data. 4***Numbers not provided Reference ranges were obtained by comparing results from previously published data and using regression equations.

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198 Lowdensity Lipoprotein Cholesterol (LDL) Male and Female Test Age n mg/dl mmol/L 1 2-12 mo 100 32-117 0.82-3.02 2-10 y 120 38-140 0.98-3.62 Male Female 2 Age n mg/dL mmol/L n mg/dL mmol/L 5-9 y * 63-129 1.63-3.34 * 68-140 1.76-3.63 10-14 y 64-133 1.66-3.44 68-136 1.76-3.52 15-19 y 62-130 1.61-3.37 59-137 1.53-3.55 3 0-90 d 20-83 0.52-2.15 15-95 0.39-2.46 91d-12 mo 35-120 0.91-3.11 45-125 1.17-3.24 13-36 mo 35-125 0.91-3.24 35-125 0.91-3.24 4-10 y 45-140 1.17-3.63 35-135 0.91-3.50 11-15 y 45-120 1.17-3.11 50-130 1.30-3.37 16-18 y 55-120 1.42-3.11 70-120 1.81-3.11

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199Specimen Type (s) 1,2Serum 3Plasma/serum Reference (s) 1Baroni S, Scribano D, Valentini P, et al. Serum apolipoprotein A1, B, CII, CIII, E, and lipoprotein (a) levels in children. Clin Biochem 1996;29: 603-5. 2Soldin SJ, Rifai N, Hicks JM, eds. Biochemical basis of pediatric disease, 3rd ed. Washington, DC: AACC Press, 1998: 468. 3Soldin OP, Bierbower LH, Choi JJ, et al. Serum iron, ferritin, transferrin, total iron binding capacity, hs-CRP, LDL cholesterol and magnesium in children; new reference intervals using the Dade Dimension Clinical Chemistry System. Clin Chem Acta 2004;342: 211-7. Method (s) 1Calculated as described in Friedewald WT, Levy RI, Frederickson DC. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18:499-502. 2See references 3Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1Results are 2.5-97.5th percentiles for healthy children. 2*Numbers of participants not provided. Results are 5-95th percentiles. 3Study use hospitalized patients and a computerized approach adapted from the Hoffman technique. Values are the 2.5-97.5th percentiles.

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200 Glucose Male and Female Test Age n mg/dL mmol/L 1 0-1 mo 207 55-115 3.1-6.4 1-6 mo 96 57-117 3.2-6.5 2 Outside the 482* 70-126 3.9-7.0 neonatal period Male Female 3 Age ** mg/dL mmol/L mg/dL mmol/L 0-1 d 36-110 2.00-6.11 36-89 2.00-4.94 1-7 d 47-110 2.61-6.11 47-110 2.61-6.11 > 7 d 54-117 3.00-6.49 54-117 3.00-6.49

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201 Specimen Type (s) 1Whole blood 2Serum 3Plasma/serum Reference (s) 1Snell J, Greeley C, Colaco A, et al. Pediatric reference ranges for arterial pH whole blood electrolytes and glucose. Clin Chem 1993;39:1173. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003; 331: 135-46. Method (s) 1Glucose oxidase YSI 2300 (Yellow Springs Instruments, Yellow Springs, OH). 2Glucose oxidase, Vitros 700 (Ortho-Clinical Diagnostics, Raritan, NJ). 3The glucose method is an adaptation of the hexokinase-glucose-6-phosphate dehydrogenase method. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1Results are 2.5-97.5th percentiles. The 0-1 mo group includes an appreciable number of premature I nfants. 2Results are 2.5-97.5th percentiles. *No significant differences were found for males and females. These ranges were therefore derived from combined data. 3**Numbers not provided. Reference ranges were obtained by comparing results from previously published data and using regression equations.

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202 Alanine Aminotransferase (ALT) Male Female Test Age n U/L n U/L 1 1-7 d 109 6-40U 84 7-40U 8-30 d 168 10-40U 71 8-32U 1-3 mo 178 13-39U 173 12-47U 4-6 mo 135 12-42U 59 12-37U 7-12 mo 130 13-45U 107 12-41U 2 1-3 y 50* 5-45U 50* 5-45U 4-6y 40* 10-25U 40* 10-25U 7-9 y 80* 10-35U 80* 10-35U 10-11 y 27 10-35U 34 10-30U 12-13 y 31 10-55U 49 10-30U 14-15 y 26 10-45U 52 5-30U 16-19 y 40 10-40U 61 5-35U 3 1-30 d 50 1-25U 51 2-25U 31-365 d 91 4-35U 76 3-30U 1-3 y 119 5-30U 115 5-30U 4-6 y 114 5-20U 101 5-25U 7-9 y 102 5-25U 109 5-25U 10-18 y 280 5-30U 269 5-20U 4 1-7 d ** 20-54 ** 21-54 8-30 d 24-54 22-46 1-3 mo 27-54 26-61 4-6 mo 26-55 26-51 7-12 mo 26-59 26-55 1-3 y 19-59 24-59 4-6 y 24-49 24-49 10-11 y 24-49 24-44 12-13 y 24-68 24-44 14-15 y 24-59 19-44 16-19 y 24-54 19-49

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203Specimen Type (s) 1-4sPlasma/serum Reference (s) 1Soldin SJ, Savwoir TV, Guo Y. Pediatric reference ranges for alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase in children less than 1 year old on the Vitros 500. Clin Chem 1997;43:S199. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Soldin SJ, Bailey J, Bjorn S, et al. Pediatric reference ranges for ALT. Clin Chem 1995;41:S92-3. (Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method (s) 1,2Vitros 500 (1) and 700 (2) (Ortho-Clinical Diagnostics, Raritan, NJ). 3Measured on the Hitachi 747 using Boehringer Mannheim reagents (Boehringer Mannheim Diagnostics, Indianapolis, IN). 4Alanine aminotransferase (ALT) catalyzes the transamination of L-alanine to -ketoglutarate ( -KG), forming L-glutamate and pyruvate. The pyruvate formed is reduced to lactate by lactate dehydrogenase (LDH) with simultaneous oxidation of reduced nicotinamide-adenine dinucleotide (NADH). Dade BehringDimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1,3Study used hospitalized patients and a computerized approach adapted from the Hoffman technique to obtain the 2.5-97.5 the percentiles. 2The study population was healthy children. Non-parametric methods were used to determine the 0.025 and 0.975 fractiles. *No significant differences were found for males and females. These ranges were therefore derived from combined data. 4**Reference ranges were obtained by comparing results from previously published data and using regression equations.

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204 Aspartate Aminotransferase (AST) Male Female Test Age n U/L n U/L 1 1-7 d 69 30-100 52 24-95 8-30 d 148 20-70 84 24-72 1-3 mo 160 22-63 131 20-64 4-6 mo 133 13-65 83 20-63 7-12 mo 131 25-55 142 22-63 2 1-3 y 50* 20-60 50* 20-60 5-6 y 40* 15-50 40* 15-50 7-9 y 80* 15-40 80* 15-40 10-11 y 27 10-60U 34 10-40U 12-13 y 31 15-40 49 10-30U 14-15 y 26 15-40 52 10-30U 16-19 y 40 15-45 61 5-30U 3 1-30 d 74 <51 57 <49 31-365 d 83 <65 71 <79 1-3 y 134 <56 108 <69 4-6 y 85 <48 84 <59 7-9 y 122 <42 96 <41 10-12 y 104 <38 62 <37 13-15 y 88 <39 86 <32 16-18 y 62 <39 78 <30 4 1-7 d ** 26-98 ** 20-93 8-30 d 16-67 20-69 1-3 mo 16-60 16-61 4-6 mo 16-62 16-60 7-12 mo 16-52 16-60 1-3 y 16-57 16-57 5-6 y 10-47 U 10-47 U 7-9 y 10-36 U 5-36 U 12-15 y 10-36 U 5-26 U 16-19 y 10-41 U 0-26

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205Specimen Type (s) 1,3,4Plasma/serum 2Plasma Reference (s) 1Soldin SJ, Savwoir TV, Guo Y. Pediatric reference ranges for alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase in children less than 1 year old on the Vitros 500. Clin Chem 1997;43:S199. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Soldin SJ, Hicks JM, Bailey J, et al. Pediatric reference ranges for AST. Clin Chem 1995;44:S94. (Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method (s) 1,2Vitros 500 (1) and 700 (2) (Ortho-Clinical Diagnostics, Raritan, NJ). 3Measured on the Hitachi 747 using Boehringer Mannheim reagents (Boehringer Mannheim Diagnostics, Indianapolis, IN). 4Aspartate aminotransferase catalyzes the transamination from L-aspartate to -ketoglutarate, forming L-glutamate and oxaloacetate. The oxaloacetate formed is reduced to malate by malate dehydrogenase (MDH) with simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1,3Study used hospitalized patients and a computerized approach adapted from the Hoffman technique to obtain the 2.5-97.5 the percentiles (Test 1) and 97.5th percentile (Test 3). 2The study population was healthy children. Non-parametric methods were used to determine the 0.025 and 0.975 fractiles. *No significant differences were found for males and females. These ranges were therefore derived from combined data. 4**Reference ranges were obtained by comparing results from previously published data and using regression equations.

