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Gender and cocaine use influence the expression of urinary markers of inflammation and oxidative stress

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Gender and cocaine use influence the expression of urinary markers of inflammation and oxidative stress
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English
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Bourgeois, Marie
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University of South Florida
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Subjects / Keywords:
Autoimmune
Interleukins
Reactive Oxygen Species
Acute Phase Reaction
Cytokines
Dissertations, Academic -- Dean's Office -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The purpose of this study was to investigate whether or not gender differences may be present in the expression of a number of urinary proteins which may serve as markers of inflammation and oxidative stress. Males and females have different patterns of illness and different life spans, suggesting basic biological traits exert significant control on the incidence of rhabdomyolysis, renal failure, atherosclerosis, myocardial ischemia, myocardial contraction band formation, autoimmune disorders and general inflammatory diseases. Men are at greater risk for cardiovascular disease; however women, particularly elderly women, have higher fatality rates due to heart failure. Renal diseases progress far more quickly in men, possibly due to testosterone. Men also have higher kidney bulk related to androgen expression. Gender disparity may be most obvious in autoimmune disorders; of the estimated 8.5 million people diagnosed with autoimmune disorders, approximately 80% are women. Hashimoto's thyroiditis, the most common form of hypothyroidism, is up to 10 times more common in women. Systemic Lupus Erythematosus (SLE), an autoimmune disease characterized by acute and chronic inflammation, is 9 times more common in women. Rheumatoid arthritis (RA), an autoimmune disease affecting approximately 1.3 million people in the United States, is four times more common in women. Diabetes mellitus (DM), affecting more than 17 million people - the majority of which are women, is linked to microvascular and macrovascular diseases such as kidney failure, strokes and atherosclerosis. These conditions are linked to physiological changes that may alter the expression of certain biomarkers of inflammation and oxidative stress. Over the past several decades, it has become increasingly clear that the role of diet, smoking, and other lifestyle choices clearly influence the etiology and pathophysiology of these diseases. The use of drugs, both licit and illicit, has been clearly linked to many of these diseases. Illicit substances, particularly cocaine, have been demonstrated to produce pathophysiological changes to many systems in the body which can greatly influence the progression of existing and drug-induced disease states leading to systemic damage. A relationship between the expression of markers of inflammation, oxidative stress, cardiac damage, or other systemic injury, gender and cocaine use has not been clearly established. Urine is an important medium for assessment of general health status. It has classically been used to monitor disease states; glucosuria as an indicator of diabetes and renal dysfunction, microorganisms signifying urinary tract or bladder infection, and biomarkers such as human chorionic gonadotropin to confirm pregnancy. Recently urine has been used to assess biomarker expression and disease states. Urine is an ideal clinical tool for toxicological screens; it is readily accessible, non invasive and typically supplied in sufficient quantity to accommodate multiple tests. In this study, urine specimens were collected and analyzed for creatinine, cocaine, total protein, aldosterone, c-reactive protein (hsCRP), myeloperoxidase (MPO), microalbumin (MAB), neutrophil gelatinase-associated lipocalin (NGAL), heat shock protein 90α (hsp90α), vascular endothelial growth factor (VEGF), myoglobin, pro atrial natriuretic peptide (proANP) and interleukins 1α, 1 β , and 6 using ELISA and colorimetric assays. Urine specimens that tested negative for all illicit substances in the standard National Institute on Drug Abuse (NIDA) 10 panel showed differences in a number of these biomarkers which strongly suggested significant differences between males and females for aldosterone, IL1α and IL1β. In addition, significance is suggested for MPO and CRP. Although sex specific differences in serum expression have been noted for some of the markers in both animal and human models, this has not been previously demonstrated in human urine. This may have implications for what is typically referred to as 'normal' values. Gender specific differences were not apparent in urine specimens that tested positive for cocaine. Also, in males only, the levels of myoglobin and aldosterone significantly increased.
Thesis:
Dissertation (PHD)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
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Statement of Responsibility:
by Marie Bourgeois.
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Title from PDF of title page.
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Document formatted into pages; contains X pages.

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ABSTRACT: The purpose of this study was to investigate whether or not gender differences may be present in the expression of a number of urinary proteins which may serve as markers of inflammation and oxidative stress. Males and females have different patterns of illness and different life spans, suggesting basic biological traits exert significant control on the incidence of rhabdomyolysis, renal failure, atherosclerosis, myocardial ischemia, myocardial contraction band formation, autoimmune disorders and general inflammatory diseases. Men are at greater risk for cardiovascular disease; however women, particularly elderly women, have higher fatality rates due to heart failure. Renal diseases progress far more quickly in men, possibly due to testosterone. Men also have higher kidney bulk related to androgen expression. Gender disparity may be most obvious in autoimmune disorders; of the estimated 8.5 million people diagnosed with autoimmune disorders, approximately 80% are women. Hashimoto's thyroiditis, the most common form of hypothyroidism, is up to 10 times more common in women. Systemic Lupus Erythematosus (SLE), an autoimmune disease characterized by acute and chronic inflammation, is 9 times more common in women. Rheumatoid arthritis (RA), an autoimmune disease affecting approximately 1.3 million people in the United States, is four times more common in women. Diabetes mellitus (DM), affecting more than 17 million people the majority of which are women, is linked to microvascular and macrovascular diseases such as kidney failure, strokes and atherosclerosis. These conditions are linked to physiological changes that may alter the expression of certain biomarkers of inflammation and oxidative stress. Over the past several decades, it has become increasingly clear that the role of diet, smoking, and other lifestyle choices clearly influence the etiology and pathophysiology of these diseases. The use of drugs, both licit and illicit, has been clearly linked to many of these diseases. Illicit substances, particularly cocaine, have been demonstrated to produce pathophysiological changes to many systems in the body which can greatly influence the progression of existing and drug-induced disease states leading to systemic damage. A relationship between the expression of markers of inflammation, oxidative stress, cardiac damage, or other systemic injury, gender and cocaine use has not been clearly established. Urine is an important medium for assessment of general health status. It has classically been used to monitor disease states; glucosuria as an indicator of diabetes and renal dysfunction, microorganisms signifying urinary tract or bladder infection, and biomarkers such as human chorionic gonadotropin to confirm pregnancy. Recently urine has been used to assess biomarker expression and disease states. Urine is an ideal clinical tool for toxicological screens; it is readily accessible, non invasive and typically supplied in sufficient quantity to accommodate multiple tests. In this study, urine specimens were collected and analyzed for creatinine, cocaine, total protein, aldosterone, c-reactive protein (hsCRP), myeloperoxidase (MPO), microalbumin (MAB), neutrophil gelatinase-associated lipocalin (NGAL), heat shock protein 90α (hsp90α), vascular endothelial growth factor (VEGF), myoglobin, pro atrial natriuretic peptide (proANP) and interleukins 1α, 1 β and 6 using ELISA and colorimetric assays. Urine specimens that tested negative for all illicit substances in the standard National Institute on Drug Abuse (NIDA) 10 panel showed differences in a number of these biomarkers which strongly suggested significant differences between males and females for aldosterone, IL1α and IL1β. In addition, significance is suggested for MPO and CRP. Although sex specific differences in serum expression have been noted for some of the markers in both animal and human models, this has not been previously demonstrated in human urine. This may have implications for what is typically referred to as 'normal' values. Gender specific differences were not apparent in urine specimens that tested positive for cocaine. Also, in males only, the levels of myoglobin and aldosterone significantly increased.
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Interleukins
Reactive Oxygen Species
Acute Phase Reaction
Cytokines
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PAGE 1

Gender and Cocaine Use Influence the Expression of Urinary Markers of Inflammation and Oxidative Stress by Marie Meagher Bourgeois A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Depart ment of Environmental and Occupational Health College of Public Health University of South Florida Ma jor Professor: Ira S. Richards, Ph.D. Steven Mlynarek, Ph.D. Raymond Harbis on, Ph.D. Jack Perman, Ph.D. Date of Approval: October 19, 2010 Keywords: Autoimmune, Interleukins, Reactive Oxygen Species, Acute Phase Reaction, Cytokines Copyright 2010, Marie Meagher Bourgeois

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Dedication Without the support, understanding and unflagging faith of my husband Bobby, and my children Jake, Shelby, Maya and Logan, this would not have been possible

PAGE 3

Acknowledgements I would like to thank my advisor Dr. Ira Richards for his encouragement and guidance not only during this research project, but also throughout my years in the graduate program. I woul d like to thank Dr. Raymond Harbison for challenging me during presentati ons; his ques tions kept me on my toes. Thanks to Dr. Steven Mlynarek who helped keep my statistics relevant. Special thanks to Dr. Jack Perman for providing me with the opportunity to work in the laboratory at the Department of Health. Additional acknow le dgement goes to Richard and Sue Brown from the Agency for Community Testing Access to their expertise and facilities proved invaluable.

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i Table of Contents List of Tables vi List of Figures v i ii List of Abbreviations x Abstract x ii Chapter 1.0 Introduction and Hypotheses 1 Chapter 2.0 Literature Review 12 2.1 Gender 12 2.2 Inflammation 15 2.3 Oxidative Stress 17 2.4 Cocaine 20 Chapter 3.0 Methods 25 3.1 EMIT 27 3.1.1 General Procedure for EMIT Assay 29 3.1.2 Cocaine / Benzoylecgonine 31

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ii 3.1.2.1 General Principle of the Assay 31 3.1 2 .2 Specificity 32 3.1.3 Creatinine 33 3.1.4 Specific Gravity 34 3.1.5 Ethanol 35 3.2 Colorimetric 36 3.2.1 Total Protein 37 3.2.1.1 General Principle of Assay 37 3.2.1.2 Assay Procedural Details 37 3.2.1.3 Interfering Substances 39 3.2.2 Creatinine 39 3.2.2.1 General Principle of Assay 39 3.2.2.2 Assay Procedural Details 40 3.3 ELISA 42 3.3.1 Cocaine/ Benzoylecgonine 53 3.3.1.1 General Principle of Assay 53 3.3.1.2 Assay Procedural Details 54 3.3.1.3 Cross Reactivity with Unrelated Drugs 55 3.3.2 Aldosterone 56 3.3.2 .1 General Principle of Assay 56 3.3.2 .2 Assay Procedural Details 57 3.3.2 .3 Urine Pretreatment 58 3.3.2.4 Cr oss Reactivity 59 3.3.3 C Reactive Protein 59 3.3.3 .1 General Principle of Assay 59 3.3.3 .2 Assay Procedural Details 60 3.3.3 .3 Cross R eactivity 62

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iii 3.3.4 Myeloperoxidase 62 3.3.4 .1 General Principle of Assay 62 3.3.4 .2 Assay Procedural Details 63 3.3.4 .3 Cross R eactivity 65 3.3.5 Microalbumin 66 3.3.5 .1 General Principle of Assay 66 3.3.5 .2 Assay Procedural Details 67 3.3.5 .3 Cross R eactivity 68 3.3.6 Myoglobin 68 3.3.6 .1 General Principle of Assay 68 3.3.6 .2 Assay Procedural Details 69 3.3.6 .3 Cross R eactivity 71 3.3.7 Heat Shock Protein 90 7 1 3.3.7 .1 General Principle of Assay 71 3.3.7 .2 Assay Procedural Details 71 3.3.7 .3 Cross Reactivity 74 3.3.8 Vascular Endothelial Growth Factor 74 3.3.8 .1 General Principle of Assay 74 3.3.8 .2 Assay Procedural Details 75 3.3.8 .3 Cross Reactivity 77 3.3.9 Neutrophil Gelatinase Associated Lipocalin 78 3.3.9 .1 General Principle of Assay 78 3.3.9 .2 Assay Procedural Details 79 3.3.10 pro Atrial Natriuretic Peptide (1 98) 81 3.3.10 .1 General Principle of Assay 81 3.3.10 .2 Assay Procedural Details 82 3.3.10 .3 Cross Reactivity 83

PAGE 7

iv 84 3.3.11 .1 Genera l Principle of Assay 84 3.3.11 .2 Assay Procedural Details 85 88 3.3.12 .1 General Principle of Assay 88 3.3.12 .2 Assay Procedural Details 89 3.3.13 Interleukin 6 91 3.3.13 .1 General Principle of Assay 91 3.3.13 .2 Assay Procedural Details 92 Chapter 4.0 Results 95 4.1 Gender 95 4.1.1 Assay results in Male and Female Control Urines 95 4.1 .2 Assay results in Male and Female Cocaine Positive Urines 96 4.2 Cocaine 98 4.2.1 Assay Results in Male Control and Cocaine Positive Urine Specimens 98 4.2.2 Assay Results in Female Control and Cocaine Positive Urine Specimens 98 4.3 Tables 101 Chapter 5.0 Discussion 103 Chapter 6.0 Conclusion 112 References 114 Bibliography 124

PAGE 8

v About the Author End Page

PAGE 9

vi List of Tables Table 1. Summary of Assays Performed 26 Table 2. Concentration of Albumin Standards 38 Table 3. Concentration of Creatinine Standards 41 Table 4. Hsp90 Serial Dilutions 73 Table 5. VEGF Serial Dilutions 76 Table 6. NGAL Serial Dilution Table 80 Table 7. Concentration of Interleukin 1 Standards 87 Table 8. Concentration of Interleukin 1 Standards 90 Table 9. Concentration of Interleukin 6 Standards 94 Table 10. Comparison of Assay results in Male and Female Control Urines 96

PAGE 10

vii Table 11. Comparison of Assay results in Male and Female Cocaine Positive Urines 97 Table 12. Comparison of Assay Results in Male Control and Cocaine Positive Urine 99 Table 13. Comparison of Assay Results in Female Control and Cocaine Positive Urine 100 Table 14. Male and Female Control and Cocaine Positive Urine Specimen Comparison 101 Table 15. Control and Cocaine Positive Male and Female Urine Specimen Comparison 102

PAGE 11

viii List of Figures Figure 1. Conditions Associated with Inflammation and Oxidative Stress 6 Figure 2. Metabolites of Cocaine 21 Figure 3. EMIT Assay Components in Action 29 Figure 4. Olympus AU640e Chemistry Immuno Analyzer 31 Figure 5. Modified Jaffe Reaction 34 Figure 6. Urine Specific Gravity Reaction Equation 35 Figure 7. Oxidation of Ethanol to Acetaldehyde 36 Figure 8 Common Steps in Direct/Competitive ELISA Assay 47 Figure 9. TECAN M200 Infinite Microplate Reader 48 Figure 10. I Control/Magellan User Interface 50

PAGE 12

ix Figure 11. Typical Magellan Microwell Absorbance Results 51 Figure 12. TECAN Columbus Strip Washer 52

PAGE 13

x List of Abbreviations ACTS Agency for Community Treatment and Services of Tampa AKI acute kidney injury AMI acute myocardial infarction ANOVA analysis of variance ANP atrial natriuretic peptide BE benzoylecgonine CE cocaethylene CKD chronic kidney disease CK MB creatine kinase MB isoenzyme CRP c reactive protein CVD cardiovascular disease DAWN Drug Abuse Warning Network DM diabetes mellitus E2 estradiol ECG electrocardiogram ED emergency department ELISA enzyme linked immunosorbent assay EME ecgonine methyl ester EMIT enzyme multiplied immunoassay technique ESRD end stage renal disease

PAGE 14

xi HCl hydrochloric acid HRP horseradish peroxidase HsCRP high sensitivity C reactive protein IL IL6 interleukin 6 MAB microalbumin MI myocardial infarction MPO myeloperoxidase N normal NGAL neutrophil gelatinase associated lipocalin NIDA National Institute on Drug Abuse NO nitric oxide NF kB nuclear factor kB (NF kB) OS oxidative stress PBS phosphate buffered saline ProANP pro atrial natriuretic peptide RA rheumatoid arthritis RAS renin angiotensin system ROS reactive oxygen species SAMSHA Substance Abuse and Mental Health Administration SLE Systemic Lupus Erythematosus THC Tetrahydrocannabinol TMB 3,3,5,5 tetramethylbenzidine