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206 Alkaline Phosphatase (ALP) Male Female Test Age n U/L n U/L 1 1-7 d 141 77-265 109 65-270 8-30 d 203 91-375 141 65-365 1-3 mo 251 60-360 234 80-425 4-6 mo 129 55-325 66 80-345 7-12 mo 113 60-300 58 60-330 2* 1-3 y 50** 129-291 50** 129-291 4-6y 40** 134-346 40** 134-346 7-9 y 80** 156-386 80** 156-386 10-11 y 27 120-488 3 116-515 12-13 y 31 178-455 49 93-386 14-15 y 26 116-483 52 62-209 16-19 y 40 58-237 61 45-116 3 1-30 d 60 75-316 75 48-406 31-365 d 132 82-383 122 124-341 1-3 y 136 104-345 111 108-317 4-6 y 113 93-309 113 96-297 7-9 y 124 86-315 104 69-325 10-12 y 111 42-362 109 51-332 13-15 y 126 74-390 105 50-162 16-18 y 112 52-171 110 47-119 4 1-7 d *** 121-351 *** 107-357 8-30 d 138-486 107-474 1-3 mo 101-467 125-547 4-6 mo 94-425 125-449 7-12 mo 101-394 101-431 1-3 y 185-383 185-383 4-6 y 191-450 191-450 7-9 y 218-499 218-499 10-11 y 174-624 169-657 12-13 y 245-584 141-499 14-15 y 169-618 103-283 16-19 y 98-317 82-169

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207Specimen Type (s) 1,3,4Plasma/serum 2Serum Reference (s) 1Soldin SJ, Savwoir TV, Guo Y. Pediatric reference ranges for alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase in children less than 1 year old on the Vitros 500. Clin Chem 1997;43:S199. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Soldin SJ, Hicks JM, Bailey J, et al. Pediatric reference ranges for alkaline phosphatase on the Hitachi 747 analyzer. Clin Chem 1997;43:S198. (Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method (s) 1,2Vitros 500 (1) and 700 (2) (Ortho-Clinical Diagnostics, Raritan, NJ). p-Nitrophenylphosphate. 3Hitachi 747 using Boehringer Mannheim reagents (p-Nitrophenylphosphate). (Boehringer Mannheim Diagnostics, Indianapolis, IN). 4Alkaline phosphatase catalyzes the transphosphorylation of pnitrophenyphosphate (p-NPP) to p-nitrophenol (p-NP). Followed spectrophotometrically. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1,3Study used hospitalized patients and a computerized approach adapted from the Hoffman technique to obtain the 2.5-97.5 the percentiles. 2The study population was healthy children. Non-parametric methods were used to determine the 0.025 and 0.975 fractiles. *Due to changes made by the manufacturer in slide performance, these results are lower than those published by the author. **No significant differences were found for males and females. These ranges were therefore derived from combined data. 4***Reference ranges were obtained by comparing results from previously published data and using regression equations.

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208 Amylase Male and Female Test Age n U/L 1 1-30 d 76 0-6 31-182 d 110 1-17U 183-365 d 54 6-44U 1-3 y 148 8-79U 4-9 y 96 16-91 10-18 y 142 19-76 2 0-0.2 y 55 <30 0.2-0.5 y 81 <50 0.5-1.0 y 170 <80 3 1-19 y 470 30-100 4 1-30 d * <18 31-182 d <43 183-365 d <81 1-3 y <106 4-9 y <106 10-18 y <106

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209Specimen Type(s) 1,2Plasma 3Serum 4Plasma/serum Reference(s) 1Soldin SJ, Hicks JM, Bailey J, et al. Pediatric reference ranges for amylase. Clin Chem 1995;41:S94. (Abstract) 2Soldin SJ, Rakotoarisoa FTS. Pediatric reference ranges for amylase and cholesterol on Kodak Ektachem 500 in the first year of life. Clin Chem 1996; 42: S308. (Abstract) 3Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 4Ghoshal AK, Soldin SJ. Evaluati on of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method(s) 1Hitachi 717 using Boehringer Mannheim reagents (Boehringer Mannheim Diagnostics, Indianapolis, IN). 2,3Amylopectin. Vitros 500 and 700 (Ortho-Clinical Diagnostics, Raritan, NJ). 4The amylase method on the Dimension® system uses a chromogenic substrate, 2-chloro-4-nitrophenol linked with maltotriose. The direct reaction of -amylase with the substrate results in the formation of 2-chloro-4 nitrophenol, which is monitored spec trophotometrically. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment(s) 1Study used hospitalized patients and a computerized approach adapted from the Hoffman technique to obtain the 2.5-97.5 the percentiles. 2Study used hospitalized patients and a computerized approach adapted from the Hoffman technique. Values are 97.5th percentiles. 3Healthy children. Non-parametric methods were used to determine the 0.025 and 0.975 fractiles. 4*Reference ranges were obtained by comparing results from previously published data and using regression equations.

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210 Lipase Male and Female Test Age n U/L 1 1-30 d 83 6-55U 31-182 d 121 4-29U 183-365 d 56 4-23U 1-3 y 148 4-31U 4-9 y 96 3-32U 10-18 y 142 4-29U Male Female Age n U/L n U/L 2 0-90 d 39* 10-85U 39* 10-85U 3-12 mo 113 13-95U 137 9-128U 1-<2 y 118 15-135U 222 15-150U 2-<7 y 323 15-175U 243 10-150U 7-<11 y 216 10-175U 209 13-150U 11-<15 y 75 10-195U 150 10-180U 15-18 y 70 10-195U 79 10-220U 3 0-90 d ** 145-174 ** 145-174 3-12 mo 146-178 144-190 1-<2 y 147-193 147-199 2-<7 y 147-209 145-199 7-<11 y 145-209 146-199 11-<15 y 145-216 145-211 15-18 y 145-216 145-226

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211Specimen Type(s) 1-3sPlasma/serum Reference(s) 1Soldin SJ, Hicks JM, Bailey J, et al. Pediatric reference ranges for lipase. Clin Chem 1995;41:S93. (Abstract) 2Soldin SJ, Ojeifo O. Pediatric reference ranges for lipase. Clin Chem 1999;45: A22. (Abstract) 3Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method(s) 1Hitachi 717 using Sigma reagents (Boehringer Mannheim Diagnostics, Indianapolis, IN). 2Vitros 500 using Vitros reagents (Ort ho-Clinical Diagnostics, Raritan, NJ). 3The LIP method technology uses a glycerol derivative esterified in the 1position with a dicarbonic acid-resorufin ester as the substrate. Lipase activity is maximized in the presence of coli pase, calclium chloride, and bile salt. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment(s) 1,2Study used hospitalized patients and a computerized approach adapted from the Hoffman technique. Values are 97.5th percentiles. *Male and female data are combined. 3**Numbers not provided. Reference ranges were obtained by comparing results from previously published data and using regression equations.

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212 Lactate Male and Female Test Age n mg/dL mmol/L 1 1-12 mo 12 10-21mg 1.1-2.3 1-7 y 27 7-14mg 0.8-1.5 7-15 y 9 5-8mg 0.6-0.9 2 0-90 d 85 30 3.3 3-24 mo 150 28 3.1 2-18 y 185 20 2.2 3 0-90 d 9-32mg 1.0-3.5 3-24 mo 9-30mg 1.0-3.3 2-18 y 9-22mg 1.0-2.4

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213Specimen Type(s) 1Whole blood precipitated with perchloric acid. 2Plasma 3Plasma/serum Reference(s) 1Bonnefont JP, Specola NB, Vassault A, et al. The fasting test in pediatrics: application to pathological hypoand hyperketotic states. Eur J Pediatr 1990; 150:80-5. 2Soldin SJ, Baumel CR. Pediatric refe rence ranges for CSF protein and plasma lactate on the Vitros 500 analyzer. Clin Chem 2001;47:A108. 3Ghoshal AK, Soldin SJ. Evaluati on of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method(s) 1Standard enzymatic procedure. See reference. 2Vitros 500 analyzer using Ortho-Clinical Diagnostics reagents (Ortho-Clinical Diagnostics, Raritan, NJ). 3The lactic acid method employs the oxidation of lactate to pyruvate. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment(s) 1The results are 10-90th percentiles and refer to 15-h fasting values. For 20and 24-h fasting values, see reference. 2Study used hospitalized patients and a computerized approach adapted from the Hoffman technique. Values are 97.5th percentiles. 3*Numbers not provided. Reference ranges were obtained by comparing results from previously published data and using regression equations.

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214 Urea Nitrogen (BUN) Male Female Test Age n mg/dL mmol/L n mg/dL mmol/L 1 1-7 d 171 2-13mg 0.7-4.6 114 2-13mg 0.7-4.6 8-30 d 209 2-16mg 0.7-5.7 154 2-15mg 0.7-5.4 1-3 mo 275 2-12mg 0.7-4.3 274 2-14mg 0.7-5.0 4-6 mo 144 1-14mg 0.4-5.0 139 1-13mg 0.4-4.6 7-12 mo 204 2-14mg 0.7-5.0 160 1-13mg 0.4-4.6 2 1-30 d 51 4-12mg 1.4-4.3 43 3-17mg 1.1-6.1 1-12 mo 69 2-13mg 0.7-4.6 60 4-14mg 1.4-5.0 1-3 y 104 3-12mg 1.1-4.3 127 3-14mg 1.1-5.0 4-6 y 140 3-16mg 1.1-5.7 122 4-14mg 1.4-5.0 7-9 y 124 4-16mg 1.4-5.7 121 4-16mg 1.4-5.7 10-12 y 133 5-18mg 1.8-6.4 125 5-16mg 1.8-5.7 13-15 y 141 7-18mg 2.5-6.4 153 4-15mg 1.4-5.4 16-18 y 111 5-20mg 1.8-7.1 120 4-15mg 1.4-5.4 Male and Female Age n mg/dL mmol/L 3 1-3y 50 5-17mg 1.8-6.0 4-6y 38 7-17mg 2.5-6.0 7-9y 72 7-17mg 2.5-6.0 10-11y 62 7-17mg 2.5-6.0 12-13y 73 7-17mg 2.5-6.0 14-15y 91 8-21mg 2.9-7.5 16-19y 107 8-21mg 2.9-7.5 4 1-7 d * 1-13mg 0.3-3.5 8-30 d 1-16mg 0.3-4.3 1-3 mo 1-12mg 0.3-3.2 4-12 mo 1-14mg 0.3-3.8 1-3 y 4-17mg 1.1-4.6 4-13 y 6-17mg 1.6-4.6 14-19 y 7-21mg 1.9-5.7

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215Specimen Type(s) 1,2,4Plasma/serum 3Plasma Reference(s) 1Soldin SJ, Savwoir TV, Guo Y. Pediatric reference ranges for alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase in children less than 1 year old on the Vitros 500. Clin Chem 1997;43:S199. (Abstract) 2Soldin SJ, Bailey J, et al. Pediatric reference ranges for blood urea nitrogen (BUN) on the Hitachi 747 analyzer. Clin Chem 1996;42:S307. 3Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method(s) 1,3Urease method, Vitros 500 & 700 (Ortho-Clinical Diagnostics, Raritan, NJ) 2Hitachi 747 with Boehringer Mannheim reagents (Boehringer Mannheim Diagnostics, Indianapolis, IN). 4The urea nitrogen method employs a urease/glutamate dehydrogenase coupled enzymatic technique. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment(s) 1,2Study used hospitalized patients and a computerized approach adapted from the Hoffman technique to obtain the 2.5-97.5 the percentiles. 3Study used normal healthy children. Values 2.5-97.5th percentiles. 4*Numbers not provided. Reference ranges were obtained by comparing results from previously published data and using regression equations.

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216 Creatinine Male and Female Test Age n mg/dL µmol/L 1 0-1 wk * 0.6-1.1 53-97 1 wk-1 mo 0.3-0.7 27-62 1-6 mo 0.2-0.4 18-35 7-12 mo 0.2-0.4 18-35 1-18 y 0.2-0.7 18-62 2 Age n mg/dL mmol/L 0-1 wk 0.7-1.2 0.06-0.11 1 mo-1 y 0.3-0.8 0.03-0.07 1-9 y 0.2-0.5 0.02-0.04 10-18 y 0.2-0.8 0.02-0.07 0.5-1.1 0.04-0.10 3 Male Female Age n mg/dL µmol/L n mg/dL µmol/L 1-30 d 42 0.5-1.2 44-106 40 0.5-0.9 44-80 31-365 d 62 0.4-0.7 35-62 59 0.4-0.6 35-53 1-3 y 103 0.4-0.7 35-62 126 0.4-0.7 35-62 4-6 y 129 0.5-0.8 44-71 116 0.5-0.8 44-71 7-9 y 121 0.6-0.9 53-80 110 0.5-0.9 44-80 10-12 y 125 0.6-1.0 53-88 117 0.6-1.0 53-88 13-15 y 135 0.6-1.2 53-106 141 0.7-1.1 62-97 16-18 y 106 0.8-1.4 71-123 114 0.8-1.2 71-106

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217Specimen Type (s) 1,3Plasma 2Plasma/serum Reference (s) 1Greeley C, Snell J, Colaco A, et al. Pediatric reference ranges for electrolytes and creatinine. Clin Chem 1993;39:1172. 2Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;31:135-46. 3Soldin SJ, Hicks JM, Bailey J, et al. Pediatric reference ranges for creatinine on the Hitachi 747 analyzer. Clin Chem 1997;43:S198. (Abstract) Method (s) 1Vitros (Ortho-Clinical Diagnostics, Raritan, NJ). 2The creatinine method employs a modification of the kinetic Jaffe reaction reported by Larsen. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). 3Jaffe method on Hitachi 747 Boehringer Mannheim reagents (Boehringer Mannheim Diagnostics, Indianapolis, IN). Comment (s) 1Study used hospitalized patients and a a computerized approach to removing outliers. *n 100. Values are 2.5-97.5th percentiles. 2**Reference ranges were obtained by comparing results from previously published data and using regression equations. 3Study used hospitalized patients and a computerized approach to removing outliers. Values reported are 2.5-97.5th percentiles.

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218 Creatinine (Urine) Male and Female Test Age n g/24h 1 3-8 y 71 0.11-0.68 9-12 y 45 0.17-1.41 13-17 y 42 0.29-1.87 Adult 104 0.63-2.50 Specimen Type (s) Urine Reference (s) Pediatric endocrine testing. Nichols Institute, 1993:35. Method (s) Kinetic, alkaline picrate Comment (s) Results are 2.5-97.5th percentiles.

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219 Potassium Male and Female Test Age n mmol/L 1 0-1 wk 100 3.2-5.5 1 wk-1 mo 100 3.4-6.0 1-6 mo 100 3.5-5.6 6 mo-1 y 100 3.5-6.1 Over 1 y 105 3.3-4.6 2 1-15 y * 3.7-5.0 16 yAdult 3.7-4.8 3 0-1 mo 207 2.5-5.4 1-6 mo 96 2.7-5.2 4 0-1 wk ** 3.2-5.7 1 wk-1 mo 3.4-6.2 1-6 mo 3.5-5.8 6 mo-1y 3.5-6.3 >1 y 3.3-4.7

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220Specimen Type (s) 1,2Plasma 3Whole blood 4Plasma/serum Reference (s) 1Greeley C, Snell J, Colaco A, et al. Pediatric reference ranges for electrolytes and creatinine. Clin Chem 1993;39:1172. (Abstract) 2Burritt MF, Slockbower JM, Forsman BS, et al. Pediatric reference intervals for 19 biologic variables in healthy children. Mayo Clinic Proceedings 1990;65:329-36. 3Greeley C, Snell J, Colaco A, et al. Pediatric reference ranges for arterial pH whole blood electrolytes, and glucose. Clin Chem 1993;39:1173.(Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;31:135-46. Method (s) 1Vitros 500 (Ortho-Clinical Diagnostics, Raritan, NJ). 2Flame Photometry-American Monitor (American Diagnostics, Inc., Indianapolis, IN). 3288 Blood Gas System (Ciba Corning Diagnostics, East Walpole, MA). 4The sodium, potassium, and chloride (Na/K/Cl) methods use indirect sample sensing with the QuikLYTE® Integrated Multisensor Technology (IMT) to develop an electrical potential proportional to the activity of each specific ion in the sample. The total carbon dioxide (TCO2) method uses a Severinghaus electrode designed to measure the liberated CO2 from an acidified sample. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1Study used hospitalized patients and a computerized approach to removing outliers. Values reported are 2.5-97.5th percentiles. 2Normal healthy children. *See reference for numbers 3Study used hospitalized patients and a computerized approach to removing outliers. Values reported are 2.5-97.5th percentiles. 4**Numbers not provided. Reference ranges were obtained by comparing results from previously published data and using regression equations. Values reported are 2.5-97.5th percentiles.