PAGE 15

xii Gender and Cocaine Use Influence the Expression of Urinary Markers of Inflammation and Oxidative Stress Marie Meagher Bourgeois Abstract The purpose of this study was to investigate whether or not gender differences may be present in the expression of a number of urinary proteins which may serve as markers of inflammation and oxidative stress. Males and females have different patterns of il lness and different life spans, suggesting basic biological traits exert significant control on the incidence of rhabdomyolysis, renal failure, atherosclerosis, myocardial ischemia, myocardial contraction band formation, autoimmune disorders and general in flammatory diseases. Men are at greater risk for cardiovascular disease; however women, particularly elderly women, have higher fatality rates due to heart failure. Renal diseases progress far more quickly in men, possibly due to testosterone. Men also ha ve higher kidney bulk related to androgen expression. Gender disparity may be most obvious in autoimmune disorders; of the estimated 8.5 million people diagnosed with comm on form of hypothyroidism, is up to 10 times more common in women. Systemic Lupus Erythematosus (SLE), an autoimmune disease characterized by acute and chronic inflammation, is 9 times more common in women. Rheumatoid arthritis (RA), an

PAGE 16

xiii autoimmune disease affecting approximately 1.3 million people in the United States, is four times more common in women. Diabetes mellitus (DM), affecting more than 17 million people the majority of which are women, is linked to microvascular and macrovascular diseases such as kidney failure, strokes and atherosclerosis. These conditions are linked to physiological changes that may alter the expression of certain biomarkers of inflammation and oxidative stress. Over the past several decades, it has become increasingly clear that the role of diet, smoking, and other lifestyle choices clearly influence the etiology and pathophysiology of these diseases. The use of drugs, both licit and illicit, has been clearly linked to many of these diseases. Illicit substances, particularly cocaine, have been demonstrated to produce pathophysiological changes to many systems in the body which can greatly influence the progression of existing and drug induced disease states leading to systemic damage. A relationship between the expression of markers of inflammation, oxidative stress, cardiac damage, or other systemic injury, gender and cocaine use has not been clearly established. Urine is an important medium for assessment of general health status. It has classically been used to monitor di sease states; glucosuria as an indicator of diabetes and renal dysfunction, microorganisms signifying urinary tract or bladder infection, and biomarkers such as human chorionic gonadotropin to confirm pregnancy. Recently urine has been used to assess bioma rker expression and disease states. Urine is an ideal clinical tool for toxicological screens; it is readily accessible, non invasive and typically supplied in sufficient quantity to accommodate multiple tests. In this study, urine specimens were collecte d and analyzed for creatinine, cocaine, total protein, aldosterone c reactive

PAGE 17

xiv protein (hsCRP), myeloperoxidase (MPO), microalbumin (MAB), neutrophil gelatinase associated lipocalin (NGAL), growth facto r (VEGF), myoglobin, pro atrial natriuretic peptide (proANP) and Urine specimens that tested negative for all illicit substances in the standard National Institute on Drug Abuse (NIDA) 10 panel showed differences in a number of these biomarkers which strongly suggested significant differences between males and females for aldosterone, In addition, significance is suggested for MPO and CRP. Although sex specific differences i n serum expression have been noted for some of the markers in both animal and human models, this has not been previously demonstrated in human urine. This may have implications for what is typically referred to ces were not apparent in urine specimens that tested positive for cocaine. Also, in males only, the levels of myoglobin and aldosterone significantly increased.

PAGE 18

1 Chapter 1.0 Introduction that should be considered in every facet of research design [1] Significant gender dimorphism has been documented in card iovascular, renal and autoimmune diseases [2 8] Higher male susceptibility to cardiovascular disease may be due to genetic, hormonal, or lifestyle factors or through a combinatio n of mechanisms [9, 10] Sex based differences in the clinical presentation, diagnosis, and treatment outcomes of cardiac disease have long been recognized [11] Gender differences in lifestyle risk factors ( e.g. smoking, exercise and diet) appear to contribute to gender disparities; however, these lifestyle factors do not comp letely account for the dimorphism. Despite the fact that there is a higher incidence of cardiovascular diseases in men in general, the total number of deaths from cardiovascular disease has been higher for women than for men [12] Coronary artery disease is the leading cause of death in women. More than twice as many women die from cardiovascular disease as from all forms of cancer combined. Men are more likel y than women to suffer from hypertension. Multiple physiological alterations have been noted in hypertensive individuals; these include renal disturbances, neurohormonal and adrenergic disruption, endothelial dysfunction, systemic

PAGE 19

2 inflammation, and increas ed oxidative stress. Increased creatinine, CRP, aldosterone and proANP are often seen in hypertension. CRP is an acute phase inflammatory marker considered indicative of atherosclerosis and AMI. ProANP is found in atrial myocytes and regulates blood pressu re by opposing aldosterone. Proteinuria is common in renal and cardiovascular disease. Elevated urine protein concentrations are linked to increased IL 6 production and heart failure. MPO is another inflammatory marker; it is used to predict the risk of AM molecule found in cardiac tissue, is linked to oxidative stress. It is inhibited by estradiol (E2). VEGF is inducible by ischemia and anoxia; it is often elevated along with microalbumin. Microalbuminuria is associated with cardiovascular disease. It is induced by vasodilation and hypoxia inducible factors. E2 promotes vasodilation. Myoglobin is are proinflammatory cytokines stimulated by hepatic acute phase cytokines such as CRP that i nduce IL6. Another example of gender influence can be seen in the prevalence and progression of many renal diseases [4, 13] The majority of chronic renal diseases occur more commonly in men and progress far more quickly. There is no discernible gender disparity in acute kidney disease rates. Chronic Kidney Disease (CKD) is one of the few c hronic renal diseases that occur more commonly in women. CKD can lead to End Stage Renal Disease (ESRD) if it is not treated. The physiology behind gender differences in renal disease is unclear, but differences in kidney size and physiology, coupled with lifestyle factors and the vasoprotective effects of estrogen, have been implicated [1, 14 19] Estrogen is thought to suppress the growth of renal scar tissue by inhibiting the production of collagen.

PAGE 20

3 Inter estingly, this female advantage in chronic kidney disease disappears when the women suffer from diabetes mellitus (DM). CKD is uncommon in premenopausal women. The rate of CKD increases as estrogen levels fall; however, this may be the result of the relati ve ratio of androgen to estrogen rather than the absolute level of estrogen. It is thought that the androgen to estrogen ratio determines the effect on the diabetic kidney. Unlike their non diabetic counterparts, women with DM are found to have similar rates of kidney disease as males. DM is an increasingly common cause of kidney failure in developed countries. Increased renin an giotensin system (RAS) activity is thought to play an important role in both the hemodynamic and nonhemodynamic pathways involved in organ damage, particularly for diabetes. RAS blockade has demonstrated nephroprotective properties in diabetic individuals. T wo mechanisms for promoting vasoconstriction are RAS activation and nitric oxide (NO) pathway disruption. Hyperglycemia disrupts NO mediated relaxation; despite this interaction, baseline renal vasodilation is common in diabetes [20] Hypertension is common in diabetics; increased aldosterone concentration in urine is a risk factor for renal disease. Creatinine can be used to asses s glomerular filtration rate. Increased concentrations are seen in DM, hypertension and impaired renal function. Normal urine does not have substantial protein; proteinuria is considered suggestive of renal disease. Microalbuminuria is often seen in renal dysfunction, as is elevated VEGF. Myoglobinuria is common in rhabdomyolysis and renal failure. CRP and MPO, inflammatory markers, are often elevated in renal disease. NGAL is the earliest responding marker in acute kidney injury; it is also elevated in CKD

PAGE 21

4 synergis tically with both IL1 subunits. The interleukins may also be elevated in autoimmune disorders. A common feature of autoimmune diseases in both humans and experimental animals is that females are far more susceptible to autoimmune conditions when compared t o males. In several animal models, estrogens promote B cell mediated autoimmune diseases. Androgens exert an inhibitory effect in the same models. Although Females display heightened immune responses not only to foreign antigens but also to self antigens. Estrogens induce T and B cells imbalances; they appear to induce hypoactivity in subsets of T cell and hyperactivity in B cells. This may be the underlyin g basis for estrogen induced autoimmunity. Of the 8.5 million Americans diagnosed with autoimmune disorders, approximately 80% are women [21] Rheumatoid arthritis (RA) is 4 times more common in women, systemic lupus erythematosus (SLE) is nine times more common in women and some forms of thyroiditis are 10 times more common in women compared with men [22, 23] Insulin dependent DM is also more common in women than it is in men. Studies have shown that a disparity exists between male and female diabetics, particularly in the control of modifiable risk factors such as blood pressure, serum glucose and cholesterol levels [16, 24] Some researchers believe this is why death due to heart disease has decreased among diabetic men but not in women. Creatinine is often elevated in DM. CRP and MPO may be elevated in cases of inflammatory immune diseases such as RA and SLE. CRP may be a surrogate marker of SLE activity and may be useful to monitor the course of the disease. NGAL often

PAGE 22

5 increases in tande inflammation, may increase with autoimmune disease. VEGF is another cytokine inducible by inflammation. Myoglobinuria may be seen in DM and other autoimmune disease. Subclinical inflammatory reaction headaches, neutrophilia and increases in ci rculating cytokines). Systemic and chronic inflammation are thought to contribute to the development of atherosclerosis and CHD. The plurality of these conditions has been linked to inflammation and oxidative stress and may result in the release of biomark ers into blood and other body fluids [2 4, 13, 22, 23, 25 30] Inflammation is one of the most prominent forms of oxidative damage. Reactive oxygen species (ROS) produced by endothelial cells, neut rophils and macrophages can stimulate the activation of proinflammatory transcription factors and induce the production of inflammatory cytokines such as IL1 and IL6 Inflammation begins with hyperemia, edema, and adherence of the circulating white blood c ells to endothelial cells. Local inflammatory response is typically accompanied by systemic changes that include hyperemia, leukocytosis and induction of acute phase reactants like CRP. Chronic inflammation is not confined to a particular tissue; it invol ves the endothelium and multiple organ systems. High levels of oxidative stress and inflammation can increase the probability of early incidence of multiple disease states, including atherosclerosis, Parkinson's disease, heart failure, myocardial infarctio n, Alzheimer's disease, fragile X syndrome and chronic fatigue syndrome (Figure 1).

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6 Figure 1. Conditions Associated with Inflammation and Oxidative Stress Oxidative stress (OS) describes the level of oxidative damage in a cell, tissue, or organ, cause d by ROS. The damage can affect everything from specific molecules to entire organisms. ROS such as free radicals and peroxides come from the metabolism of oxygen and are endogenous in all aerobic organisms. The rate at which damage occurs is primarily det ermined by the clearance of generated ROS by antioxidants. The rate of damage depends on the level of repair enzyme. Most are by products of essential metabolic reactions such as mitochondrial energy generation and hepatic cytochrome P 450 detoxification r eactions. Exogenous sources of ROS include lifestyle choices

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7 (cigarette smoking, alcohol consumption, dietary, etc.), environmental exposures (automotive emissions, industrial pollutants, asbestos, etc.), bacterial, fungal or viral infections and radiation Exercise has the potential to induce free radical formation leading to OS [31] Much of the damage associated with cocaine toxicity stems from OS [32] In addition to environmental and lifestyle exposures, the determinants of oxidative stress are reg ulated by hereditary factors. Oxidative stress is also implicated in the ischemic cascade in reperfusion injuries. Myoglobin from myolysis, common with syndromes like rhabdomyolysis, may cause OS [33] ROS are not uniformly destructive; they are used by the immune system as a way to attack and kill pathogens and in redox signaling. Cocaine (benzoylmethylecgonine) is an alkaloid derived from the leaves of Erthr oxylon coca a shrub indigenous to South America [34] Despite restrictions on importation and distribution, cocaine has become one of the most commonly used illicit drugs [35] According to the 2007 National Survey on Drug Use and Health, nearly 1.6 million Americans met Diagnostic and Statistical Manual of Mental Disorders criteria for dependence or abuse of cocaine (in any form) in the past 12 months. The 2005 Drug Abuse Warning Network (DAWN) report stated that cocaine was involved in 448,481 of the total 1,449,154 (31%) visits to emergency departments (ED) for drug misuse or abuse. Cocaine is the m ost frequently reported illicit drug assoc iated with ED admissions [36] Cocaine is a powerful sympathomimetic capable of vasoconstriction and increasing heart rate, blood pressure, contractility, respiration, myocardial oxygen demand and body temperature [37] Hyperpyrexia can lead to rhabdomyolysis, myoglobulinuria, renal failure, liver damage and disseminated intravascular coagulation [38] Cocaine use is

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8 linked to atherosclerosis, myocardial ischemia, myocardial contraction band formation and sudden death [36, 39] Cocaine induced chest pain is the most common symptom reported by patients; an estimated 64,000 patients were evaluated for myocardial ischemia in 1995 subsequent to cocaine related chest pain [40, 41] Some of these patients exhibited elevated CK MB levels or electrocardiogram (ECG) changes consistent with acute myocardial infarction (AMI); however, the majority reported recur rent chest pain subsequent to cocaine use following discharge [41, 42] The metabolism of cocaine is primarily hepatic, with only about 1% excreted unchanged in the urine [43] Cocaine has two primary me tabolic pathways in humans, deesterification and demethylation. Benzoylecgonine (BE) is the primary metabolite and is not considered an important source of ROS. Ecgonine methyl ester (EME) is the second most common metabolite. Neither BE nor EME are biolog ically active. Transmethylation of cocaine is a minor pathway; metabolites of this pathway include norcocaine, norcocaine nitroxide, n hydroxynorcocaine and formaldehyde. Microsomes in the brain and liver oxidize norcocaine to nitroxide. Microsomal reducti on of norcocaine nitroxide in the brain generates superoxide ( SO ) [32] Hepatic microsomes incubated with nitroxide or the N hydroxy derivative results in lipid peroxidation [27] Transesterification of cocaine oc curs in the presence of alcohol and produces the biologically active intermediate cocaethylene. Various cocaine related hepatotoxicity studies indicate the generation of ROS, including lipid peroxides [44, 45] Cocaine has been shown to induce the release of SO which has been implicated in cardiac and cerebrovascular dysfunction [32]

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9 The vasoconstriction associated with cocaine usage is thought to result from its blockade of norepinephrine and dopamine reuptake at preganglionic sympathetic nerve endings; the extended catecholamine presence increases heart rate and blood pressure [40, 46] Cocaine also disrup ts catecholamine metabolism by inhibiting monoamine oxidase (MAO). B y blocking the neuronal plasma membrane transporter cocaine increases extracellular levels of monoamines [47] Hypertension is associated with disruptions in endothelial cell function and oxidative stress. MPO promotes endothelial dysfunction [48] Microalbumin is inducible by endothelial dysfunction and oxidative stress; cocaine use may impact expression of this marker [49] by increased body temperature [50] Cocaine is a known hyperpyrexic so it is possible that usage could result in increases in the concentrations of these markers [51] Oxidative stress has also been implicated as an early triggerin g event of cocaine induced cardiomyopathy and atherosclerosis [52] IL6, CRP and MPO a re being considered for validation as cardiac biomarkers [48, 53] MPO has also been linked to the pathogenesis of renal injury [54, 55] Increased expression of these cytokines may result from cocaine induced atherosclerosis and other cardiac disruptions. Fluctuations in blood pressure activat e the renin angiotensin system RAS which may increase proANP expression along with aldosterone production. RAS activation contributes to OS and vascular inflammation; it can also change the baseline filtering activity of the kidneys. Aldosterone stimulates inflammation through ROS generation. Cocaine use is linked to both renal dysfunction and rhabdomyolysis [34, 56] NGAL is a validated marker for acute kidney damage and inflammatory processes [57] Myoglobinuria, proteinuria and

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10 increased expression of creatinine are associated with rhabdomyolysis and cardiac dysfunction; both conditions may result from cocaine use [53, 58] The link between bi omarker expression in urine and gender or cocaine based inflammation, cardiac damage, or other systemic injury has not been elucidated. Urine samples were assayed for benzoylecgonine (BE), total protein aldosterone c reactive protein (CRP), myeloperoxida se (MPO), microalbumin (MAB), neutrophil gelatinase associated lipocalin (NGAL), growth factor (VEGF), myoglobin, pro atrial natriuretic peptide (proANP) and colorimetric assays; these markers were selected for their biological plausibility and assay kit availability. Urine is a non invasive specimen typically provided in sufficient quantity to allow multiple tests. As most toxicological screens primarily utili ze urine specimens in clinical settings, we examined the possibility of establishing relationships between the expression of several urinary biomarkers associated with oxidative stress and inflammation and gender and cocaine use. The following hypotheses were tested in this study: 1. The expression of some urinary markers of inflammat ion and/or oxidative stress is influenced by gender. Creatinine and myoglobin are related to lean muscle mass; therefore, males should have higher urinary mean concentrations. Total protein, microalbumin and NGAL may be elevated in fema les as they suffer far more urinary tract infections than males. IL1 I L1 IL6, MPO, VEGF and CRP may be higher in females as they suffer disproportionately from autoimmune/inflammatory diseases

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11 2. The expression of these markers is influenced by the use of cocaine. Creatinine and myoglobin are increased by hyperthermia and rhabdomyolysis; therefore, cocaine positive specimens should have a higher mean values than control urine specimens. Cocaine induces oxidative stress and inflammation; aldosterone, myoglobin, VEGF, NGAL, IL1 IL1 IL6, MPO and CRP may be higher in cocai ne positive specimens. Hsp90 and proANP are associated with cardiac dysfunction; they may be higher in cocaine positive specimens.