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221 Phosphorus Male Female Test Age n mg/dL mmol/L n mg/dL mmol/L 1 1-30 d 62 3.9-6.9 1.25-2. 25 66 4.3-7.7 1.40-2.50 31-365 d 83 3.5-6.6 1.15-2. 15 66 3.7-6.5 1.20-2.10 1-3 y 126 3.1-6.0 1.00-1.95 119 3.4-6.0 1.10-1.95 4-6 y 112 3.3-5.6 1.05-1.80 107 3.2-5.5 1.05-1.80 7-9 y 117 3.0-5.4 0.95-1.75 107 3.1-5.5 1.00-1.80 10-12 y 135 3.2-5.7 1.05-1.85 115 3.3-5.3 1.05-1.70 13-15 y 109 2.9-5.1 0.95-1.65 110 2.8-4.8 0.90-1.55 16-18 y 95 2.7-4.9 0.85-1.60 122 2.5-4.8 0.80-1.55 2 0-5 d 50 4.6-8.0 1.50-2. 60 50 4.6-8.0 1.50-2.60 (<2.5 kg) 1-3 y 50 3.9-6.5 1.25-2.10 50 3.9-6.5 1.25-2.10 4-6 y 38 4.0-5.4 1.30-1.75 38 4.0-5.4 1.30-1.75 7-9 y 72 3.7-5.6 1.20-1.80 72 3.7-5.6 1.20-1.80 10-11 y 62 3.7-5.6 1.20-1.80 62 3.7-5.6 1.20-1.80 12-13 y 73 3.3-5.4 1.05-1.75 73 3.3-5.4 1.05-1.75 14-15 y 91 2.9-5.4 0.95-1.75 91 2.9-5.4 0.95-1.75 16-19 y 107 2.8-4.6 0.90-1.50 107 2.8-4.6 0.90-1.50 3 0-30 d 181 2.7-7.2 0.87-2. 33 140 3.0-8.0 0.97-2.58 31-90 d 84 3.0-6.8 0.97-2. 20 87 3.0-7.5 0.97-2.42 3-12 mo 109 3.0-6.9 0.97-2. 23 119 2.5-7.0 0.81-2.26 13-24 mo 69 2.5-6.4 0.81-2. 07 78 3.0-6.5 0.97-2.10 2-<13 y 148 3.0-6.0 0.97-1. 94 254 2.5-6.0 0.81-1.94 13-<16 y 175 3.0-5.4 0.97-1. 74 72 3.0-5.6 0.97-1.81 16-<18 y 72 3.0-5.2 0.97-1. 68 196 3.0-4.8 0.97-1.55 4 0-30 d * 2.8-7.0 0.90-2. 26 3.1-7.7 1.00-2.49 31-90 d 3.1-6.6 1.00-2.13 3.1-7.2 1.00-2.32 3-12 mo 3.1-6.6 1.00-2. 13 3.1-6.8 1.00-2.20 13-24 mo 3.1-6.2 1.00-2.00 3.1-6.3 1.00-2.03 2-<13 y 3.1-5.9 1.00-1. 90 3.1-5.9 1.00-1.90 13-<16 y 3.1-5.3 1.00-1. 71 3.1-5.5 1.00-1.78 16-18 y 3.1-5.1 1.00-1.65 3.1-4.8 1.00-1.55

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222Specimen Type (s) 1-4s Plasma/serum Reference (s) 1Soldin SJ, Hicks JM, Bailey J, et al. Pediatric reference ranges for creatinine on the Hitachi 747 analyzer. Clin Chem 1997;43:S198. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Soldin SJ, Hunt C, Hicks JM. Pediatric reference ranges for Phosphorus on the Vitros 500; Analyzer. Clin Chem 1999;45:A22. (Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;31:135-46. Method (s) 1Hitachi 747 using ammonium molybdate method (Boehringer Mannheim, Diagnostics, Indianapolis, IN). 2,3Vitros 700 (2) and 500 (3) using ammonium molybdate method (Ortho-Clinical Diagnostics, Raritan, NJ). 4The phosphorus method uses a mixture of p-methylaminophenol sulfate and bisulfite to reduce the phosphomolybdate. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1,3Study used hospitalized patients and a computerized approach to removing outliers. Values reported are 2.5-97.5th percentiles. 2Study used normal healthy children. Values are 2.5-97.5th percentiles. 4*Numbers not provided. Reference ranges were obtained by comparing results from previously published data and using regression equations.

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223 Carbon dioxide (Total) Male and Female Test Age mmol/L 1 0-1 wk 17-26 1 wk-1 mo 17-27 1-6 mo 17-29 6 mo-1 y 18-29 1 y 20-31 2 Infants 13-29 Adults 24-30 3 0-1 wk 13-21 1 wk-1 mo 13-22 1-6 mo 13-23 6 mo-1 y 14-23 >1 y 16-25

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224Specimen Type (s) 1Plasma 2Cord blood 3Plasma/serum Reference (s) 1Greeley C, Snell J, Colaco A, et al. Pediatric reference ranges for electrolytes and creatinine. Clin Chem 1993;39:1172. (Abstract) 2Chemistry analytes in arterial and venous umbilical cord blood. Clin Chem 1993;39: 1041-4. 3Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;31:135-46. Method (s) 1Vitros 700 (Ortho-Clinical Diagnostics, Raritan, NJ). 2Hitachi 737, Boehringer Mannheim reagents (Boehringer Mannheim, Montreal, Canada). 3The total carbon dioxide method uses a Severinghaus electrode designed to measure liberated CO2 from an acidified sample. Dade Behring Dimenson RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1Study used hospitalized patients and a a computerized approach to removing outliers. N 100. Values are 2.5-97.5th percentiles. 2Study used 397 infants (209 girls and 188 boys delivered between 37 and 41 weeks gestation). Results are 2.5-97.5th percentiles. 3Reference ranges were obtained by comparing results from previously published data and using regression equations.

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225 Urine Volume Male and Female Test Age mL L 1 Newborn 50-300 0.05-0.30 Infant 350-550 0.35-0.55 Child 500-1000 0.50-1.00 Adolescent 700-1400 0.70-1.40 Thereafter 600-1800 0.60-1.80 Specimen Type (s) Urine (24 h) Reference (s) Behrman RE, ed. Nelson textbook of pediatrics, 14th ed. Philadelphia, PA: WB Saunders Company, 1992:1824. Method (s) Not given. Comment (s) Varies with intake and other factors.

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226 Hemoglobin (Hgb) Male Female Test Age n g/dL g/L n g/dL g/L 1 1-3 d 217 12.2-17.9 122-179 144 13.2-18.4 132-184 4-7 d 209 12.8-16.9 128-169 132 11.8-17.7 118-177 8-14 d 224 11.8-16.8 118-168 151 11.5-16.3 115-163 15-30 d 447 10.6-15.4 106-154 339 10.9-15.3 109-153 31-60 d 82 9.0-12.1 90-121 197 9.2-14.4 92-144 61-180 d 98 10.0-13.2 100-132 123 9.8-13.7 98-137 0.5-<2 y 199 9.8-13.4 98-134 143 9.6-13.1 96-131 2-<6 y 200 9.6a-12.8 96a-128 144 10.9a-13.4 109a-134 6-<12 y 135 10.7a-13.5 107a-135 143 10.9a-13.7 109a-137 12-18 y 147 9.5a-14.8 95a-148 174 9.5a-13.3 95a-133 2 1-3 d 437 12.5-16.6 125-166 362 12.7-16.4 127-164 4-7 d 276 12.5-16.3 125-163 333 12.6-15.3 126-153 8-14 d 304 11.9-15.7 119-157 356 12.7-14.9 127-149 15-30 d 344 11.6-14.2 116-142 417 11.6-14.3 116-143 31-60 d 486 10.2-12.7 102-127 407 11.1-13.7 111-137 61-180 d 1197 10.5-13.0 105-130 111310.7-13.4 107-134 0.5<2 y 2916 10.4-12.5 104-125 237710.8-12.6 108-126 2-<6 y 4123 11.0-12.8 110-128 371411.1-12.9 111-129 6-<12 y 4183 11.0-13.3 110-133 336611.3-13.4 113-134 12-<18 y 5095 11.0-14.3 110-143 534911.3-13.4 113-134 18 y 2526 11.4-15.1 114-151 387410.9-13.4 109-134 3 1-14 d 13.9-19.1 139-191 13.4-20.0 134-200 15-30 d 10.0-15.3 100-153 10.8-14.6 108-146 31-60 d 8.9-12.7 89-127 9.2-11.4 92-114 61-180 d 9.6-12.4 96-124 9.9-12.4 99-124 0.5-<2 y 10.1-12.5 101-125 10.2-12.7 102-127 2-<6 y 10.2-12.7 102-127 10.2-12.7 102-127 6-<12 y 10.7-13.4 107-134 10.6-13.2 106-132 12-<18 y 11.0-14.5 110-145 10.8-13.3 108-133 18 y 11.9-15.4 119-154 10.6-13.5 106-135

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227 Specimen Type (s) 1,3 Whole blood (K3/EDTA anticoagulant) 2 Whole blood (K3/EDTA anticoagulant) Reference (s) 1Soldin SJ, Brugnara C, Wond ED, eds. Pediatri reference ranges, 4th ed. Washington, DC: AACC Press, 2003 2 Brugnara C. Unpublished data 3 Children's National Medical Center, Washington DC. Unpublished Data Method (s) 1 Coulter STKS (Beckman Coulter Inc, Brea, CA). 2 Bayer ADVIA 120 (Bayer Diagnostics, Tarrytown, NY). 3 Sysmex XE-2100 (Sysmex Corporation, Kobe, Japan). Comment (s) 1Data from hospitalized patients (excluding hematology/oncology and intensive care unit patients). A computerized approach adapted from the Hoffmann technique was used to remove outliers and obtain the w.5th and 97.5th percentiles.aTo obtain the 2.5th percentile, the statistical technique was modified to use the central 60% of the data for the Hoffmann plot. 2Data from inpatients, excludi ng hematology/oncolgy patients. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion (1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffmann--the top and bottom 25% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 50% of the data. 1. Young HD. Statistical treatment of experimental data. New York:McGraw-Hill, 1962:76-9, 162. 2. Hoffman RG. Statistics in the practice of medicine. JAMA 1963;185:864-73. 3Performed on outpatients and ER pat ients. Hematology/oncology clinic patients were excluded. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion(1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffman-the top and bottom 10% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 80% of the data.

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228 Hematocrit (Hct) Male Female Test Age n % Volume Fraction n % Volume Fraction 1 1-3 d 216 33.5-52.7 .335-.527 143 38.4-56.0 .384-.560 4-7 d 211 36.9-50.1 .369-.501 132 34.4-50.8 .344-.508 8-14 d 226 33.4-49.0 .334-.490 151 32.9-47.5 .329-.475 15-30 d 445 30.7-44.9 .307-.449 338 31.9-45.2 .319-.452 31-60 d 81 27.0-35.8 .270-.358 197 27.1-41.9 .271-.419 61-180 d 86 29.9-39.0 .299-.390 109 28.6-42.3 .286-.423 0.5-<2 y 198 28.3-40.0 .283-.400 139 28.1-39.1 .281-.391 2-<6 y 204 28.5a-37.9 .285a-.379 147 32.0a-39.8 .320a-.398 6-<12 y 141 31.6a-39.5 .316a-.395 148 32.0a-40.0 .320a-.400 12-18 y 155 27.3a-43.6 .273a-.436 172 27.9a-39.6 .279a-.396 2 1-3 d 438 36.4-47.4 .364-.474 362 36.5-47.7 .365-.477 4-7 d 276 35.9-46.6 .359-.466 333 36.1-44.0 .361-.440 8-14 d 304 34.4-45.4 .344-.454 356 36.6-43.2 .366-.432 15-30 d 346 33.6-41.0 .336-.410 418 34.1-41.8 .341-.418 31-60 d 485 29.1-36.6 .291-.366 409 32.0-39.9 .320-.399 61-180 d 119530.5-37.7 .305-.377 1113 30.5-38.6 .305-.386 0.5<2 y 290030.5-36.4 .305-.364 2381 30.9-36.4 .309-.364 2-<6 y 412431.5-36.8 .315-.368 3712 31.8-37.0 .318-.370 6-<12 y 418131.5-38.0 .315-.380 3366 32.3-38.3 .323-.383 12-<18 y 509431.4-41.0 .314-.410 5351 32.1-38.7 .321-.387 18 y 252733.0-43.4 .330-.434 3869 31.7-39.1 .317-.391 3 0-14 d 39.8-53.6 .398-.536 39.6-57.2 .396-.572 15-30 d 30.5-45.0 .305-.450 32.0-44.5 .320-.445 31-60 d 26.8-37.5 .268-.375 27.7-35.1 .277-.351 61-180 d 28.6-37.2 .286-.372 29.5-37.1 .295-.371 0.5-<2 y 30.8-37.8 .308-.378 30.9-37.9 .309-.379 2-<6 y 31.0-37.7 .310-.377 31.2-37.8 .312-.378 6-<12 y 32.2-39.8 .322-.398 32.4-39.5 .324-.395 12-18 y 33.9-43.5 .339-.435 33.4-40.4 .334-.404 > 18 y 36.2-46.3 .362-.463 32.9-41.2 .329-.412

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229Specimen Type (s) 1,3 Whole blood (K3/EDTA anticoagulant) 2 Whole blood (K3/EDTA anticoagulant) Reference (s) 1Soldin SJ, Brugnara C, Wond ED, eds. Pediatri reference ranges, 4th ed. Washington, DC: AACC Press, 2003 2 Brugnara C. Unpublished data 3 Children's National Medical Center, Washington DC. Unpublished Data Method (s) 1 Coulter STKS (Beckman Coulter Inc, Brea, CA). 2 Bayer ADVIA 120 (Bayer Diagnostics, Tarrytown, NY). 3 Sysmex XE-2100 (Sysmex Corporation, Kobe, Japan). Comment (s) 1Data from hospitalized patients (excluding hematology/oncology and intensive care unit patients). A computerized approach adapted from the Hoffmann technique was used to remove outliers and obtain the w.5th and 97.5th percentiles.aTo obtain the 2.5th percentile, the statistical technique was modified to use the central 60% of the data for the Hoffmann plot. 2Data from inpatients, excluding hematology/oncolgy patients. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion (1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffmann--the top and bottom 25% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 50% of the data. 1. Young HD. Statistical treatment of experimental data. New York:McGraw-Hill, 1962:76-9, 162. 2. Hoffman RG. Statistics in the practice of medicine. JAMA 1963;185:864-73. 3Performed on outpatients and ER patients. Hematology/oncology clinic patients were excluded. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion(1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffman-the top and bottom 10% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 80% of the data.

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230 Mean Corpuscular Volume (MCV) Test n Male n Female Age µm3 fL µm3 fL 1 1-3 d 174 93.0-113.4 93.0-1 13.4 117 92.4-115.4 92.4-115.4 4-7 d 155 88.1-106.5 88.1-106. 5 108 90.6-108.3 90.6-108.3 8-14 d 182 85.1-104.5 85.1-104. 5 124 87.9-101.8 87.9-101.8 15-30 d 393 83.4-97.6 83.4-97.6 291 84.5-99.7 84.5-99.7 31-60 d 74 82.0-89.8 82.0-89.8 185 85.5-95.3 85.5-95.3 61-180 d 75 76.5-87.8 76.5-87.8 102 77.5-89.4 77.5-89.4 0.5-<2 y 177 72.3-88.7 72.3-88.7 119 70.6-87.9 70.6-87.9 2-<6 y 190 73.6-90.0 73.6-90.0 135 75.6-88.0 75.6-88.0 6-<12 y 128 77.3-88.3 77.3-88.3 138 80.7-95.6 80.7-95.6 12-18 y 140 76.3-90.1 76.3-90.1 154 79.1-91.7 79.1-91.7 2 1-3 d 439 94.0-106.3 94.0-1 06.3 366 89.7-105.4 89.7-105.4 4-7 d 276 87.1-96.5 87.1-96.5 334 86.5-93.8 86.5-93.8 8-14 d 304 87.1-94.8 87.1-94.8 350 87.4-92.2 87.4-92.2 15-30 d 348 88.0-95.2 88.0-95.2 415 88.4-93.3 88.4-93.3 31-60 d 483 86.5-92.1 86.5-92.1 410 85.7-91.6 85.7-91.6 61-180 d 119279.6-86.3 79.6-86.3 110382.0-87.0 82.0-87.0 0.5-<2 y 298975.6-83.1 75.6-83.1 236676.6-83.2 76.6-83.2 2-<6 y 412676.8-83.3 76.8-83.3 369877.7-84.1 77.7-84.1 6-<12 y 409778.2-83.9 78.2-83.9 331779.5-85.2 79.5-85.2 12-<18 y 504680.8-86.6 80.8-86.6 525782.1-87.7 82.1-87.7 18 y 247783.5-90.2 83.5-90.2 381582.7-89.4 82.7-89.4 3 0-14 d 91.3-103.1 91.3-1 03.1 92.7-106.4 92.7-106.4 15-30 d 89.4-99.7 89.4-99.7 90.1-103.0 90.1-103.0 31-60 d 84.3-94.2 84.3-94.2 83.4-96.4 83.4-96.4 61-180 d 74.1-87.5 74.1-87.5 74.8-88.3 74.8-88.3 0.5-<2 y 69.5-81.7 69.5-81.7 71.3-82.6 71.3-82.6 2-<6 y 71.3-84.0 71.3-84.0 72.3-85.0 72.3-85.0 6-<12 y 74.4-86.1 74.4-86.1 75.9-87.6 75.9-87.6 12-18 y 76.7-89.2 76.7-89.2 76.9-90.6 76.9-90.6 > 18 y 80.0-93.6 80.0-93.6 77.7-93.7 77.7-93.7

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231Specimen Type (s) 1,3 Whole blood (K3/EDTA anticoagulant) 2 Whole blood (K3/EDTA anticoagulant) Reference (s) 1Soldin SJ, Brugnara C, Wond ED, eds. Pediatri reference ranges, 4th ed. Washington, DC: AACC Press, 2003 2 Brugnara C. Unpublished data 3 Children's National Medical Center, Washington DC. Unpublished Data Method (s) 1 Coulter STKS (Beckman Coulter Inc, Brea, CA). 2 Bayer ADVIA 120 (Bayer Diagnostics, Tarrytown, NY). 3 Sysmex XE-2100 (Sysmex Corporation, Kobe, Japan). Comment (s) 1Data from hospitalized patients (excluding hematology/oncology and intensive care unit patients). A computerized approach adapted from the Hoffmann technique was used to remove outliers and obtain the w.5th and 97.5th percentiles. 2Data from inpatients, excluding hematology/oncolgy patients. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion (1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffmann--the top and bottom 25% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 50% of the data. 1. Young HD. Statistical treatment of experimental data. New York:McGraw-Hill, 1962:76-9, 162. 2. Hoffman RG. Statistics in the practice of medicine. JAMA 1963;185:864-73. 3Performed on outpatients and ER patients. Hematology/oncology clinic patients were excluded. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion(1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffman-the top and bottom 10% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 80% of the data.