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12 C hapter 2.0 Literature Review 2.1 Gender There are significant gender based differences in the etiology of disease states including cardiac and vascular disease, metabolic disorders, autoimmune disorders and drug abuse toxicology. The basis of the gender dimorphism is unclear; however, cardiac muscle and vascular tissue are influenced by hormones such as estrog en and testosterone [1, 9, 10, 12, 59 61] Premenopausal women appear to be partially protected to some extent from car diovascular and kidney diseases; however, this protection weakens after menopause [62] Gender differences may exist not only in atherogenesis but also in post ischemic/ infarction cardiovascular repair Angiogenesis is essential to cardiovascular repair and regeneration. The effect of estrogen on angiogenesis has been extensively studied but the role of androgens remains unexplored [63] Estrogens induce vasodilation by increasing the activity of endothelial nitric oxide synthase (eNOS) E strogens a nd androgens regulate the RAS which in turn regulates t he cardiovascular system and the kidneys [64] In general, estrogen decreases renin

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13 production thereby decreasing aldosterone production. Progesterone competes with aldosterone for mineralocorticoid receptor. Aldosterone is linked to inf lammation via ROS generated oxidative stress. Aldosterone is a renal hormone in the RAS secreted in response to hypotension. Aldosterone has been implicated in renal disease progression; the majority of renal diseases are far more common in men. Increased cardiac concentrations have been linked to heart failure. Creatinine is also a general marker of muscle and kidney disease [18, 19] Because creatinine is related to lean muscle mass, men tend to have slightly higher serum creatinine levels than do women Increased va scular oxidative stress leads to endothelial dysfunction and hypertension I nhibition of th e RAS results in a decline in ROS production [65, 66] Estrogen also activates natriuretic peptides such as proANP; these peptides are a counterpart of the RAS. Testosterone seems to exert the opposite effect on renin levels. These effects of sex hormones on the RAS may explain some of the gender differences in cardiovascular and kidney diseases [67] Exposure to sex hormones modulates many endocrine factors involved in atherosclerosis Increased vascula r oxidative stress leads to endothelial dysfunction and hypertension. It has been noted that inhibition of the RAS results in a decline in ROS production [65, 66] Studies suggest females are at lower risk of developing cardiovascular disease (CVD) as compared to males [68] Striking sex differences exist not only in the incidence of cardiovascular disease, but also in the clinical outcomes. Cardiovascular events occur earlier in men than in women; however, it appears wome n have poorer short term and long term outcomes following these events compared to men.

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14 Autoimmune disorders may contribute to the gender based differences in cardiac outcomes; for example, RA, far more common in women, is associated with a higher prevalen ce and higher severity of atherosclerosis [69] Autoimmune diseases are the third most common category of disease in the United States after cancer and heart disease. Male and female immune response differs; women mount more vigorous immune responses with increased antibody production [70] Women also have far higher autoimmune disease rates [71, 72] When men develop autoimmune diseases they are often more severe Many animal models of autoimmune d isease have shown a similar sex bias, with a higher incidence of disease in females [73 77] E strogen, testosterone, and progesterone are thought to mediate most of the sex biased differ ences in the immune response [78, 79] Cytokine receptors such as interleukin 1 receptor ( IL 1R ) have been discovered on hormone producing tissues P roinflammatory cytokines such as IL 1 glucocorticoids which regu lates the inflammatory process along wi th androgens and estrogens. E strogen significantly increases proinflammatory cytokine production of IL 6 [6, 27, 29, 30, 80 82] Interleukins act specif ically as mediators between leuk ocytes. Activated leukocytes can generate ROS, which contributes to hyperten sion and atherosclerosis [83] Many autoimmune disorders are strongly associated with cardiac and metaboli c disorders. W omen tend to have slightly higher serum CRP than men with the same body mass index (BMI) and age; however, these differences are not pronounced enough to warrant gender dependent reference ranges [84, 85] The role of gender in predisposition

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15 to oxidative stress has yet to be determined. CRP is an acute phase inflammatory marker considered to be a reliable prognostic indicator of atherosclerosis and acute myocardial infarction (AMI) [61, 82, 86] Urinary levels peak approximately 24 hours following the inflammatory event and are und etectable within 13 to 16 hours As a measure of leukocyte infiltration, MPO is an inflammator y marker used to predict the risk of AMI in the absence of cardiac necrosis [54, 87] The predictive value of this marker has not been fully characterized; however, it is associated with long term adverse cardiac events. E 2 has been suggested as a poten tial endogenous substrate for MPO in plasma [88] Recognition and elaboration of the innate differences of gender physiology may result i n better treatment adaptation s for women and better outcomes [89] 2.2 Inflammation Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response to harmful stimuli and is achieved by mobilization of plasma and leukocytes, particularly granulocytes, from the blood into the injured tissues. It is characterized by vasodilation, increased permeability and reduced blood flow; these changes are induced by multiple inflammatory mediators [90] Trauma, inflammation, or infection leads to the activation of the inflammatory cascade. A progression of biochemical events including components of the local vasculature, the immune system, and various cells triggers the inflammatory response. Unlike acute inflammation, chronic

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16 inflammation is mediated by cells such as monocytes and lymphocytes. C hronic i nflammation leads to a progressive shift in the type of cells present at the site of infl ammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process [91] Tissue macrophages, monocytes, mast cells, platelets, and endothelial cells are able to produce a multitude of cytokines. The release of as IL leads to cleava ge of the nuclear factor kB (NF kB) inhibitor. Removal of the inhibitor allows NF kB to initiate mRNA production thereby inducing production of other proinflammatory cytokines like IL 6. IL 1 is responsible for fever and the release of stress hormones (norepinephrine, vasopressin, activation of the renin angiotensin aldosterone system). Cytokines like IL 6 stimulate the release of acute phase reactants such as CRP. Infection has been show n to elicit a stronger response than trauma, which translates to a greater release of IL 6. Estrogen has been shown to directly inhibit IL 6 production [92] Inflammation is a highly regulated process that recruits the immune system to sites of infection and injury and to facilitate tissue repair processes. Prolonged inflammation produces local and systemic damage associated with a loss of norma l physiological functions. Activation of proinflammatory cytokines results in vasodilatation, release of cytotoxic compounds like MPO, generation of ROS and damage to the vasculature. Proinflammatory cytokines like IL 6 upregulate endothe lial enzyme expression which play s an important role in atherogenesis. Some autoimmune diseases, such as RA, SLE and Sjogren's syndrome are characterized by chronic inflammation.

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17 Chronic i nflammation is a crucial element in the development of multiple disease processes, including renal dysfunction, cardiovascular disease autoimmune disorders, cancer and DM [53, 91] Assessment of inflammatory markers like CRP, MPO and the interleukins can lead to improved risk stratification in disease Estrogen and testosterone can affect the expression of proinflammatory markers in macrophages T estosterone may reduce the e xpression and secretion of IL but it does not appear to affect the expression of IL 6 or CRP Estrogen elicits a vari able response in CRP expression [93] Aldoste rone causes tissue inflammation; this may result in fibrosis and remodeling in the heart, vasculature, and kidney [94] Aldosterone triggers endothelial cell exocytosis, which is a crucial step in leukocyte mobilization [95] D uring active in metabolic shifts may cause in hypoxia in affected membranes Activation of hypoxia inducible factor (HIF) subsequent to hypoxia has been shown to upregul ate production of hsp90 and VEGF [96] VEGF increases endothelial permeability and is associated with microalbuminuria and myoglobinuria [97] 2.3 Oxidative stress Oxidative stress is determined by the balance between the rate at whi ch oxidative damage in a cell, tissue, or organ is induced and the rate at which it is repaired or removed Inactivation of ROS by endogenous antioxidants like superoxide dismutase and

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18 glutathione peroxidase determines the rate at which damage is induced [67, 9 8] Repair enzymes determine the rate at which damage is repaired Most ROS come from the endogenous sources as by p roducts of normal and essential metabolic reactions. Lifestyle and environmental exposures also contribute to ROS. While there are many biomarkers of inflammation, oxidative stress is difficult to measure in vivo. Traditional indices of oxidative stress i nclude markers of oxidative damage to lipids, proteins and DNA. Oxidative stress is also indirectly assessed by me asuring markers of inflammation such as MPO [99] ROS include free radicals and peroxides such as hydrogen peroxide, hypochlorous acid and peroxynitrite When converted by oxidoreduction reactions, superoxid e can be transformed into more aggressive radical species capable of causing extensive cellular damage. Long term effects of ROS exposure can include damage to DNA. Oxidative stress is implicated in diseases such as atherosclerosis, Parkinson's disease, he art failure, myocardial infarction, Alzheimer's disease, fragile X syndrome and chronic fatigue syndr ome Oxidative stress is linked to certain cardiovascular disease s; oxidation of low density lipoprotein in the vascul ar endothelium is a precursor of athe rosclerotic plaque formatio n. Oxidative stress plays a role in the ischemic cascade due to oxygen reperfusion injury following hypoxia. It is also associated with the detrimental effects of aging. Aging induces a pro inflammatory state characterized by in creasing levels of inflammatory cytokines such as IL 6 [100, 101] A ging is ass ociated with a declining serum testosteron e levels. Studies suggest a close relationship exists

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19 between the development of a pro inflamm atory state and the decline in testosterone levels, two trends that are often observed in aging men. Additionally, E2 is weakly associated with IL 6 in older men, independent of testosterone. Myoglobin is a molecular radical scavenger activated by ischemia and myolysis; serum and urine myoglobin concentrations increase with OS [33, 102] Creatinine conce ntrations are associated with muscle mass. Activity of creatinine and myoglobin correlate to stress induced neutrophil response common in exertional and injury induced rhabdomyolysis [103] Aldosterone is linked to generation of ROS and the induction of oxidative stress [104, 105] Atrial natriuretic peptide (ANP) is a hormone, primar ily produced by cardiomyocytes, which regulate s blood pressure. Studies have linked ANP with the generation of ROS ANP may produce either antioxidant or prooxidant effects, depending on experimental conditions and cell context [106] ROS generation occurs in hepatocytes and in the respiratory burst of Kupffer cells, triggering a proinflammatory casc ade upregulating multiple interleukins. S pecific receptors in hepatocytes stimulate the exp ression of antioxidant enzymes such as manganese superoxide dismutase and acute phase protein s These responses help protect the liver a gainst ischemia reperfusion i njury [10 7] OS plays an important role in pathogenesis of hepatic diseases like alcoholic liver injury. Cardiac surgery associated acute kidney injury (AKI) is common and is associated with increased expression of NGAL [105] Increased OS is considered a causative factor of DM, particularly diabetic cardiovascular and renal sequelae. Hyperglycemia is thought to induce the overproduction of ROS. Oxidative stress is also a factor in the pathogenesis of

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20 hypertension and target organ damage, largely due to its effects on the vasculature Oxidative metabolites of cocaine generate significant ROS; much of the pathophysiological damage associated with cocaine use stems from oxidative stress and electron transfer [32] 2.4 Cocaine C o caine C 17 H 21 NO 4, is a potent cardiac and central nervous system stimulant It is fat soluble and freely crosses the blood brain barrier. Cocaine is rapidly hydrolyzed by serum cholinesterase in plasma and carboxylesterases in the liver following use; the primary metabolites are BE and EME (Figure 2 ) Lesser metabolites include cocaethylene (CE) norcocaine and anhydrous ecgonine methyl ester (only seen in crack cocaine use) Cocaine and ethanol are independently cardiotoxic; together they exhibit synergistic cardiotoxicity. In the presence of alcohol, a nonspecific carboxylesterase catalyzes ethyl transesterification of cocaine to CE. Animal studies have demonstrated that co a dministration results in prolonged cardiac toxicity and dysrhythmias [108] It has also b een demonstrated that ED patients with detectable CE concentrations are more likely to be admitted to intensive care units than those patients testing negative for CE [109] The half lives of cocaine, CE and BE are 40 minutes, 2.5 hours and 5 to 8 hours respectively. This may explain why cocaine related symptoms can continue for some time after cocaine is last used. Cocaine has only one biologically active metabolite,

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21 norcocaine. The oxidative metabolism of cocaine to norcocaine nitroxide is postulated to be essential for cocaine hepatotoxicity [110] Cocaine induces local vasoconstriction and an anesthetic effect by inhibiting fast sodium channels. Cocaine is a strong sympathomimetic that blocks catecholamine reuptake, effectively flooding the synaptic space with norepinephrine thereby stimulating the central and peripheral nervous systems [56] The effect of cocaine is especially pronounced in the limbic system where it potentiates dopaminergic transmission in the Figure 2. Metabolites of Cocaine

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22 ventral basal nuclei. The potentiation of dopamine is responsible for the pleasurable behavioral effects that make cocaine a popular drug of abuse C ocaine causes moderate release and reuptake blockade of serotonin and dopamine [111 113] Cocaine induced hyperpyrexia, a potentially fatal elevation in body temperature exceeding 41 C, is thought to be associated with dopamine receptor blockade in the temperature regulation region of the hypothalamus. The sodium channel blockade decreases the resting membrane potential a nd action potential amplitude while simultaneously prolonging the duration of the action potential. Cocaine also blocks potassium channels. In some cellular membranes, it may block sodium calcium exchange. Almost every organ system is affected by cocaine use. My ocardial infarction, arrhythmia s, renal failure, hypertension, atherosclerosis, and rhabdomyolysis are all in the spectrum of acute and chronic cocaine toxicity [114, 115] Cocaine is associated with an increased vascular risk; the brain, heart, kidney, liver and lungs are all susceptible to i ts vasculotoxicity It can cause abrupt changes in blood pressure, embolism via infective endocarditis, and hemostatic and hematologic abnormalities that can result in increased blood viscosity and platelet aggregation. Most ED patients with cocaine associated chest pain have normal cardiac profiles at the time of presentation. The n egative inotropic effects of cocaine observed in animal models do not appear to be present in patients who develop chest pain after using recreational doses of cocaine [116] Cocaine is known to have specific, dose dependent effects on brain and body temperatures, th ese effects are strongly modulated by an individual's activity state and environmental conditions, and change dramatically during the development of drug self

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23 administration. Environmental conditions potentiate the thermal effects of cocaine which may resu lt in pathological brain overheating. Hyperthermia can exaggerate the toxicity of cocaine; environmental conditions that impair heat loss can result in acute life threatening complications and chronic destructive CNS changes [117] Deleterious effects of hyperthermia include activation stress response systems, decreased immune efficiency, increased blood viscos ity, vasoconstriction, renal and hepatic insufficiency and rhabdomyolysis. Rhabdomyolysis is a disorder in which injury to muscle results in leakage of myocyte intracellular contents into the plasma. Dissolution of muscle fibers and leakage of muscle enzy mes, myoglobin, potassium, calcium, and other intracellular constituents, can occur. For this reason, m yoglobinuria and albuminuria are common clinical markers of rhabdomyolysis. Serum proinflammatory and inflammatory cytokines like IL RP are similarly elevated. Cocaine has been shown to diminish IL 6 response to proinflammatory challenges [118] IL 6 is a multifunctional cytokine implicated in many age related dis eases, including postmenopausal osteoporosis and [119] The most likely etiology of rhabdomyolysis in patients presenting to the emergency department is ingestion of drugs of abuse such as cocaine [58] The hemodynami c actions of cocaine contribute to renal injury. Cocaine abuse has been linked with acute renal failure and acid base and/or electrolyte disorders and may trigger the progression of chronic renal failure to end stage renal disease. Cocaine use is associat ed with frontal cortex and glial injury in both frontal gray and white matter. Women showed equivalent responses to drug administration with the

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24 exception of the limbic system; their perception of well being was significantly increased [120] These findings may have imp lications for differential risk for acute and chronic toxicity in women. Some animal studies suggest estradiol increases sensitivity of the brain reward system [121] Progesterone may enhance cocaine craving and relapse susceptibility in women [122] Research also suggests adolescents are more sensitive than adults to interaction s between testosterone and cocaine [123] There are significant gaps in the literature that make it difficult to define the relationship between gender and cocaine. Social parameters have been described; males are more lik ely than females to use cocaine and males tend t o use cocaine at an earlier age than do fema les. The pathophysiological gender based differences cannot be elaborated until study populations begin to include females. The bulk of research performed in recent years has focused on psychosocial and neurobiol ogical differences between cocaine using females and males [124 126] Given the stark physical contrasts between the genders, particularly in immune response and autoimmunity, the focus may need to be widened to include gender based differences in the inflammatory and oxidative response to cocaine use.

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25 Chapt er 3 .0 Materials and Methods The Agency for Community Testing Services (ACTS) Laboratories provided urine specimens with all information blinded apart from gender, creatinine and the results of the NIDA 10 drug screen. 41 specimens were donated by males, 39 specimens by females. 40 specimens tested negative to all substances on the NIDA 10 panel (THC, opiates, amphetamines, barbiturates, cocaine, ethanol, benzodiazepines, propoxyphene, methadone and oxycodone) and 40 specimens tested positive for cocaine metabolites alone. Limited demographic information was available for some of the donors; for the mean donor age was 34 years old. Donor weight was available for 46 of the 80 donors. The female weights ranged from 113 to 327 lbs and the mean female donor weight was 162.5 lbs. The male weights ranged from 120 to 240 lbs and the mean male donor weight was 166.6 lbs.

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26 Table 1. Summary of Assays Performed Analyte Method Manufacturer Total Protein Colorimetric Pierce Creatinine Colorimetric Cayman Cocaine/Benzoylecgonine (BE) ELISA Immunalysis Aldosterone ELISA ALPCO C Reactive Protein (CRP) ELISA ALPCO Myeloperoxidase (MPO) ELISA ALPCO Microalbumin (MAB) ELISA ALPCO Myoglobin (MGB) ELISA Life Diagnostics Heat Shock Protein 90 90 ELISA Assay Designs Vascular Endothelial Growth Factor (VEGF) ELISA Raybiotech Neutrophil Gelatinase Associated Lipocalin (NGAL) ELISA Quantikine Pro Atrial Natriuretic Peptide (proANP) ELISA ALPCO Interleukin 1 1 ELISA Cayman Interleukin 1 1 ELISA Cayman Interleukin 6 ELISA Cayman

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27 3.1 EMIT Enzyme Multiplied Immunoassay Technique (EMIT) is a common method for screening urine and blood for drugs, legal and illicit. This inexpensive technique is fast, relatively sensitive and specific, and has been adapted to various automated analyzers. The non linear relationship between the change in absorbance and analyte/drug concentration in the specimen is the rea son the assay is only useful for qualitative results for cutoff value calibrators to indicate a positive or negative result. This qualitative procedure may be followed with c onfirmator y (quantitative) GC/MS testing as necessary. An EMIT drug test contains antibodies that target specific drugs in the urine specimen. If an antibody does not become attached to the drug in the urine specimens, it attaches itself to the chemicall y tagged drug in the EMIT reagent. ACTS uses DRI/Microgenics (Thermo Fisher Scientific Corporation, Waltham, MA 02454) reagents and controls for the NIDA 10 EMIT panel. The NIDA 10 panel consists of Cocaine (COC), Amphetamine (AMP), Methamphetamine (M AMP) Tetrahydrocannabinol (THC), Methadone (MTD), Opiates (OPI), Phencyclidine (PCP), Barbiturates (BAR), Benzodiazepines (BZD) and Oxycodone (OXY) in human urine. Chemically tagged drugs without an attached antibody ( i.e. the reagent) are transformed by a r eaction that changes the absorbance of the test sample. Attachment of an antibody inhibits this reaction. The Olympus AU640e Chemistry ImmunoAnalyzer (OLYMPUS America Inc.,

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28 Center Valley, PA 18034 0610) used by ACTS Laboratory measures the changes in absor bance in the test sample; the change in absorbance is then compared to the absorbance of the calibrators. If the absorbance of the test sample is equal to or exceeds if the absorbance of the test sample is less than that of the calibrator, the specimen is considered negative for the drug. In 1988, the American Association for Clinical Chemistry (AACC) conducted a study of the testing accuracy of laboratories with reg ard to drugs of abuse. This study was blinded and used cutoff concentrations very close to the guidelines of the Substance Abuse and Mental Health Services Administration (SAMHSA). Their overall accuracy rate was 97%. There were a small number of false ne gative results (2 4%) on samples with drug concentrations close to the cutoff. There were no false positive results. Components of the EMIT Assay Method (Figure 3) : 1. Drug 2. Antibody 3. Substrate 4. Drug enzyme complex

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29 Figure 3 EMIT Assay Components in Action 3.1.1 General Procedure for EMIT Assay 1. Mix sample containing drug with fixed quantity of enzyme bound drug, and antibody 2. Add substrate 3. Measure absorbance at 15 and 45 seconds after substrate addition 4. Quantitate by measuring enzyme substrate reac tion (by UV visible spectroscopy) 5. 6.

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30 7. Determine standard curve ACTS laboratory uses the Olympus AU640e for drugs of abuse analysis (Fig ure 4). The analyzer screens urine specimens for the NIDA 10 panel of drugs. The cocaine test analyzes urine specimens for the presence of the primary cocaine metabolite, benzoylecgonine, rather than the parent compound (cocaine). As previously stated, EMI T does not measure the amount of the drug present; it simply detects its presence or absence as determined by an established cut labeled with an enzyme such as glucose 6 phosphate dehydrogenase (G6PDH). The enzyme ligand antibody complex is inactive thereby allowing determination of unlabeled ligand.

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31 Figure 4 Olympus AU640e Chemistry Immuno Analyzer 3.1.2 Cocaine/Benzoylecgonine 3.1.2.1 General Principle of the Assay The DRI Cocaine Metabolite Assay used (DRI Cocaine Metabolite Assay #0056, Microgenics Corporation Fremont, CA 94538) by ACTS Laboratory is intended for the qualitative and semiquantitative determination of benzoylecgonine in human urine. This assay provid es only a preliminary analytical test result. Quantitative determination

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32 requires alternative methods; gas chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method. The DRI Cocaine Metabolite Assay is a homogeneous enzyme immunoassa y using ready to use liquid reagents. The assay uses a specific antibody to detect benzoylecgonine in urine specimens. The assay is based on the competition of an enzyme glucose 6 phosphate dehydrogenase (G6PDH) labeled BE and any BE from the urine sample for a finite number of specific antibody binding sites. In the absence of BE in the sample, the specific antibody binds to the G6PDH labeled BE and the enzyme activity is inhibited. This means there is a direct relationship between the BE concentration in the urine and the enzyme activity. The enzyme G6PDH activity is determined by measuring its ability to convert nicotinamide adenine dinucleotide (NAD) to NADH Immuno Analyzer. 3.1.2. 2 Specificity Benzoylecgonine, cocaine and other compounds that are concurrently present in the urine were tested for cross reactivity in the assay. Cocaine and EME are the only compounds that demonstrated crossreactivity. Urine specimens arrive in steril e containers from a number of remote collection sites, including jails, hospitals and medical offices. Possible adulteration of urine specimens is an important consideration for drug testing facilities. Accordingly, samples undergo several tests to determi ne the likelihood of adulteration. Color and odor are assessed for evidence of adulteration, either by dilution

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33 or the addition of an oxidant such as bleach. The tests used to check for adulteration include creatinine concentration, pH and specific gravity Urine specimens with values outside normal reference ranges are discarded. The reference ranges for: Urine creatinine is 25 350 mg/dL Urine pH is 5.0 to 6.5 Urine specific gravity is 1.003 to 1.030 Dilution of the specimen may result in abnormal lows for one or more of the markers. In some instances the specimens are highly concentrated and dilution may make creatinine levels normal. Substitution with a liquid other than urine will be revealed by the absence of creatinine. Commercial masking agents may include components such as glutaraldehyde to interfere with the drug screen; for this reason, nitrites levels are occasionally used to establish the legitimacy of a specimen. This is not foolproof as bacterial infection and contamination may increase urin e nitrite levels. 3.1.3 Creatinine Creatinine is routinely included in urinary analysis as a control for dilute or adulterated specimens. It is a metabolic by product of creatine degradation in muscle tissue. Most methods for determining the creatinine c oncentration of a specimen, in urine

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34 or serum, rely on the Jaffe reaction in which creatinine reacts with alkaline picrate to form a red yellow solution that is then measured photometrically (Figure 5 ). NaOH Creatinine + Picric Acid Ja novski Complex Figure 5 Modified Jaffe Reaction Since urine contains other substances which can react with picrate, the Olympus uses reagents from Microgenics for a modified version of the Jaffe reaction with improved specificity. The color intensity of the creatinine picric acid complex is directly proportional to the concentration of creatinine in the urine specimen and is measured spectrophotometrically at 505 nm. Each laboratory establishes its own reference range for creatinine levels but they avera ge from 25 to 300 mg/dL. When the value obtained exceeds 300 mg/dL, the specimen should be diluted with physiological saline and re assayed. The established cutoff value for creatinine is 20 mg/dL and the assay is said to be linear to 400 mg/dL. 3.1.4 Specific Gravity Specific gravity simply refers to the dissolved solutes present in urine as compared to the value set by convention, for pure water (1.000). Values obtained for

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35 specific gravity are affected by the number, size and weight of molecules in u rine. For this reason, it is considered an approximation of solute concentration. The urine specific gravity assay relies upon the use of a linear relationship between urinary chloride ion concentration and measured specific gravity. Determination of the c hloride ion is colorimetric; in an aqueous medium, ferric perchlorate and chloride ion form a ferrous chloride complex that has an absorbance maximum of 340 nm (Figure 6 ). Absorbance at 340 nm is directly proportional to concentration of chloride ion in the specimen. Urine specific gravity is extrapolated from this value. Cl + Fe 3+ FeCl 2+ Figure 6 Urine Specific Gravity Reaction Equation 3.1.5 Ethanol The ethanol assay is based on the high specificity of alcohol dehydrogenase (ADH) for ethyl alc ohol. In the presence of ADH and nicotinamide adenine dinucleotide (NAD), ethanol is readily oxidized to acetaldehyde and NADH (Figure 7 ). The enzymatic reaction is monitored spectrophotometrically at 340nm. The ethanol in the sample is in direct proporti on to the rate of change of absorbance at 340 nm.

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36 Figure 7 Oxidation of Ethanol to Acetaldehyde 3.2 Colorimetric Analysis Colorimetry relies on colored solutions absorbing light of a particular wavelength; however the technique can also be used to a nalyze colorless substances if they react with a dye. The TECAN M200 Infinite Microplate Reader (TECAN US Inc., Durham NC 27703) can be used for simple colorimetric analyses such as the creatinine assay and total protein assessment. The absorbance of the u rine specimens is determined by comparing them to a calibration curve generated from known standards.

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37 3.2.1 Total Protein 3.2.1.1 General Principle of Assay The Coomassie Protein Assay kit used (Coomassie Protein Assay Kit #23200, Pierce Biotechnology, Rockford, IL 61105) is a typical colorimetric assay. The Pierce Coomassie Protein reagent is a modification of the Bradford Coomassie Dye protein binding colorimetric method of protein quantitation. When Coomassie dye binds to protein in an acidic medium, the color in the well s changes from brown to blue as the absorbance maximum shifts. f Coomassie Reagent in micro plate wells for 10 minutes. The plate is read at 595 nm to obtain the absorbance. 5 concentration of protein in the sample. A set of standar ds is used to plot a standard curve from which the amount of total protein in samples and controls can be directly read. 3.2.1.2 Assay Procedural Details Kit Contents: 1. Coomassie Protein Assay Reagent ready to use 2. Albumin Standard Ampules 2mg/ml re quires serial dilution

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38 Phosphate Buffered Saline (PBS) is the diluent chosen for the assay. PBS is a commonly used isotonic buffer solution in biological research. The osmolarity and ion concentrations of the solution usually match those of the human body fluids. The 1x PBS stock solution used for the Total Protein Assay was prepared by dissolving 8.00 g of NaCl, 0.20 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 in 800 ml of Barnstead Nanopure Water. If necessary, the pH can be calibrated to 7.4 with th e addition of HCl or NaOH. The final volume is adjusted to 1 liter with additional Barnstead Nanopure Water. The albumin standards were prepared as described in the table below (Table 2) : Table 2. Concentration of Albumin Standards Tube Volume of Diluent Volume and Source of BSA Final Concentration A 0 300 of stock 2000 B 125 375 of stock 1500 C 325 325 of stock 1000 D 175 175 of tube B 750 E 325 325 of tube C 500 F 325 325 of tube E 250 G 325 325 of tube F 125 H 400 100 of tube G 25 I 400 0 0 (blank)

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39 All reagents must reach room temperature before use. Mix the Coomassie Reagent immediately prior to use by gently inverting the bottle several times. Calibrators, controls and specimen samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. correspondingly labe pipetted into each well. The plate was incubated on a plate shaker (approximately 200 rpm) for 10 minutes at room temperature. The plate was read at 595 nm to calculate the absorbance. A standard curve was produced and the concentration of protein in each of the specimens was determined from the blank adjusted absorbance. 3.2.1.3 Interfering Substances Most ionic and nonionic detergents 3.2.2 Creatinine 3.2.2.1 General Principle of Assay The Creatinine kit used (Creatinine Assay Kit #500701, Cayman Chemical Company, Ann Arbor, MI 48108) is a typical colorimetric assay. It is not appropriate for a yellow/orange color is formed when the metabolite is exposed to alkaline picrate. The color derived from creatinine is

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40 destroyed at acidic pH. The difference in the color before and after acidification is measured at 500 nm and is proportional to the cre atinine concentration in the urine specimen. Picrate Solution in micro plate wells for 10 minutes. The plate is read at 500 nm; the incubating for 20 minutes, the Final Absorbance is obtained by reading the plate at 500 nm. The difference between the initi al and final absorbance is directly proportional to the concentration of creatinine in the sample. A set of standards is used to plot a standard curve from which the amount of creatinine in samples and controls can be directly read. The dynamic range of th e Cayman Creatinine assay is 0 15 mg/dl. 3.2.2.2 Assay Procedural Details Kit Contents: 1. Creatinine Standard requires dilution 2. Creatinine Color Reagent (1.2% picric acid) ready to use 3. Creatinine Sodium Hydroxide (0.1M NaOH) ready to use 4. Creatinine Acid Solution ready to use

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41 5. Creatinine Sodium Borate ready to use 6. Creatinine Surfactant ready to use To prepare Alkaline Picrate Solution sufficient for a 96 well plate, mix together 2 ml sodium borate, 6 ml surfactant, 10 ml color rea gent and 3.6 ml NaOH. This resultant solution is stable for one week stored in the dark at room temperature. Urine specimens were diluted 1:20 with Barnstead Nanopure water prior to use. The creatinine standards were prepared as described in Table 3 : Table 3. Concentration of Creatinine Standards Tube Creatinine Standard Nanopure Water Final Concentration (mg/dl creatinine) A 0 500 0 B 50 450 2 C 100 400 4 D 150 350 6 E 200 300 8 F 250 250 10 G 300 200 12 H 375 125 15

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42 All reagents must reach room temperature before use. Calibrators, controls and specimen samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. ed cocaine positive urine alkaline picrate solution was pipetted into each well to initiate the reaction. The plate was incubated on a plate shaker (approximately 200 rpm) for 10 minutes at room solution was added to all wells and the plate was incubated for 20 minutes on a plate shaker at room temperature. The final absorbance was calculated by reading the plate at 500 nm. A standard curve was produced and the concentration of creatinine in each of the specimens was determined from the adjusted absorbance. 3.3 ELISA Immunoassays are biochemical tests that measure the level of a su bstance in a biological liquid, in our case urine. The assay utilizes the specific binding relationship of antibody and antigen. Monoclonal antibodies are often used because they typically bind a single site of a particular molecule; this property enhances specificity and accuracy. Polyclonal antibodies have also been successfully applied for various immunoassays.