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232 Red Blood Cell Male Female Count x 106/ µ L x 1012/L x 106/ µ L Test Age n n x 1012/L 1 1-3 d 174 3.3-5.0 3.35.0 115 3.3-5.3 3.3-5.3 4-7 d 175 3.7-5.3 3.75.3 108 3.1-5.2 3.1-5.2 8-14 d 182 3.5-5.2 3.55.2 124 3.6-4.9 3.6-4.9 15-30 d 397 3.3-4.9 3.34.9 292 3.6-4.9 3.6-4.9 31-60 d 74 3.1-4.1 3.14.1 185 3.1-4.7 3.1-4.7 61-180 d 75 3.6-5.2 3.65.2 102 3.3-5.0 3.3-5.0 0.5-<2 y 177 3.4-5.0 3. 4-5.0 119 3.6-5.3 3.6-5.3 2-<6 y 190 3.3a-4.8 3.3a-4.8 135 3.8a-4.9 3.8a-4.9 6-<12 y 128 3.7a-4.9 3.7a-4.9 138 3.6a-4.9 3.6a-4.9 12-18 y 140 3.3a-5.4 3.3a-5.4 154 3.4a-4.7 3.4a-4.7 2 1-3 d 439 3.69-4.75 3.69-4. 75 360 3.79-4.76 3.79-4.76 4-7 d 276 3.98-5.08 3.98-5. 08 335 4.05-4.83 4.05-4.83 8-14 d 304 3.75-4.93 3.75-4. 93 355 4.01-4.73 4.01-4.73 15-30 d 347 3.61-4.46 3.61-4. 46 416 3.70-4.59 3.70-4.59 31-60 d 484 3.24-4.08 3.24-4. 08 409 3.55-4.57 3.55-4.57 61-180 d 1195 3.67-4.61 3.67-4. 61 11093.63-4.61 3.63-4.61 0.5-<2 y 2930 3.81-4.74 3.814.74 23983.83-4.67 3.83-4.67 2-<6 y 4140 3.92-4.72 3.92-4.72 37293.89-4.67 3.89-4.67 6-<12 y 4183 3.85-4.75 3.85-4. 75 33713.88-4.72 3.88-4.72 12-<18 y 5092 3.74-4.93 3.74-4. 93 53493.79-4.61 3.79-4.61 18 y 2526 3.75-5.07 3.75-5.07 38493.75-4.54 3.75-4.54 3 0-14 d 59 4.10-5.55 4.10-5. 55 47 4.12-5.74 4.12-5.74 15-30 d 53 3.16-4.63 3.16-4. 63 39 3.32-4.80 3.32-4.80 31-60 d 111 3.02-4.22 3.02-4. 22 72 2.93-3.87 2.93-3.87 61-180 d 282 3.43-4.80 3.43-4. 80 214 3.45-4.75 3.45-4.75 0.5-< 2 y 1210 4.03-5.07 4.03-5. 07 10493.97-5.01 3.97-5.01 2-<6 y 1198 3.89-4.97 3.89-4.97 10873.84-4.92 3.84-4.92 6-<12 y 1278 3.96-5.03 3.96-5. 03 10843.90-4.96 3.90-4.96 12-< 18 y 1467 4.03-5.29 4.03-5. 29 17623.93-4.90 3.93-4.90 18 y 264 4.18-5.48 4.18-5.48 467 3.70-4.87 3.70-4.87

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233Specimen Type (s) 1,3 Whole blood (K3/EDTA anticoagulant) 2 Whole blood (K3/EDTA anticoagulant) Reference (s) 1 Soldin SJ, Brugnara C, Wond ED, eds. Pediatri reference ranges, 4th ed. Washington, DC: AACC Press, 2003 2 Brugnara C. Unpublished data 3 g Unpublished Data Method (s) 1 Coulter STKS (Beckman Coulter Inc, Brea, CA). 2 Bayer ADVIA 120 (Bayer Diagnostics, Tarrytown, NY). 3 Sysmex XE-2100 (Sysmex Corporation, Kobe, Japan). Comment (s) 1Data from hospitalized patients (excluding hematology/oncology and intensive care unit patients). A computerized approach adapted from the Hoffmann technique was used to remove outliers and obtain the 5th and 97.5th percentiles.aTo obtain the 2.5th percentile, the statistical technique was modified to use the central 60% of the data for the Hoffman plot. 2Data from inpatients, excluding hematology/oncolgy patients. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion (1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffmann--the top and bottom 25% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 50% of the data. 1. Young HD. Statistical treatment of experimental data. New York:McGraw-Hill, 1962:76-9, 162. 2. Hoffman RG. Statistics in the practice of medicine. JAMA 1963;185:864-73. 3Performed on outpatients and ER patients. Hematology/oncology clinic patients were excluded. Data was analyzed using SAS statistical software. Outliers were removed according to the Chauvenet's Criterion (1). This criterion uses an exclusion factor based on the number of values in the sample set and is reapplied until no more outliers are excluded. The remaining data was truncated based on a method described by Hoffman-the top and bottom 10% of data, based on an assumed normal distribution, was discarded (2). The method was modified to allow for computerized handling of the data. The 2.5th and 97.5th percentiles were calculated on the remaining 80% of the data.

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234 Albumin Male Female Test Age n g/dL g/L n g/dL g/L 1 1-7 d 1612.3-3.8 23-38 132 1.8-3.9 18-39 8-30 d 2522.0-4.5 20-45 124 1.8-4.4 18-44 31-90 d 1992.0-4.8 20-48 178 1.9-4.2 19-42 91-180 d 1352.1-4.9 21-49 121 2.2-4.4 22-44 181 d-1 y 2952.1-4.7 21-47 267 2.2-4.7 22-47 2 0-5 d (<2.5 kg) 30 2.0-3.6 20-36 30 2.0-3.6 20-36 0-5 d (>2.5 kg) 93 2.6-3.6 26-36 93 2.6-3.6 26-36 1-3 y 50 3.4-4.2 34-42 50 3.4-4.2 34-42 4-6 y 38 3.5-5.2 35-52 38 3.5-5.2 35-52 7-9 y 74 3.7-5.6 37-56 74 3.7-5.6 37-56 10-19 y 3323.7-5.6 37-56 332 3.7-5.6 37-56 3 1-30 d 73 2.6-4.1 26-41 51 2.7-4.3 27-43 31-182 d 58 2.8-4.6 28-46 30 2.9-4.2 29-42 183-365 d 29 2.8-4.8 28-48 42 3.3-4.8 33-48 1-18 y 6523.2-4.7 32-47 626 2.9-4.2 29-42 4 1-7 d * 2.4-3.9 24-39 * 1.9-4.0 19-40 8-30 d 2.1-4.5 21-45 1.9-4.4 19-44 31-90 d 2.1-4.8 21-48 2.0-4.2 20-42 91-180 d 2.2-4.9 22-49 2.3-4.4 23-44 181 d1 y 2.2-4.7 22-47 2.3-4.7 23-47 1-3 y 3.5-4.2 35-42 3.5-4.7 35-47 4-6 y 3.6-5.2 36-52 3.6-5.2 36-52 7-9 y 3.8-5.6 38-56 3.8-5.6 38-56 10-19 y 3.8-5.6 38-56 3.8-5.6 38-56

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235Specimen Type (s) 1-4sPlasma/serum Reference (s) 1Soldin SJ, Morse AS. Pediatric reference ranges for albumin and total protein in children < 1 year old using the Vitros 500 Analyzer. Clin Chem 1998;44: A16. (Abstract) 2Lockitch G, Halstead AC, Albersheim S, et al. Ageand sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer. Clin Chem 1988; 34: 1622-5. 3Soldin SJ, Bjorn S, Beatey J, et al. Ageand sex-specific pediatric reference ranges for albumin and total protein on the Hitachi 747. Clin Chem 1995;41: S93. (Abstract) 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. Method (s) 1Bromcresol green method. Vitros 500 (Ortho-Clinical Diagnostics, Raritan, NJ). 2Bromcresol green method. Vitros 700 (Ortho-Clinical Diagnostics, Raritan, NJ). 3Boehringer Mannheim albumin reagent (bromcresol green). Albumin was measured on the Hitachi 747 (Boehringer Mannheim Diagnostics, Indianapolis, IN). 4The albumin method is an adaptation of the bromcresol purple (BCP) dyebinding method. Dade Behring Dimensi on RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1,3Study used hospitalized patients and a computerized approach adapted from the Hoffman technique. Values are 2.5-97.5 percentiles. 2Healthy normal children. Values are 2.5-97.5 percentiles. 4*Reference ranges were obtained by comparing results from previously published data and using regression equations.