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43 Assay success depends on antibody affinity for the antigen. It is possible to measure both the presence of antigen and antibodies. Enzyme linked Immunosorbent Assay (ELISA) uses antibodies (or antigens) coupled with an enzyme to produce an antibody specific assay with the sensitivity of simple enzyme assays. This allows an ELISA to provide a useful measurement of antigen or antibody concentration. ELISA plates consist of 96 or 480 polyvinyl chloride microwells containing enzymes, antigens or antibodies immobilized on a solid phase and the substance. Depending on the type of ELISA, an antibody capable of detecting an antigen or an antigen that will elicit the response of a particular antibody is allowed to bind to the immobilized protein. There are 5 types of ELISA: direct, indirect, sandw ich, competitive and multiplex: 1. Direct ELISA: This method directly labels the antibody. The target antigen is applied to the microwells of an ELISA plate; bound antigen may be quantitated using colorimetric, chemiluminescent or fluorescent measurement. This method provides relatively quick results with limited cross reactivity. There is little opportunity for sig nal amplification with this method. 2. Indirect ELISA: This two step method uses a second antibody for detection. The primary antibody is incubated with the antigen; the specimens in the wells then incubate with a second labeled antibody capable of recognizing the primary antibody. This method allows for considerable signal amplification.

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44 between two layers of antibodies. The sandwich ELISA requires the antigens to be measured contain two or more antigenic sites capable of binding the capture and detection antibodies; therefore, these assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs are excellent for quantita ting antigens when the concentration of antigens is low or the concentration of contaminating protein is high. Briefly, the capture antibody is purified and bound to a solid phase (usually attached to the bottom of a plate well). Specimens thought to cont ain the antigen of interest are added to the microwells and allowed to complex with the capture antibody. The plates are washed to remove unbound products and the labeled detection antibody is allowed to bind to the antigen. This completes the The assay is quantitated using a colorimetric substrate to measure the amount of detection antibody bound to the matrix. 4. Competitive ELISA: This method requires one reagent be conjugated to a detection enzyme (horseradish peroxidase is one of the most commonly used). This detection enzyme may be linked to the antigen or the primary antibody. The microwells of the assay plate are coated with a purified unlabeled primary antibody. Following this, the plate is incubated with unlabeled standards and unknown s. The reaction is allowed to proceed to equilibrium; labeled conjugate antigen is then added to the microwells. The conjugate will bind to the available binding sites on the primary antibody. The plate is then developed with substrate and color change is measured. In a competitive ELISA, there is an inverse

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45 relationship between the signal obtained and the concentration of the analyte in the sample i.e. the more unlabeled antigens in the sample or standard, the lower the amount of conjugated antigen bound. 5. Multiplex ELISA: This assay allows simultaneous detection of multiple analytes arrayed in a single well. Detection method can be direct or indirect, sandwich or competitive, labeling or non labeling, depending upon antibody array technologies. There a re further assay variations depending upon the labeling and signa l detection methodology The basic approach stays the same: fixation of either antigen or antibody and detection of the antibody antigen complex. The general steps in direct/competitive ELISA s can be seen in Figure 8. Step 1: The antibody is attached to the walls of the microtiter plate.

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46 Step 2: Urine is added to the well to test for the presence of the antigen Step 3: If the antigen is present in the urine, it will bind to the antibodies attached to the microtiter walls. Step 4: The plates are rinsed to remove test fluid and unbound antigen. Step 5: A solution of modified antibodies is added. These antibodies carry a reporter enzyme designed to elicit a color change when the antigen antibody complex forms.

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47 Step 6: The sample is rinsed again to remove unbound antibodies. If the antigen is present, a complex consisting of antigen, plate bound antibody and enzyme conjugated antibody forms. Step 7: The substrate of the enzyme is added. A color change indicates the presence of the enzyme labeled antibody and the antigen. Figure 8 Common Steps in Direct/Competitive ELISA Assay ELISAs were performed using a TECAN M200 Infinite Microplate Reader (TECAN US Inc., Durham NC 27703). The TECAN M200 (Figure 9 ) is an automated microplate reader is capable of UV & VIS Absorbance (ABS), Fluorescence (FL) Intensity Top with Time Resolved Fluorescence, Fluorescence Resonance Energy Transfer, Fluorescence Intensity Bottom, Spectrally Enhance d PMT, Photon counting Luminescence, Temperature Incubation and Cuvette Port Module, Single and Dual Injectors. The M200 can measure wavelength range of in tunable 1nm increments, Injector Module for fast kinetics in FL, Abs or Luminescent modes with one o r two injector(s). Available features include heated incubation to 420C, linear and orbital shaking. The M200 accommodates 6, 12, 24, 48, 96 and 384 well microplate formats. It is packaged with i Control and Magellan software for instrument measurement con trol,

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48 spectral scanning, plate definition creation and raw data acquisition saved in a MS Excel format. Figure 9 TECAN M200 Infinite Microplate Reader A monochromator is an optical instrument that enables any wavelength to be selected from a defined op tical spectrum. It operates like a tunable optical filter, allowing both the wavelength and bandwidth to be adjusted. A monochromator consists of an entrance slit, a dispersive element and an exit slit. The dispersive element diffracts the

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49 light into the o ptical spectrum and projects it onto the exit slit. The dispersive element may be a glass prism or an optical grating. The infinite M200 is designed with optical gratings. Rotation of the optical grating moves the spectrum across the exit slit so that onl y a small part of the spectrum (band pass) passes through the exit slit. This means that when the monochromator entrance slit is illuminated with white light, only light with a specific wavelength (monochromatic light) passes through the exit slit. The wav elength of this light is set by the rotation angle of the optical grating and the bandwidth is set by the width of the exit slit. The bandwidth can be defined as full width at half maximum (FWHM). Monochromators block undesired wavelengths. This means when the monochromator is set for light with a wavelength of 500 nm and the detector detects a signal of 10,000 counts, light with different wavelengths is effectively dampened. Double monochromators setups permit a higher level of blocking; two monochromators are connected in series so that the exit slit of the first monochromator acts as the entrance slit of t he second monochromator This arrangement boosts the blocking count by a factor of 10 3 In the infinite M200, a double monochromator is installed on bot h the excitation and detection side. This opens the opportunity for easy selection of excitation and fluorescence wavelengths with no limitations by cut off filters. The i control and Magellan software packaged with the TECAN M200 make operation of the mic roplate reader relatively simple (Figure 10 ). The user is able to select

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50 Figure 10 I Control/Magellan User Interface from a variety of common microplate conformations. The injectors can be programmed to automate reagent injection at specific stages of the assay, reducing operator error such as faulty pipetting. The operator can set the absorbance wavelengths, measured and reference, and determine the read pattern in the same panel. The Magellan software is a wizard based interface; users can start a mea surement wizard, evaluate results wizard, create and edit sample ID lists wizard, and create and edit methods (analysis protocols) wizard. Users can edit plate geometry to design the assay that suits their research. Results

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51 can be displayed as absorbance o r concentration (calculated from the standard curve/calibrator data) in each microwell (Figure 1 1 ). Results can also be displayed as averages when specimens are assayed in duplicate. Figure 11 Typical Magellan Microwell Absorbance Results

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52 Figure 12 TECAN Columbus Strip Washer Use of an automated plate washer enhances assay accuracy by ensuring complete well aspiration and washing. The laboratory used the Columbus Strip Washer (Figure 12 ). It is a fully automated microplate strip washer for 96 and 384 well plates. Its features include manifolds of 8, 12 or 16 channels, crosswise aspiration, overflow and bottom washing. Assay protocols can either be programmed on board or via PC interface using the WinWash software. Thirty distinct methods can be pr ogrammed and stored simplified operation. A liquid sensor system monitors the liquid level of waste, wash buffer and rinse solution. Rinsing is fully automated, ensuring easy maintenance and operation.

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53 3.3.1 Cocaine/Benzoylecgonine 3.3.1.1 General Principle of Assay The principle of the Cocaine/Benzoylecgonine ELISA Kit used (Cocaine/Benzoylecgonine Direct ELISA Kit #212 0480, Immunalysis Corporation, Pomona, California 91767) is based upon the competitive binding to an tibody of enzyme labeled antigen and unlabeled antigen, in proportion to their concentration in the reaction mixture. Unmetabolized cocaine urine concentration is far lower than that of its major metabolite BE. Cocaine is undetectable (at a 50 ng/ml cut of f) 12 hours after administration in comparison with BE which persists for up to 48 hours after use. ). It has been suggested that a BE/cocaine ratio of less than 100 is indicative of use within the past 10 hours. Results are expressed in BE equivalents per ml. horseradish peroxidase labeled BE derivative in micro plate wells, coated with fixed amounts of oriented high affinity purified polyclonal antibody. The wells are washed 6 times thoroughly and a chromogenic substrate added. The color produced is stopped using a dilute acid stop solution and the wells read at 450 nm. The intensity of the co lor developed is inversely proportional to the concentration of drug in the sample. The technique is sensitive to 1 ng/ml. The precision of the Immunalysis Cocaine/Benzoylecgonine Direct ELISA Kit has been verified by assessment of the mean, standard devia tion (SD) and coefficients of variation (CV) in data resulting from repetitive assays. Assay sensitivity based on the

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54 minimum BE concentration required to produce a four standard deviation from assay Ao is 1 ng/ml. 3.3.1.2 Assay Procedural Details Kit con tents: 1. 96 well micro plate coated with polyclonal anti benzoylecgonine and polyclonal anti cocaine 2. Cocaine/Benzoylecgonine Enzyme Conjugate solution 3. Positive Reference Standard containing 50 ng/ml of BE dissolved in a synthetic urine 4. Negative Standard 5. TMB chromogenic substrate 6. Stop Reagent containing 1 N hydrochloric acid All reagents were brought to room temperature prior to use. Positive reference standards of 0, 10, 25 and 50 ng/ml were prepared. Dilution of the urine specimens wit h dded to

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55 each well. After tapping the sides of the plate holder to ensure proper mixing, the plate was incubated in the dark for 60 minutes at room temperature. water. Following incubat ion, the microplate was inverted and slapped vigorously on absorbent paper to ensure all residual moisture was removed. This step was critical to well and the sides of pl ate holder tapped to ensure proper mixing. The plate was added to each well, changing the blue color to yellow. Absorbance was measured at a dual wavelength of 450 nm and 650 nm. A standard curve was produced and the concentration of BE in each of the specimens was determined. 3.3.1.3 Cross Reactivity Aliquots of a human urine matrix were spiked with the following compounds at a concentration of 5000 ng/ml. None of these compounds gave values in the assay that were equal to or greater than the assay sensitivity level (<1 ng/ml). Acetaminophen, Acetylsalicylic acid, Amphetamine, Aminopyrine, Ampicillin, Amobarbital, Ascorbic acid, Atropine, Barbital, Butabarbital, Caffeine, Carbamazepine, Codeine, Chloroquine, Chloropromazine, Carbromal ,Desipramine, Dextromethorphan, Dextropropoxyphene, 5,5 Diphenylhydantoin, 10 11 Dihydrocarbamazepine, Diazepam, Ethosuximide, Estriol, Estrone, Estradiol, Ethotoin, Glutethimide, Hexobarbit al,

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56 Ibuprofen, Imipramine, Lidocaine, LSD, MDA, MDMA, Methadone, Methadone Methyl propylsuccinimide, Mephobarbital, Methyl PEMA, Methsuximid e 4 Methylprimidone, Morphine Me peridine, Niacinamide, Norethindrone, N Normethsuximide, Phenobarbital Phensuximide, PEMA, Primidone, Phencyclidine, Pentobarbital, Phenothiazine, Phenylpropanolamine, Procaine, Quinine, Secobarbital, Tetracycline, Tetrahydrozoline, THCCOOH 3.3.2 Aldoste rone 3.3.2.1 General Principle of Assay The Aldosterone EIA kit used (Aldosterone Direct EIA Kit #11 ALDHU E01, ALPCO Diagnostics, Salem, NH 03079) follows the typical competitive binding scenario. Unlabeled antigen (presen t in standards, control and samples) and an enzyme labeled antigen (conjugate) compete for a limited number of antibody binding sites on the microplate. The kit can be used with urine specimens with significant pretreatment (hydrolysis, neutralization and dilution). Approximately 1 ml of urine is required per duplicate determination. horseradish pe roxidase labeled aldosterone derivative in micro plate wells, coated with fixed amounts of oriented high affinity purified polyclonal antibody. The wells are

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57 washed 3 times thoroughly and a chromogenic substrate added. The color produced is stopped using a dilute acid stop solution and the wells read at 450 nm. The intensity of the color formed is inversely proportional to the concentration of aldosterone in the sample. A set of standards is used to plot a standard curve from which the amount of aldosterone in samples and controls can be directly read. The lower detection limit is calculated from the standard curve by determining the resulting concentration of the mean OD of Calibrator A (based on 10 replicate analyses) minus 2 SD. Therefore, the sensitivity of the Direct Aldosterone ELISA kit is 15pg/ml. 3.3.2.2 Assay Procedural Details Kit Contents: 1. Rabbit Anti Aldosterone Antibody Coated Microwell Plate 2. Aldosterone Biotin: Avidin Horse Radish Peroxidase (HRP) Conjugate 3. Aldosterone Calibrators 4. Control Ready To Use. 5. Wash Buffer Concentrate 6. Assay Buffer 7. TMB Substrate 8. Stopping Solution

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58 3.3.2.3 Urine Pretreatment 1 ml of each urine sample was pipetted into an appropriately labeled glass or polypropylene tube 0.1 ml of 3.2 N HCl was added to every tube. 3.2 N HCl can be made by adding 1 ml of concentrated HCl (12N) to 2.75 ml distilled water. Tubes were then capped securely and heat for 1 hour at 60oC in the dark. Urine specimens were neutralized by adding 0.1 ml o f 3.2 N NaOH to every tube. 3.2 N NaOH can be made by dissolving 1.28 grams of NaOH pellets into 10 ml distilled water. Tubes were then mixed gently and thoroughly. The final step in pretreatment requires the dilution of samples 1:50 with calibrator A. All reagents must reach room temperature before use. Positive reference standards of 0, 20, 80, 300, 800 and 2000 pg/ml are provided ready to use. Calibrators, controls and specimen samples should be assayed in duplicate. Once the procedure has been s tarted, all steps should be completed without interruption. The conjugate was prepared by diluting the aldosterone biotin: avidin HRP concentrate 1:50 in assay buffer wa sh buffer concentrate 1:10 with Barnstead Nanopure water (50 ml of the wash buffer concentrate in 450 ml of water). were pipetted into correspondingly labeled wells in dup conjugate working solution was pipetted into each well. The plate was incubated on a plate shaker (approximately 200 rpm) for 1 hour at room temperature. The wells were ll. The plate was tapped firmly

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59 into each well at timed intervals and the plate was incubated on a plate shaker for 10 15 minutes at room temperature. An alternate e ndpoint is to allow calibrator A to attain a same timed intervals as in the TMB step. Finally, the plate was read at 450nm within 20 minutes after addition of the st opping solution. A standard curve was produced and the concentration of aldosterone in each of the specimens was determined. 3.3.2.4 Cross Reactivity The following compounds cross reacted at less than 0.001%: Androsterone, Cortisone, 11 Deoxycortisol, 21 Deoxycortisol, Dihydrotestosterone, Estradiol, Estriol, Estrone and Testosterone. 3.3.3 C Reactive Protein 3.3.3.1 General Principle of the Assay The high sensitivity C Reactive Protein EIA kit used (high sensitivity C Re active Protein #30 9710s, ALPCO Diagnostics, Salem, NH 03079) was a sandwich enzyme immunoassay intended for the quantitative determination of C reactive protein in plasma, serum, stool and urine. The combination of two specific antibodies in the CRP ELI SA significantly reduces the possibility of false negatives. It is for in vitro diagnostic use only. The wells of the microplate are coated with polyclonal antibodies directed against C

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60 reactive protein. The kit may be used with plasma, serum, stool and ur ine with little pretreatment. previously determined by EMIT) urine specimen is added to the appropriate wells in duplicate and incubated for an hour. The plate is then washed 5 times to remove all labeled CRP conjugate antibody is added and the plate is incubated for another hour. The plate is washed 5 times and a chromogenic substrate is added. After allowing the plate to incu bate for 20 minutes, an acidic stopping solution is then added. The intensity of the color that forms is directly proportional to the concentration of CRP in the sample. A dose response curve of the absorbance (at 450 nm) unit vs. concentration is generate d. CRP, present in the patient samples, is determined directly from this calibration curve. The assay is sensitive to 0.124 ng/ml. 3.3.3.2 Assay Procedural Details Kit Contents: 1. Microplate with precoated strips 2. Wash buffer concentrate 3. Antibody, (rabbit anti CRP, Peroxidase labeled) 4. Calibrators, ready to use, (0; 1.9; 5.6; 16.7; 50; 150 ng/ml)

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61 5. Control 6. Sample buffer 7. TMB chromogenic substrate 8. Stop solution containing sulfuric acid All reagents and samples should be at room temperature (18 26 C) prior to use. Positive reference standards of 0, 1.9, 5.6, 16.7, 50 and 150 ng/ml are provided ready to use. Urine specimens must be diluted 1:5 with sample dilution buffer. The wash buffer concentrate was diluted 1:10 with Barnstead Nanopure Water before use (100 ml concentrated wash buffer + 900 ml Nanopure Water). Crystals formed in the wash buffer concentrate (a common occurrence due to high salt concentration in the stock solutions); this necessitated soaking the concentrate in a 37C using a water bath before dilution. Calibrators, controls and specimen samples should be assayed in duplicate. The pl pipetted to each well in duplicate. After incubating at room temperature on a plate s haker for an hour, the contents of the wells were discarded and the plate was washed 5 times each well. After tapping the sides of the plate holder to ensure proper mixing, the plate

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62 was covered tightly and incubated on a plate shaker for 60 minutes at room temperature. of Substrate reagent was added to each well and the sides of plate hold er tapped to ensure proper mixing. The plate was incubated in the dark for 15 minutes at room temperature. at 450 nm against 620 nm as a reference. 3.3.3.3 Cross R eactiv ity Alpha 1 Antitrypsin 0 % Lysozyme 0 % Albumin 0 % Other acute phase proteins 0 % No cross reactivity with CRP in mouse serum was observed. 3.3.4 Myeloperoxidase 3.3.4.1 General Principle of the Assay The Myeloperoxidase EIA kit used (Myeloperoxidase #30 6630, ALPCO Diagnostics, Salem, NH 03079)is a sandwich enzyme immunoassay intended for the quantitative determination of MPO in plasma, serum, stool and urine. The combination of

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63 two specific antibodie s in the MPO ELISA significantly reduces the possibility of false negatives. It is for in vitro diagnostic use only. The wells of the microplate are coated with polyclonal antibodies directed against MPO. The kit may be used with urine with little pretreat ment aside from dilution. This ELISA is suitable for the quantitative determination of MPO in urine and stool. In the first incubation step, the MPO in the samples is bound to available antibodies against MPO immobilized on the surface of the microwells. To remove all unbound substances, a washing step is carried out. In a second incubation step, a peroxidase labeled antibody against MPO is added. After another washing step to remove all unbound substances, the solid phase is incubated with the chromogenic substrate TMB. An acidic stop solution is then added to stop the reaction. The intensity of the yellow color is directly proportional to the concentration of MPO in the sample. A dose response curve of the absorbance unit vs. concentration is generated, u sing results obtained from the calibrators. MPO, present in the samples, is determined directly from this curve. 3.3.4.2 Assay Procedural Details Kit Contents: 1. Microplate with precoated strips 2. Wash buffer concentrate 3. Detection Antibody (biotinylated) 4. Standards, lyophilized

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64 5. Control, lyophilized 6. Conjugate, (streptavidin peroxidase labeled) 7. Sample Dilution buffer 8. TMB chromogenic substrate 9. Stop solution containing sulfuric acid All reagents and samples should be at room temperature (18 26 C) prior to use. The Control and Positive reference standards of 0, 3.6, 11, 33 and 100 ng/ml are Urine specimens must be diluted 1:10 wi th sample dilution buffer. The wash buffer concentrate was diluted 1:10 with Barnstead Nanopure Water before use (100 ml concentrated wash buffer + 900 ml Nanopure Water). Crystals formed in the wash buffer concentrate (a common occurrence due to high sal t concentration in the stock solutions); this necessitated soaking the concentrate in a 37C using a water bath before dilution. 10 ml wash buffer). The Antibody requ concentrated antibody + 10 ml wash buffer). Calibrators, controls and specimen samples should be assayed in duplicate. The

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65 pipetted to each well in duplicate. After incubating at room temperature on a plate shaker for an hour, the contents of the wells were discarded and the plate was washed 5 times well. After tapping the sides of the plate holder to ensure proper mixing, the plate was covered tightly and incubated on a plate shaker for 60 minutes at room temperature. The the diluted conjugate was added to each well. After tapping the sides of the plate holder to ensure proper mixing, the plate was covered tightly and i ncubated on a plate shaker for sides of plate holder tapped to ensure proper mixing. The plate was incubated in the dark the absorption was read immediately at 450 nm against 620 nm as a reference. 3.3.4.3 Cross R eactivity No cross reactivity to other pl asma proteins in stool. Alpha 1 Antitrypsin Albumin CRP Lysozyme sIgA PMN Elastase Calprotectin

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66 3.3.5 Microalbumin 3.3.5.1 General Principle of the Assay The Microalbumin EIA kit used (Microalbumin # 24 MABHU E01 ALPCO Diagnostics, Salem, NH 03079)is a competitive enzyme immunoassay intended for the quantitative determination of MAB in urine. The microwells of the assay plate are coated with a purified unlabeled primary antibody. Following this, the plate is inc ubated with unlabeled standards and unknowns. The reaction is allowed to proceed to equilibrium; labeled conjugate antigen is then added to the microwells. The conjugate will bind to the available binding sites on the primary antibody. The plate is then de veloped with substrate and color change is measured. After incubation for a fixed time, separation of bound albumin from free albumin is achieved by simple decantation and plate washing. The enzyme activity on the plate is measured using enzyme substrate and a chromogen. The absorbency of the color developed is read in an EIA colorimetric reader. In a competitive ELISA, there is an inverse relationship between the signal obtained and the concentration of the analyte in the sample i.e. the more unlabeled an tigens in the sample or standard, the lower the amount of conjugated antigen bound. The concentration of albumin in the urine is determined from a calibration curve. The assay literature indicates that sensitivity was calculated by processing ten zero cal ibrator (maximum binding) wells along with a calibration curve. Mean and standard deviation were calculated for the absorbance of the ten zero calibrator wells.

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67 Apparent sensitivity was found to be 0.24 mg/ml. The antiserum is highly specific for human alb umin. 3.3.5.2 Assay Procedural Details Kit Contents: 1. Microplate with precoated strips 2. Albumin Calibrators (5) 3. Albumin Enzyme Conjugate 4. Controls (2) 5. TMB chromogenic substrate 6. Stop solution containing sulfuric acid All reagents and samples should be at room temperature (18 26 C) prior to use. The Control and Positive reference standards packaged ready to use. Urine specimens may be used without pretreatment or dilution. Calibrators, controls and specimen samples sh was added to every well. After incubating at room temperature on a plate shaker for an hour, the contents of the wells were discarded and the plate was washed 5 times with 250

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68 tapping the sides of the plate holder to ensure proper mixing, the pl ate was covered tightly and incubated on a plate shaker for 60 minutes at room temperature. The the diluted conj ugate was added to each well. After tapping the sides of t he plate holder to ensure proper mixing, the plate was covered tightly and incubated on a plate shaker for to each well and the sides of plate holder tapped to ensure proper mixing. The plate was incubated in the dark the absorption was read immediately at 450 nm against 620 nm as a reference. 3.3.5.3 Cross R eactivity No cross reactivity to bovine serum albumin, myoglobin, hemoglobin, and alpha fetoprotein were noted at significantly high concentration. 3.3.6 Myoglobin 3.3.6.1 General Princip le of the Assay The Myoglobin EIA kit used (Myoglobin # 2110 Life Diagnostics, Inc., P.O. Box 5205, West Chester, PA, 19380) was a sandwich enzyme immunoassay intended for the quantitative determination of myoglobin in serum and plasma. The combination of two

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69 specific antibodies in the myoglobin ELISA significantly reduces the possibility of false negatives. It is for in vitro diagnostic use only. The wells of the microplate are coated with polyclonal antibodies directed against myoglobin. This ELISA is su itable for the quantitative determination of myoglobin in serum and plasma. Although not labeled for use with urine specimens, the technical support staff at Life Diagnostics ( www.lifediagnostics.com 610 431 770 7) asserted the assay was cocaine positive urine specimens (as previously determined by EMIT) are incubated simultaneously with two antibodies, resulting in myoglobin mole cules being linked antibodies. To remove all unbound substances, the plate is washed with Barnstead Nanopure Water. The chromogenic substrate TMB is added and the plate is incubated for 20 minutes. An acidic stop solution is then added to stop the reaction. The intensity of the yellow color is directly proportional to the concentration of myoglobin in the sample. A dose response curve of the absorbance unit vs. concentration is generated, using results obtaine d from the calibrators. Myoglobin, present in the samples, is determined directly from this curve. The lowest detectable level of myoglobin by this assay is estimated to be 5 ng/ml. 3.3.6.2 Assay Procedural Details Kit Contents: 1. Murine Monoclonal Anti My oglobin coated microtiter wells, 96 wells 2. Myoglobin Reference Standards (0, 25, 100, 250, 500 and 1000 ng/ml)

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70 3. Sample Diluent 4. Enzyme Conjugate Reagent 5. TMB Reagent 6. Stop Solution (1N HCl) All reagents and samples should be at room temperature (18 26 C) prior to use. The myoglobin reference standards of 0, 25, 100, 250, 500 and 1000ng/ml are provided agent to each well in duplicate and thoroughly mixed for 30 seconds. After incubating at room temperature for 45 minutes, the contents of the wells were discarded and the plate was washed 5 times Substrate reagent was added to each well and the sides of plate holder tapped for 5 seconds to ensure proper Solution was added to each well and the plate was gently mixed f or 30 seconds. It is important to make sure the blue color is completely changed to yellow before reading the absorption. Finally, the absorption was read immediately at 450 nm against 620 nm as a reference.

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71 3.3.6.3 Cross R eactivity No cross reactivity i s noted in the assay kit literature. 3.3.7.1 General Principle of Assay EKS 895 Assay Designs, Ann Arbor, MI 48108 ) is a quantitative sandwich immunoassay or cocaine positive (as previously determined by EMIT) urine specimen is pipetted into the wells by the immobilized antibody. The wells are washed and the Detection Antibody is added. After washing away unbound Detection Antibody, HRP conjugated streptavidin is pipetted to the wells. The wells are washed again, a TMB Substrate Reagent is add ed to the wells and color color from blue to yellow, and the intensity of the color is measured at 450 nm. 3.3.7.2 Assay Procedural Details Kit contents: 1. Anti roplate 2. 20X Wash Buffer Concentrate

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72 4. Sample Diluent 5. HRP Conjugate 6. HRP Conjugate Diluent 7. TMB Substrate 8. Stop Solution All reagents were brought to room temperature prior to use. Dilute 10 0 ml of Wash Buffer Concentrate into 1900 ml Barnstead Nanopure Water t o yield 200 0 ml of 1x Wash Buffer. HRP Conjugate diluent to prepare diluted HRP Conjugate Mix thoroughly The sta ndar ds are pre pared by g/ml stock sta ndard to 50 Sample Diluent and performing serial dilutions according to Table 4

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73 Tube Volume and Source Sample Diluent Concentration (ng/ml ) A standard vial 500 4 B 250 from Tube A 250 2 C 250 from Tube B 250 1 D 250 from Tube C 250 0.5 E 250 from Tube D 250 0.25 F 250 from Tube E 250 0.125 G 250 from Tube F 250 0.0625 H 0 250 0 Urine speci mens do not require dilution. 10 specimens were pipetted to each well in duplicate and thoroughly mixed for 30 seconds. After incub ating at room temperature for 60 minutes, the contents of the wells were discarded and the plate was w ashed 6 times with 30 Wash Buffer HRP Conjugate was added to each well except H 1 and H 2 After incubating at room temperature for 60 minutes, the contents of the wells were discarded and the plate was added to each well and the sides of plate holder tapped for 5 seconds to ensure proper Solution was added to each well and the plate was gently mixed for 30 seconds. It is important to make sure the blue color is completely changed to yellow before reading the

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74 absorption. Finally, the absorption was read immediately at 450 nm against 620 nm as a reference. 3.3.7.3 Cross Reactivity cross react with 100ng/mL of Grp94, Hsp60, Hsp70. The 3.3.8 Vascular Endothelial Growth Factor 3.3.8.1 General Principle of Assay The principle of Vascula r Endothelial Growth Factor ELISA Kit used (Vascular Endothelial Growth Factor ELISA Kit #ELH VEGF 001, RayBiotech Corporation, Norcross, GA 30092) is intended for the quantitative measurement of human VEGF in serum, plasma, cell culture supernatants and urine. The assay is based upon the competitive binding to antibody of enzyme labeled antigen and unlabeled antigen, in proportion to their concentration in the reaction mixture. previously determined by EMIT) urine specimen is pipetted into the wells and VEGF present in a sample is bound to the wells by the immobilized antibody. The wells are washed and the Detection Antibody is added. After washing away unbound Detection

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75 Antibody HRP conjugated streptavidin is pipetted to the wells. The wells are washed again, a TMB Substrate Reagent is added to the wells and color develops in proportion to the amount of VEGF bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. 3.3.8.2 Assay Procedural Details Kit contents: 1. VEGF Microplate 2. Wash Buffer Concentrate 3. Recombinant human VEGF Standard 4. Assay Diluent A (serum/plasma) 5. Assay Diluent B (urine and other body fluids) 6. Detection Antibody VEGF ( biotinylated anti humanVEGF) 7. HRP Streptavidin concentrate 8. TMB One Step Substrate Reagent 9. Stop Solution (2 M sulfuric acid) All reagents were brought to room temperature prior to use. Assay Diluent B is use d for dilution of culture supernatants and urine; Assay Diluent B is prepared by