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236 Prealbumin (Transthyretin) Male and Female Test Age n mg/dL mg/L 1 0-1 mo 63 7.0-39.0 70-390 1-6 mo 55 8.3-34.0 83-340 6 mo-4 y 159 2.0-36.0 20-360 4-6 y 59 12.0-30.0 120-300 6-19 y 189 12.0-42.0 120-420 2 0-5 d 69 6.0-21.0 60-210 1-5 y 68 14.0-30.0 140-300 6-9 y 68 15.0-33.0 150-330 10-13 y 61 20.0-36.0 200-360 14-19 y 70 22.0-45.0 220-450 3 0-4 d 118 7.3-14.4 73-144 1 mo-4 y 116 6.7-17.1 67-171 5-11 y 149 9.1-22.0 91-220 12-20 y 207 12.4-30.2 124-302 4 0-5 d * 8.6-23.2 86-232 6 d-24 mo 9.6-32.0 96-320 2-5 y 16.4-32.0 164-320 6-9 y 17.3-34.9 173-349 10-13 y 22.2-37.8 222-378 14-19 y 24.2-46.6 242-466 Male Female 5 Age n mg/dL mg/L n mg/dL mg/L 1-40 d 156 3.2-15.9 32-159.0 104 4.2-14.4 42-144 41-90 d 134 2.7-17.6 27-176.0 90 2.5-21.9 25-219 3-9 mo 114 7.3-27.9 73-279.0 94 5.3-25.0 53-250.0 10-24 mo 106 6.7-28.5 67-285.0 80 7.3-33.7 73-337 2-10 y 325 6.9-31.2 69-312.0 138 8.0-35.2 80-352 11-15 y 153 6.3-33.5 63-335.0 169 8.6-40.7 86-407 16-18 y 67 8.0-41.6 80-416.0 89 13.7-44.1 137-441

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237 Specimen Type (s) 1-3,5Serum 4Plasma/serum Reference (s) 1Davis ML, Austin C, Messmer BL, et al. IFCC-standardized pediatric reference intervals for 10 serum proteins using the Beckman Array 360 system. Clin Biochem 1996;29:489-92. 2Lockitch G, Halstead AZ, Quigley G, et al. Age-and sex-specific pediatric reference intervals: study design and methods illustrated by measurement of serum proteins with the Behring LN nephelometer. Clin Chem 1988;34: 1618-21. 3Hamlin CR, Pankowsky DA. Turbidimetric determination of transthyretin (prealbumin) with a centrifugal analyzer. Clin Chem 1987;33:144-6. 4Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003;331: 135-46. 5Soldin S, Zhang M, Schaub JR, Ghoshal A. Pediatric reference ranges for prealbumin and retinol binding protein. Clin Biochem 2003 (in press). Method (s) 1Rate nephelometry. Beckman Array 360 (Beckman Instruments, Brea, CA) 2,5Nephelometry using the Behring LN nephelometer (Behring Diagnostics, Westwood, MA). 3Immunoturbidimetric procedure usin g a Cobas-Bio centrifugal analyzer (Roche Analytical Instruments, Inc., Nutley, NJ) and rabbit antiserum (Behring Diagnostics, La Jolla, CA). 4The prealbumin method is based on a particle-enhanced turbidimetric immunosassay (PETIA) technique. Dade Behring Dimension RxL Analyzer (Dade Behring Inc., Newark, DE). Comment (s) 1Samples were obtained from children attendind outpatient clinics. Results are 2.5-97.5th percentiles. 2Healthy children. Results are 2.5-97.5th percentiles. No significant differences were found for males and females. These ranges were therefore derived from combined data. 3Healthy children. Results are 2.5-97.5th percentiles. 4*Numbers not provided Reference ranges were obtained by comparing results from previously published data and using regression equations. 5Study used hospitalized patients in a computerized approach to removing outliers.

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238 APPENDIX DD DIVISION OF AIDS TABLE FOR GRAD ING THE SEVERITY OF ADULT AND PEDIATRIC ADVERSE EVENTS PUBLISH DATE: DECEMBER, 2004 Quick Reference The Division of AIDS Table for Grading th e Severity of Adult and Pediatric Adverse Events (“DAIDS AE grading table”) is a de scriptive terminology which can be utilized for Adverse Event (AE) reporting. A grading (severity) scale is provided for each AE term. General Instructions Estimating Severity Grade If the need arises to grade a clinical AE th at is not identified in the DAIDS AE grading table, use the category “Estimating Severity Gr ade” located at the top of Page 3. For AEs that are not listed in the ta ble but will be collected systematically for a study/trial, protocol teams are highly encouraged to define study-specific severity scales within the protocol or an appendix to the protocol . (Please see “Template Wording for the Expedited Adverse Event Reporting Section of DAIDS-sponsored Protocols”.) This is particularly for laboratory values because th e “Estimating Severity Grade” category only applies to clinical symptoms. Grading Adult and Pediatric AEs The DAIDS AE grading table includes parame ters for grading both Adult and Pediatric AEs. When a single set of parameters is not appropriate for grading specific types of AEs for both Adult and Pediatric populations, separate sets of parameters for Adult and/or Pediatric populations (with specified respective age ranges ) are given in the table. If there is no distinction in the table between Adult and Pediatric values for a type of AE, then the single set of parameters listed is to be used for grading the severity of both Adult and Pediatric events of that type. Determining Severity Grade If the severity of an AE could fall under eith er one of two grades (e .g.., the severity of an AE could be either Grade 2 or Grade 3), sele ct the higher of the tw o grades for the AE.

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239 Definitions Basic Self-care Functions Adult Activi ties such as bathin g, dressing, toileting, transfer/movement, continence, and feeding. Young Children Activities that are age and culturally appropriate (e.g., feeding self with culturally appropriate eating implement. LLN Lower limit of normal Medical Intervention Use of pharmacologic or biologic agent(s) for treatment of an AE. NA Not Applicable Operative Intervention Surgical OR ot her invasive mechanical procedures. ULN Upper limit of normal Usual Social & Functional Adult Activities Adaptive tasks and desirable activities, such as going to work, shopping, cooking, use of transportation, pursuing a hobby, etc. Young Children Activities that are age and culturally appropriate (e.g., social interactions, play activities, learning Tasks, etc.).

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240 EXCERPTED FROM DIVISION OF AIDS (DAIDS) TABLE FOR GRADING THE SEVERITY OF ADULT AND PEDIATRIC ADVERSE EVENTS PUBLISH DATE: DECEMBER, 2004 PARAMETERGRADE 1GRADE 2GRADE 3GRADE 4 ESTIMATING SEVERITY GRADE Clinical adverse event Symptoms causingSymptoms causingSymptoms causingSymptoms causing inability NOT identified no or minimal greater than minimalinability to performto perform basic self-care elsewhere in thisinterference with interference withusual social &functions OR Medical or operative DAIDS AE gradingusual social & us ual social &functional activities .intervention indicated to prevent tablefunctional activitiesfunctional activitiespermanent impairment, persistent disability, or death. HEMATOLOGY‡Hemoglobin 1-7 days old13.0-14.0g/dL12.0-12.9g/dL<12.0g/dLCardiac failure 2ndary to anemia 8-21 days old12.0-13.0g/dL10.0-11.9g/dL<10.0g/dLCardiac failure 2ndary to anemia 22-35 days old9.5-10.5g/dL8.0-9.4g/dL<8.0g/dLCardiac failure 2ndary to anemia 36-56 days old8.5-9.4g/dL7.0-8.4g/dL<7.0g/dLCardiac failure 2ndary to anemia 57-90 days old9.0-9.9g/dL7.0-8.9g/dL<7.0g/dLCardiac failure 2ndary to anemia Hemoglobin *36-56 days8.5-9.4g/dL7.0-8.4g/dL6.0-6.9g/dL<6.0 g/dL (HIV Positive or1.32-1.46mmol/L1.09-1.31mmol/L0.93-1.08mmol/L<0.93mmol/L Negative) >57 days (HIV8.5-10.0g/dL7.5-8.4g/dL6.5-7.4g/dL<6.5g/dL Positive Adult1.32-1.55mmol/L1.16-1.31mmol/L1.01-1.15mmol/L<1.01mmol/L and Pediatric) >57 days (HIV10.0-10.9g/dL9.0-9.9g/dL7.0-8.9g/dL<7.0g/dL Negative)1.55-1.69mmol/L1.40-1.54mmol/L1.09-1.39mmol/L<1.09mmol/L or any decrease of: 2.5-3.40.39-0.533.5-4.40.54-0.68 4.5 0.69 Creatinine 1.1-1.3 x ULN†1.4-1.8 x ULN†1.9-3.4 x ULN† 3.5 x ULN† ‡Creatinine < 7 days old1.0-1.7mg/dl1.8-2.4mg/dl2.5-3.0mg/dl>3.0mg/dl 7-60 days old0.5-0.9mg/dl1.0-1.4mg/dl1.5-2.0mg/dl>2.0mg/dl 61-90 days old0.6-0.8mg/dl0.9-1.1mg/dl1.2-1.5mg/dl>1.5mg/dl‡Cr Clearance < 7 days old35-40 ml/min30-34ml/min25-29ml/min<25ml/min 7-60 days old45-50ml/min40-44ml/min35-39ml/min<35ml/min 61-90 days old60-75ml/min50-59ml/min35-49ml/min<35ml/min * Values are for termed infants.† Use age and sex appropriate values, including preterm infants.‡ Excerpted from April 2004 DAIDS table