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76 diluting the contents of the vial 5 fold with Barnstead Nanopure Water. Dilute 20 ml of Wash Buffer Concentrate into Barnstead Nanopure Water to yield 400 ml of 1x Wash Buffer Assay Diluent B to prepare a 100 fold diluted HRP Streptavidin. Mix thoroughly and then pip fold diluted solution into a tube with 15 ml 1x Assay Diluent B to prepare a final 15,000 fold diluted HRP Streptavidin solution. The standards and performing serial dilutions according to Table 5 : Table 5. VEGF Serial Dilutions Tube Volume and Source Assay Diluent B Final VEGF Concentration (pg/ml ) A 60 from the VEGF standard vial 440 6000 B 200 from Tube A 400 2000 C 200 from Tube B 400 666.7 D 200 from Tube C 400 222.2 E 200 from Tube D 400 74.07 F 200 from Tube E 400 24.69 G 200 from Tube F 400 8.23 H 0 400 0

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77 of the specimens were pipetted in duplicate to the appropriate wells. The microplate was covered and incubated for 2.5 hours at room temperature or over night at 4C with gentle shaking. Wash the plate 4 times with 1x Wash Solution. After the last wash, t he plate was inverted and blotted against clean paper towels. Next, incubated for 1 hour at room temperature with gentle shaking. The wash cycle was red Streptavidin solution was added to each cell. After tapping the sides of the plate holder to ensure proper mixing, the plate was incubated for 45 minutes at room temperature on a plate shaker. The wash cycle was repeated a final time. bstrate Reagent was added to each well and the plate was incubated for 30 minutes at room temperature in the dark with on a plate shaker. The reaction was 450 nm immedi ately. A standard curve was generated and the concentration of VEGF in each of the specimens was determined. 3.3.8.3 Cross Reactivity This ELISA kit shows no cross reactivity with any of the following cytokines: human Angiogenin, BDNF, BLC, ENA 78, FGF 4, IL 2, IL 3, IL 4, IL 5, IL 7, IL 8, IL 9, IL 10, IL 11, IL 12 p70, IL 12 p40, IL 13, IL 15, IL 309, IP 10, G CSF, GM CSF, IFN 1, MCP 2, MCP 3, MDC, MIP

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78 1, TIMP 2, TNF TPO 3.3.9 Human Matrix Metalloproteinase 9/ Neutrophil Gelatinase Associated Lipocalin 3.3.9.1 General Principle of Assay The principle of Human Matrix Metalloproteinase 9/ Neutrophil Gelatinase Associated Lipocalin ELISA Kit used (Human Matrix Metalloproteinase 9/ Neutrophil Gelatinase Associated Lipocalin Direct ELISA Kit #DM9L20, R&D Systems, Minneapolis, Minnesota 55413) a sandwich enzyme immunoassay intended for the quantitative determination of MMP 9/N GAL in cell culture supernates, serum, plasma, urine and saliva. The combination of two specific antibodies in the MMP 9/NGAL ELISA significantly reduces the possibility of false negatives. It is for research use only. The wells of the microplate are coate d with polyclonal antibodies directed against MMP 9/NGAL. at room temperature. The wells 9/NGAL conjugate is added. The plate is incubated and washed again before a chromogenic substrate added. The color produced is stopped using a dilute acid stop solution and the wells read at 450 nm. The inten sity of the color developed is inversely

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79 proportional to the concentration of drug in the sample. The technique is sensitive to 0.013 ng/ml. 3.3.9.2 Assay Procedural Details Kit contents: 1. 96 well micro plate coated with mouse monoclonal anti MMP 9/NGAL 2. MMP 9/NGAL Conjugate solution 3. Standard containing 20 ng/ml of MMP 9/NGAL lyophilized 4. Assay Diluent RD1 87 5. Calibrator Diluent RD5 10 6. Color Reagent A 7. Color Reagent B 8. Wash Buffer Concentrate 9. Stop Reagent containing 2 N sulfuric acid All reagents were br ought to room temperature prior to use. The Wash Buffer was prepared by diluting 20 ml of Wash Buffer Concentrate in 480 ml Barnstead Nanopure Water. Color Reagents A and B were mixed together in equal volumes to prepare the Substrate Solution. It has to be used within 15 minutes of preparation. The lyophilized MMP 9/NGAL standard was reconstituted with 1 ml Barnstead Nanopure Water to yield a 20 ng/ml MMP 9/NGAL stock solution. Calibrators were prepared from the stock solution according to Table 6 :

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80 Table 6. NGAL Serial Dilution Table Tube Volume and Source of MMP 9/NGAL Calibrator Diluent RD5 10 Final Concentration (ng/ml MMP 9/NGAL) A 200 from Stock Solution 200 10 B 200 from Tube A 200 5 C 200 from Tube B 200 2.5 D 200 from Tube C 200 1.25 E 200 from Tube D 200 0.625 F 200 from Tube E 200 0.312 G 0 200 0 standards, control urine or cocaine positive urine specimens were pipetted in duplicate to the appropriate wells. After tapping the sides of the plate holder to ensure proper mixing, the plate was incubated for 3 hours at room temperature. The microplate wells were incubati on, the microplate was inverted and slapped vigorously on absorbent paper to each well and the plate was incubated for 1 hour at room temperature. The aspiration and wash the sides of plate holder tapped to ensure proper mixing. The plate was incubated in the each well, changing the blue color to yellow.

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81 Absorbance was measured at a wavelength of 450 nm. A standard curve was produced and the concentration of MMP 9/NGAL in each of the specimens was determined. No significant cross reactivity has been noted. 3. 3.10 Pro atrial natriuretic peptide (1 98) 3.3.10.1 General Principle of Assay The pro atrial natriuretic peptide (1 98) EIA kit used (proANP Direct EIA Kit # 04 BI 20892, ALPCO Diagnostics, Salem, NH 03079) follows the typ ical direct binding scenario. It is appropriate for research use only. The wells of the microplate are coated with polyclonal antibodies directed against proANP. The kit may be used with urine with no pretreatment. rine or cocaine positive (as previously Conjugate is added to all the wells, except the blanks, and incubated in the dark for 3 hours at room temperature. The wells are wa Substrate is added. The plate is incubated the reaction is stopped using a dilute acid stop solution. Finally, measure the absorbance at 450 nm with a reference of 620 nm. The intensity of the color developed is propor tional to the concentration of drug in the sample.

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82 3.3.10.2 Assay Procedural Details Kit Contents: 1. Polyclonal sheep anti proANP Coated Microwell Plate 2. Conjugate (polyclonal anti proANP antibody HRPO) 3. ProANP standards, lyophilized 4. Control, lyophilized 5. Wash Buffer Concentrate 6. Assay Buffer 7. TMB Substrate 8. Stop Solution (Sulfuric Acid) All reagents must reach room temperature before use. The Control and Positive reference standards of 0, 0.63, 1.25, 2.5, 5.0 and 10.0 nmol/l are lyophilized and required samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. The conj ugate (polyclonal anti proANP antibody HRPO) was provided ready to use. The wash buffer was prepared by diluting the wash buffer concentrate 1:20 with Barnstead Nanopure water (50 ml of the wash buffer concentrate in 950 ml of water).

PAGE 100

83 brator, treated control urine and treated cocaine positive urine (as determined by EMIT) were pipetted into correspondingly labeled wells in duplicate. blanks. The pla te was incubated for 3 hours at room temperature in the dark. The wells substrate was pipetted into each well and the plate was incubated on a plate shaker for 30 well and the plate was read at 450nm with a reference of 620 nm. A standard curve was produced and the concentration of proANP in each of the specimens was determined. 3.3.10.3 Cross Reactivity proANP (1 30) < 1% proANP (31 67) <1% proANP (79 98) <1% alpha ANP (99 126 ) < 1% proBNP (8 29) <1% proBNP (32 57) <1% proCNP (1 19) <1% proCNP (30 50) <1%

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84 proCNP ( 51 97) <1% The assay also detects mouse and rat proANP (1 98). 3.3.11.1 General Principle of the Assay The EIA kit used ( #583301, Cayman Chemicals, Ann Arbor, MI 48108) is a sandwich enzyme immunoassay intended for the quantitative determination of in serum and plasma. Cayman Chemical Technical Support verified the assay was compatible with urine specimens so long as the standards are diluted with an appropriate sample matrix blank (SMB). Synthetic Urine from Immunalysis (Synthetic Urine with Creatinine Dry Powder #SUP 5, Immunalysis Corporation, Pomona, CA 91767 ) was used for the sample matrix blank. The com bination of two specific antibodies in the ELISA significantly reduces the possibility of false negatives. It is for research use only. The wells of the microplate are coated with polyclonal antibodies directed against A 100 previously determined by EMIT) urine specimen is added to the appropriate wells in 1 is added to all well s except the blanks and incubated for overnight at 4 C. The plate is then Reagent is added to each well and the plate is incubated in the dark. The reaction is

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85 monitored for several hours until the S1 wells are visibly yellow. The intensity of the color that forms is directly proportional to the concentration of Interleukin 1 in the sample. A dose response curve of the absorbance (at 405 nm) unit vs. concentration is gen erated. Interleukin 1 present in the patient samples, is determined directly from this calibration curve. The assay is sensitive to 1.5 pg/ml Interleukin 1 3.3.11.2 Assay Procedural Details Kit Contents: 1. Microplate with precoated strips 2. Interleukin 1 3. Non specific Mouse Serum 4. Sample Matrix Blank for plasma, serum (cannot be used for this assay) 5. Acetylcholinesterase: Interleukin 1 6. EIA Buffer Concentrate 7. Wash Buffer Concentrate 8. Twee n

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86 All reagents and samples should be at room temperature (18 26 C) prior to use. The EIA Buffer concentrate was reconstituted with 90 ml of Barnstead Nanopure water. The Wash Buffer was prepared by bringing 5 ml of wash buffer conc entrate to a total volume of 2 L with Barnstead Nanopure water and then adding 1 ml Tween. The Acetylcholinesterase: Interleukin 1 was reconstituted with 10 ml of EIA buffer. The urine specimens were used without any dilution. Interleukin 1 standards required reconstitution with 2 ml EIA Buffer prior to dilution with SMB. This initial dilution yielded a stock solution of 5 ng/ml Interleukin 1 The Immunalysis Synthetic Urine served as the SMB; the lyophilized urine was reconstituted wi th Barnstead Nanopure water prior to use. 4.5 L of Nanopure water were added to the contents of the pouch. The solution was stirred until the lyophilized urine dissolved completely. The total volume was adjusted to 5 L with Nanopure water. The synthetic ur ine is stable for 1 year when stored at 4C. The Interleukin 1 standards were pr epared as described in Table 7 :

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87 Table 7. Concentration of Interleukin 1 Standards Tube Volume and Source of Interleukin 1 Sample Matrix Final Concentration (pg/ml Interleukin 1 ) A 50 from Stock Solution Dilute to 1 ml total 250 B 500 from Tube A 500 125 C 500 from Tube B 500 62.5 D 500 from Tube C 500 31.25 E 500 from Tube D 500 15.6 F 500 from Tube E 500 7.8 G 500 from Tube F 500 3.9 H 0 500 0 Calibrators, controls and specimen samples should be assayed in duplicate. 100 1 is added to all wells except the blanks and incubated for overnight at 4 C. The plate is n the dark. The reaction is monitored for several hours until the S1 wells are visibly yellow. The intensity of the color that forms is directly proportional to the concentration of Interleukin 1 in the sample. The absorption was read immediately at 405 nm.

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88 3.3.12.1 General Principle of the Assay The EIA kit used ( #583311, Cayman Chemicals, Ann Arbor, MI 48108) is a sandwich enzyme immunoassay intended for the quantitative determination of in serum and plasma. Cayman Chemical Technical Support verified the assay was compatible with urine specimens so long as the standards are diluted with an appropriate sample matrix blank (SMB). Synthetic Urine from Immunalysis (Synthetic Urine with Creatinine Dry Powder #SUP 5, Immunalysis Corporation, Pomona, CA 91767 ) was used for the sample matrix blank. The com bination of two specific antibodies in the ELISA significantly reduces the possibility of false negatives. It is for research use only. The wells of the microplate are coated with polyclonal antibodies directed against A 100 previously determined by EMIT) urine specimen is added to the appropriate wells in 1 is added to all well s except the blanks and incubated for overnight at 4 C. The plate is then Reagent is added to each well and the plate is incubated in the dark. The reaction is monitored for several hours until the S1 ( 250 pg/ml) wells are visibly yellow. The intensity of the color that forms is directly proportional to the concentration of Interleukin 1 in the sample. A dose response curve of the absorbance (at 405 nm) unit vs. concentr ation is generated. Interleukin 1 present in the patient samples, is

PAGE 106

89 determined directly from this calibration curve. The assay is sensitive to 1.5 pg/ml Interleukin 1 3.3.12.2 Assay Procedural Details Kit Contents: 1. Microplate with precoated s trips 2. Interleukin 1 3. Non specific Mouse Serum 4. Sample Matrix Blank for plasma, serum (cannot be used for this assay) 5. Acetylcholinesterase: Interleukin 1 6. EIA Buffer Concentrate 7. Wash Buffer Concentrate 8. Tween All reagents and samples should be at room temperature (18 26 C) prior to use. The EIA Buffer concentrate was reconstituted with 90 ml of Barnstead Nanopure water. The Wash Buffer was prepar ed by bringing 5 ml of wash buffer concentrate to a total volume of 2 L with Barnstead Nanopure water and then adding 1 ml Tween. The

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90 Acetylcholinesterase: Interleukin 1 was reconstituted with 10 ml of EIA buffer. The urine specimens were used without any dilution. Interleukin 1 standards required reconstitution with 2 ml EIA Buffer prior to dilution with SMB. This initial dilution yielded a stock solution of 5 ng/ml Interleukin 1 The Immunalysis Synthetic Urine served as the SMB; the lyophilized urine was reconstituted with Barnstead Nanopure water prior to use. 4.5 L of Nanopure water were added to the contents of the pouch. The solution was stirred until the lyophilized urine dissolved completely. The total volume was adjusted to 5 L with Nanopure water. The synthetic urine is stable for 1 year when stored at 4C. The Interleukin 1 standards were prepared as described in Table 8 : Table 8. Concentration of Interleukin 1 Standards Tube Volume and Source of Interleukin 1 Sample Matrix Final Concentration (pg/ml Interleukin 1 ) A 50 from Stock Solution Dilute to 1 ml total 250 B 500 from Tube A 500 125 C 500 from Tube B 500 62.5 D 500 from Tube C 500 31.25 E 500 from Tube D 500 15.6 F 500 from Tube E 500 7.8 G 500 from Tube F 500 3.9 H 0 500 0

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91 Calibrators, controls and specimen samples should be assayed in duplicate. 100 Acetylcholinesterase: Interleukin 1 is added to all wells except the blanks and incubated for overnight at 4 C. The plate is h well and the plate is incubated in the dark. The reaction is monitored for several hours until the S1 wells are visibly yellow. The intensity of the color that forms is directly proportional to the concentration of Interleukin 1 in the sample. The abso rption was read immediately at 405 nm. 3.3.13 Interleukin 6 3.3.13.1 General Principle of the Assay The Interleukin 6 EIA kit used ( Interleukin 6 #583361, Cayman Chemicals, Ann Arbor, MI 48108) is a sandwich enzyme immuno assay intended for the quantitative determination of Interleukin 6 in serum and plasma. Cayman Chemical Technical Support verified the assay was compatible with urine specimens so long as the standards are diluted with an appropriate sample matrix blank ( SMB). Synthetic Urine from Immunalysis (Synthetic Urine with Creatinine Dry Powder #SUP 5, Immunalysis Corporation, Pomona, CA 91767 ) was used for the sample matrix blank. The combination of two specific antibodies in the Interleukin 6 ELISA significantly reduces the possibility of false negatives. It is for research use only. The wells of the microplate are coated with polyclonal antibodies directed against Interleukin 6.