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241 PARAMETERGRADE 1GRADE 2GRADE 3GRADE 4 HYPERTENSION Adult > 17 years >140-159mmHg>160-179mmHg>180mmHgLife-threatening (with repeat testingsystolicsystolicsystolicconsequences (e.g. at same visit) malignant hypertension) OROROROR >90-99mmHg>100-109mmHg>110mmHgHospitalization indicated diastolicdiastolicdiastolic(other than emergency room visit) Pediatric 17 NA 91st-94th percentile 95th percentile Life-threatening years adjusted for age, adjusted for age, consequences (e.g. (with repeat testingheight, and gender height, and gender malignant hypertension) at same visit)(systolic and/or (systolic and/or OR diastolic)diastolic)Hospitalization indicated (other than emergency room visit) PancreatitisNASymptomatic ANDSymptomatic ANDLife-threatening Hospitalization not Hospitalization consequences (e.g., indicated (other than indicated other thancirculatory failure, hemorrhage, ER visit)ER visit)sepsis) Bone Mineral Loss Adult 21 yearsBMD t-scoreBMD t-scorePathological fracturePathological fracture -2.5 to -1.0< -2.5(including loss of causing life-threatening vertebral height)consequences Pediatric < 21 yearsBMD z-scoreBMD z-scorePathological fracturePathological fracture -2.5 to -1.0< -2.5(including loss of causing life-threatening vertebral height)consequences URINARY Urinary tract NASigns or symptomsSigns or symptomsObstruction causing obstruction (e.g.of urinary tract of urinary tract life-threatening stone)obstuction without obstruction withconsequences hydronephrosis orhydronephrosis or renal dysfunctionrenal dysfunction ENDOCRINE/METABOLIC Abdominal fat Detectable by study Detectable on Disfiguring OR NA accumulation (e.g.participant (or byphysical exam byObvious changes on back of neck, breasts,caregiver for younghealth care providercasual visual abdomen)children and disabledinspection adults) Lipoatrophy (e.g., fatDetectable by study Detectable on Disfiguring OR NA loss from the face, participant (or byphysical exam byObvious changes on extremities, buttocks)caregiver for younghealth care providercasual visual children and disabledinspection adults) Diabetes mellitusNANew onset withoutNew onset with Life-threatening need to initiateinitiation of consequences (e.g., medication ORmedication indicatedketoacidosis, hyperosmolar Modification of current OR Diabetes non-ketotic coma) medications to regainuncontrolled despite glucose controltreatment modification

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242 CHEMISTRIES Albumin, serum, low3 g/dl2.0-2.9 g/dl<2.0 g/dlNA 30 g/L20-29 g/L<20 g/LNA Alkaline Phosphatase 1.25-2.5 x ULN†2.6-5.0 x ULN†5.1-10.0 x ULN†> 10.0 x ULN†ALT1.25-2.5 x ULN2.6-5.0 x ULN5.1-10.0 x ULN> 10.0 x ULN AST1.25-2.5 x ULN2.6-5.0 x ULN5.1-10.0 x ULN> 10.0 x ULN Bicarbonate,serum, low16.0 mEq/L < LLN11.0-15.9 mEq/L8.0-10.9 mEq/L< 8.0 mEq/L 16.0 mmol/L < LLN11.0-15.9 mmol/L8.0-10.9 mmol/L< 8.0 mmol.L Cholesterol (total/fasting) Adult 18 years200-239 mg/dL240-300 mg/dL>300 mg/dLNA 5.18-6.19 mmol/L6.20-7.77 mmol/L>7.77 mmol/LNA Pediatric < 18 years170-199 mg/dL200-300 mg/dL>300 mg/dLNA 4.40-5.15 mmol/L5.16-7.77 mmol/L>7.77 mmol/LNA LDL cholesterol (fasting) Adult 18 years130-159 mg/dL160-190 mg/dL 190 mg/dLNA 3.37-4.12 mmol/L4.13-4.90 mmol/L 4.91 mmol/LNA Pediatric > 2 < 18110-129 mg/dL130-189 mg/dL 190 mg/dLNA years2.85-3.34 mmol/L3.35-4.90 mmol/L 4.91 mmol/LNA Glucose, serum, high Nonfasting116-160 mg/dL161-250 mg/dL251-500 mg/dL> 500 mg/dL 6.44-8.88 mmol/L8.89-13.88 mmol/L13.89-27.75 mmol/L> 27.75 mmol/L Fasting110-125 mg/dL126-250 mg/dL251-500 mg/dL> 500 mg/dL 6.11-6.94 mmol/L6.95-13.88 mmol/L13.89-27.75 mmol/L> 27.75 mmol/L Glucose, serum, low Adult and Pediatric55-64 mg/dL40-54 mg/dL30-39 mg/dL< 30 mg/dL 1 month3.05-3.55 mmol/L2.22-3.06 mmol/L1.67-2.23 mmol/L< 1.67 mmol/L Infant*†, < 1 month 50-54 mg/dL40-49 mg/dL30-39 mg/dL< 30 mg/dL 2.78-3.00 mmol/L2.22-2.77 mmol/L1.67-2.21 mmol/L< 1.67 mmol/L Triglycerides (fasting)NA500-750 mg/dL751-1200 mg/dL> 1200 mg/dL 5.65-8.48 mmol/L8.49-13.56 mmol/L> 13.56 mmol/L Lactate<2.0 x ULN without 2.0 x ULN withoutIncreased lactateIncreased lactate with acidosisacidosiswith pH < 7.3pH < 7.3 with lifewithout life-threatening consequences. threatening consequences * Values are for termed infants.† Use age and sex appropriate values, including preterm infants.

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243 PARAMETERGRADE 1GRADE 2GRADE 3GRADE 4 CHEMISTRIES Cont. Lipase1.1-1.5 x ULN1.6-3.0 x ULN3.1-5.0 x ULN Pancreatic Amylase1.1-1.5 x ULN1.6-2.0 x ULN2.1-5.0 x ULN> 5.0 x ULN Phosphate, serum, low Adult and Pediatric2.5 mg/dL < LLN2.0-2.4 mg/dL1.0-1.9 mg/dL< 1.00 mg/dL > 14 years0.81 mmol/L -< LLN0.65-0.80 mmol/L0.32-0.64 mmol/L< 0.32 mmol/L Pediatric 1 year 143.0-3.5 mg/dL2.5-2.9 mg/dL1.5-2.4 mg/dL< 1.50 mg/dL years0.97-1.13 mmol/L0.81-0.96 mmol/L0.48-0.80 mmol/L< 0.48 mmol/L Pediatric < 1 year3.5-4.5 mg/dL2.5-3.4 mg/dl1.5-2.4 mg/dL<1.50 mg/dL 1.13-1.45 mmol/L0.81-1.12 mmol/L0.48-0.80 mmol/L<0.48 mmol/L Potassium,serum, high5.6-6.0 mEq/L6.1-6.5 mEq/L6.6-7.0 mEq/L>7.0 mEq/L 5.6-6.0 mmol/L6.1-6.5 mmol/L6.6-7.0 mmol/L>7.0 mmol/L Potassium,serum, low3.0-3.4 mEq/L2.5-2.9 mEq/L2.0-2.4 mEq/L<2.0 mEq/L 3.0-3.4 mmol/L2.5-2.9 mmol/L2.0-2.4 mmol/L<2.0 mmol/L URINALYSIS Hematuria 6-10 RBC/HPF> 10 RBC/HPFGross, with or Transfusion indicated (microscopic)without clots OR RBC casts Proteinuria, random1+2-3+4+NA collection Proteinuria, 24 hour collection Adult and Pediatric200-999 mg/24 h1000-1999 mg/24 h2000-3500 mg/24 h>3500 mg/24 h 10 years0.200-0.999 g/d1.000-1.999 g/d2.000-3.500 g/d>3500 g/d Pediatric > 3 mo -< 201-499 mg/m2/24 h500-799 mg/m2/24 h800-1000 mg/m2/24h> 1000 mg/m2/24 h 10 years0.201-0.499 g/d0.500-0.799 g/d0.800-1.000 g/d> 1000 g/d

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257 BIOGRAPHICAL SKETCH Marie Esther Dajuste was born on Septem ber 13, 1980, in St. Maarten, Netherlands Antilles, to Jean and Marie A. Dajuste. She graduated from the University of Florida with a Bachelor of Science in food scie nce and human nutrition in August 2002. She then participated in Notre Dame AmeriCor ps and subsequently worked in the ESOL department at Paul Laurence Dunbar Middle School in Fort Myers, Florida. In the fall of 2004, she began her Master of Science in food science and human nutrition working under Dr. Peggy Borum, a long-time advocate of mitochondrial dysfunction as one of the key factors in the emergence of metabolic dys function. Her thesis interest was in the metabolic disturbances present within the pediatric HIV positive population at Shands Immunology Clinic. In her spare time she enj oys the company of family and friends, singing, dancing, listening to music, and exer cising. Upon graduation she plans to return to Fort Myers and enter the nonprofit sector as a public health nutrition educator.