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92 positive (as previously determined by EMIT) urine specimen is added to the appropriate wells in 6 is added to all wells except the blanks and incubated for overnight at 4 C. The plate is then washed is added to each well and the plate is incubated in the dark. The reaction is monitored for several hours until the S1 (250 pg/ml) wells are visibly yellow. The intensity of the color that forms is directly proportional to the concentration of Interleukin 6 in the sample. A dose response curve of the absorbance (at 405 nm) unit vs. concentration is generated. Interleukin 6 present in the patient sample s is determined directly from this calibration curve. The assay is sensitive to 7.8 pg/ml Interleukin 6. 3.3.13.2 Assay Procedural Details Kit Contents: 1. Microplate with precoated strips 2. Interleukin 6 standard 3. Non specific Mouse Serum 4. Sample Matrix Blank for plasma, serum (cannot be used for this assay) 5. Acetylcholinesterase: Interleukin 6 6. EIA Buffer Concentrate

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93 7. Wash Buffer Concentrate 8. Tween 20 All reagents and samples should be at room temperature (18 26 C) prior to use. The EIA Buffer concentrate was reconstituted with 90 ml of Barnstead Nanopure water. The Wash Buffer was prepared by bringing 5 ml of wash buffer concentrate to a total volume of 2 L with Barnstead Nanopure w ater and then adding 1 ml Tween. The Acetylcholinesterase: Interleukin 6 was reconstituted with 10 ml of EIA buffer. The urine specimens were used without any dilution. Interleukin 6 standards required reconstitution with 2 ml EIA Buffer pr ior to dilution with SMB. This initial dilution yielded a stock solution of 5 ng/ml Interleukin 6 The Immunalysis Synthetic Urine served as the SMB; the lyophilized urine was reconstituted with Barnstead Nanopure water prior to use. 4.5 L of Nanopure wate r were added to the contents of the pouch. The solution was stirred until the lyophilized urine dissolved completely. The total volume was adjusted to 5 L with Nanopure water. The synthetic urine is stable for 1 year when stored at 4C. The Interleukin 6 standards were prepared as described in the table below (Table 9 ) :

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94 Table 9. Concentration of Interleukin 6 Standards Tube Volume and Source of Interleukin 6 Sample Matrix Final Concentration (pg/ml Interleukin 6) A 50 from Stock Solution Dilute to 1 ml total 250 B 500 from Tube A 500 125 C 500 from Tube B 500 62.5 D 500 from Tube C 500 31.3 E 500 from Tube D 500 15.6 F 500 from Tube E 500 7.8 G 500 from Tube F 500 3.9 H 0 500 0 Calibrators, controls and specimen samples should be assayed in duplicate. 100 6 is added to all wells except the blanks and incubated for overnight at 4 C. The plate is then Reagent is added to each well and the plate is incubated in the dark. The reaction is monitored for several hours until the S1 (250 pg/ml standard) wells are visibly yellow. The intensity of the color that forms is directly proportional to the concentration of Interleukin 6 in the sample. The absorption was read immediately at 405 nm. Crossreactivity with the other interleu kins is limited.

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95 Chapter 4.0 Results Results were analyzed using a Statistics, Release 4.0.4 SAS Institute, 2001) was used to compare biomarker concentration in the male and female control and cocaine positive specimens. Because the data was not parametric, the Wilcoxon Rank Sums and Mann Whitney U tests were also used to test the me ans. In addition, the Tukey Kramer HSD test was also used to analyze the means. The results were the same regardless of th e analysis performed. 4.1 Gender 4.1.1 Assay results in Male and Female Control Urines A summary of the differences in marker expression between male and female control urine specimens is presented in Table 10 The individual results are standardized and expressed per milligram creatinine. The mean aldosterone concentration was statistically significantly (p=0.0223) in female urine when compared to male urine hsCRP (p=. 0662) and MPO (p=.0562) are suggestive of statistical significance. There was no statistically significant difference in the other biomarkers.

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96 Table 10. Comparison of Assay results in Male and Female Control Urines Assay Control Male Female p Total Protein (pg) 32.2 + 12. 1 65. 9 + 20. 8 .2294 Aldosterone (pg) 1907.7 + 356.7 4917.96 + 1159.9 .0223* hsCRP (ng) 0.78 + 6 202. 3 + 103.0 .0662 Myeloperoxidase (ng) 20. 5 + 7.9 187.1 + 81.3 .0562 Microalbumin (ug) 18. 4 + 7.3 41.2 + 14.4 .1763 0.9 2 + .4 0.3 + 2 .2133 VEGF (pg) 2453.0 + 972.5 3286.2 + 1402.6 .6205 proANP (nmol) 0. 3 + 1 0.3 4 + 1 .5482 Myoglobin (ng) 15.5 + 9. 8 26.5 + 9. 1 .393 NGAL (ng) 0.08 + 1 12.5 + 9.8 .2213 IL6 (pg) 0.86 + .4 7.52 + 4.4 .1532 3.8 + 3.8 56.9 + 12.5 .0007* 2.2 + 1. 0 6.6 + 1.4 .0198* *significant at p<0.05 values expressed as mean + standard error of the mean (SEM) 4.1.2 Assay results in Male and Female Cocaine Positive Urines A summary of the differences in marker expression between male and female cocaine positive urine specimens is presented in Table 11 The individual results are

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97 are sugges tive of statistical significance. There was no statistically significant difference in the other biomarkers. Table 11. Comparison of Assay results in Male and Female Cocaine Positive Urines Assay Cocaine Positive Male Female p Total Protein (pg) 66. 9 + 22.4 47.4 + 3.2 .4612 Aldosterone (pg) 3331. 8 + 399.8 3463.6 + 723.2 .8600 hsCRP (ng) 42.1 + 44.4 47.4 + 34.3 .9275 Myeloperoxidase (ng) 25.1 5 + 9.7 52.1 + 22.1 .2140 Microalbumin (ug) 32.9 + 9. 7 40. 5 + 10.2 .5995 0.9 + .3 0.02 + .1 .0650 VEGF (pg) 2900. 9 + 1168 1308.4 + 351.9 .2535 proANP (nmol) 0. 5 + .1 0.3 + 1 .3612 Myoglobin (ng) 36. 9 + 4. 8 45.9 + 9.8 .3586 NGAL (ng) 0.3 + .12 20.2 + 12.0 .1534 IL6 (pg) 3. 8 + 8. 9 6. 3 + 4.6 .6521 1.74 + 1.07 26.2 + 12.3 .0907 2.5 + 5 9. 5 + 3.4 .0846 significant at p<0.05 values expressed as mean + standard error of the mean (SEM)

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98 4.2 Cocaine 4.2.1 Assay Results in Male Control and Cocaine Positive Urine Specimens A summary of the differences in marker expression between control and cocaine positive m ale urine specimens is presented in Table 12 The individual assay results are standardized and expressed per milligram creatinine. The mean aldosterone concentration was significantly higher (p=0.0095) in cocaine positive urine when compared to control ur ine specimens. The mean myoglobin concentration was significantly higher (p=0.0332) in cocaine positive urine when compared to control urine specimens. There was no statistically significant difference in the other biomarkers. 4.2.2 Assay Results in Fem ale Control and Cocaine Positive Urine Specimens A summary of the differences in marker expression in control and cocaine positive female urine specimens is presented in Table 13. The individual results are standardized and expressed per milligram creatin ine. There were no statistically significant differences between the control and cocaine positive means for any of the assays.

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99 Table 12. Comparison of Assay Results in Male Control and Cocaine Positive Urine Assay Male Control Cocaine Positive p Total Protein (pg) 32.2 + 12.1 66.9 + 22.4 0.2377 Aldosterone (pg) 1907.7 + 356.7 3331.8 + 399.8 0.0095* hsCRP (ng) 0.78 + .6 42.1 + 44.4 0.3668 Myeloperoxidase (ng) 20.5 + 7.9 25.15 + 9.7 0.6985 Microalbumin (ug) 18.4 + 7.3 32.9 + 9.7 0.2474 0.92 + .4 0.9 + .3 0.967 VEGF (pg) 2453.0 + 972.5 2900.9 + 1168 0.7555 proANP (nmol) 0.3 + .1 0.5 + .1 0.1276 Myoglobin (ng) 15.5 + 9.8 36.9 + 4.8 0.0332* NGAL (ng) 0.08 + 1 0.3 + .12 0.0984 IL6 (pg) 0.86 + .4 3.8 + 8.9 0.1909 3.8 + 3.8 1.74 + 1.07 0.6247 (pg) 2.2 + 1.0 2.5 + .5 0.8124 significant p < .05 values expressed as mean + standard error of the mean (SEM)

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100 Table 13. Comparison of Assay Results in Female Control and Cocaine Positive Urine Assay Female Control Cocaine Positive p Total Protein (pg) 65.9 + 20.8 47.4 + 3.2 0.4443 Aldosterone (pg) 4917.96 + 1159.9 3463.6 + 723.2 0.3321 hsCRP (ng) 202.3 + 103.0 47.4 + 34.3 0.2147 Myeloperoxidase (ng) 187.1 + 81.3 52.1 + 22.1 0.1684 Microalbumin (ug) 41.2 + 14.4 40.5 + 10.2 0.9677 0.3 + .2 0.02 + .1 0.5478 VEGF (pg) 3286.2 + 1402.6 1308.4 + 351.9 0.2366 proANP (nmol) 0.34 + .1 0.3 + .1 0.9436 Myoglobin (ng) 26.5 + 9.1 45.9 + 9.8 0.1657 NGAL (ng) 12.5 + 9.8 20.2 + 12.0 0.6309 IL6 (pg) 7.52 + 4.4 6.3 + 4.6 0.8475 56.9 + 12.5 26.2 + 12.3 0.0963 (pg) 6.6 + 1.4 9.5 + 3.4 0.461 significant p < .05 values expressed as mean + standard error of the mean (SEM)

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101 4.3 Tables Table 14. Male and Female Control and Cocaine Positive Urine Specimen Comparison Assay Male Female Control Cocaine Positive p Control Cocaine Positive p Total Protein (pg) 32.2 + 12.1 66.9 + 22.4 0.2377 65.9 + 20.8 47.4 + 3.2 0.4443 Aldosterone (pg) 1907.7 + 356.7 3331.8 + 399.8 0.0095* 4917.96 + 1159.9 3463.6 + 723.2 0.3321 hsCRP (ng) 0.78 + .6 42.1 + 44.4 0.3668 202.3 + 103.0 47.4 + 34.3 0.2147 Myeloperoxidase (ng) 20.5 + 7.9 25.15 + 9.7 0.6985 187.1 + 81.3 52.1 + 22.1 0.1684 Microalbumin (ug) 18.4 + 7.3 32.9 + 9.7 0.2474 41.2 + 14.4 40.5 + 10.2 0.9677 0.92 + .4 0.9 + .3 0.967 0.3 + .2 0.02 + .1 0.5478 VEGF (pg) 2453.0 + 972.5 2900.9 + 1168 0.7555 3286.2 + 1402.6 1308.4 + 351.9 0.2366 proANP (nmol) 0.3 + .1 0.5 + .1 0.1276 0.34 + .1 0.3 + .1 0.9436 Myoglobin (ng) 15.5 + 9.8 36.9 + 4.8 0.0332* 26.5 + 9.1 45.9 + 9.8 0.1657 NGAL (ng) 0.08 + 1 0.3 + .12 0.0984 12.5 + 9.8 20.2 + 12.0 0.6309 IL6 (pg) 0.86 + .4 3.8 + 8.9 0.1909 7.52 + 4.4 6.3 + 4.6 0.8475 3.8 + 3.8 1.74 + 1.07 0.6247 56.9 + 12.5 26.2 + 12.3 0.0963 2.2 + 1.0 2.5 + .5 0.8124 6.6 + 1.4 9.5 + 3.4 0.461 significant p < .05 values expressed as mean + standard error of the mean (SEM)

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1 02 Table 15. Control and Cocaine Positive Male and Female Urine Specimen Comparison Assay Control Cocaine Positive Male Female p Male Female p Total Protein (pg) 32.2 + 12.1 65.9 + 20.8 .2294 66.9 + 22.4 47.4 + 3.2 .4612 Aldosterone (pg) 1907.7 + 356.7 4917.96 + 1159.9 .0223* 3331.8 + 399.8 3463.6 + 723.2 .8600 hsCRP (ng) 0.78 + .6 202.3 + 103.0 .0662 42.1 + 44.4 47.4 + 34.3 .9275 Myeloperoxidase (ng) 20.5 + 7.9 187.1 + 81.3 .0562 25.15 + 9.7 52.1 + 22.1 .2140 Microalbumin (ug) 18.4 + 7.3 41.2 + 14.4 .1763 32.9 + 9.7 40.5 + 10.2 .5995 0.92 + .4 0.3 + .2 .2133 0.9 + .3 0.02 + .1 .0650 VEGF (pg) 2453.0 + 972.5 3286.2 + 1402.6 .6205 2900.9 + 1168 1308.4 + 351.9 .2535 proANP (nmol) 0.3 + .1 0.34 + .1 .5482 0.5 + .1 0.3 + .1 .3612 Myoglobin (ng) 15.5 + 9.8 26.5 + 9.1 .393 36.9 + 4.8 45.9 + 9.8 .3586 NGAL (ng) 0.08 + 1 12.5 + 9.8 .2213 0.3 + .12 20.2 + 12.0 .1534 IL6 (pg) 0.86 + .4 7.52 + 4.4 .1532 3.8 + 8.9 6.3 + 4.6 .6521 3.8 + 3.8 56.9 + 12.5 .0007* 1.74 + 1.07 26.2 + 12.3 .0907 2.2 + 1.0 6.6 + 1.4 .0198* 2.5 + .5 9.5 + 3.4 .0846 significant p < .05 values expressed as mean + standard error of the mean (SEM)

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103 Chapter 5.0 Discussion There are well documented gender based differences in the etiology and pathophysiology in many disease states [62, 64, 67, 70, 71, 73 75, 78, 93, 127 142] Gender differences are well known to exist for many important markers of disease including triglycerides, e nzymes such as AST and SGOT, HDL, uric acid, creatine and hormones. While blood has remained the medium of clinical preference, toxicological screens however are frequently done on urine. Unlike serum and plasma, urine typically lacks interference from clo tting and compounds such as proteases [143] It is well documented that many proteins are expressed in urine, particularly during c ertain disease states; in fact, the human urinary proteome contai ns more than 1500 proteins [144] Little is known about the differences in urinary marker expression between males and females. Recently, the use of urine as a medium to analyze the expression of many proteins in the body has become more acceptable [145] Urinary analysis takes a dvantage of protein cell and tissue specificity and their alteration over time in response to different situations [146] The proteins and peptides in urine are referred to as the urinary proteome; variation in the levels of these protein biomarkers has been associated with

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104 certain pathological states. Markers in the urinary proteome have been validated for inflammation, oxidative stress, cardiovascular dysfunction and renal disorders [144, 145] Inflammation is a very common, non specific form of pathophysiology; myeloperoxidase, C reactive protein, and IL 6 are markers of acute and systemic inflammation [147, 148] MPO is also commonly used as an endpoint of oxidative stress [149] IL are immunoregulatory cytokines that favor inflammation; increased expression is associated with fever, inflammation, tissue destruction, shock and death. The d ifference between male and female CRP and MPO were close to statistical significance in control urine. IL expression is also associated with autoimmune disorders. It is possible that the gender disparity seen in autoimmune disorders could refl ect a chronic, low level activation of the immune system that might be seen in elevated interleukins [150] In control urine, female IL igher than male values by a statistically significant amount. Elevated concentrations of interleukins upregulate acute phase proteins such as IL 6 and CRP. Albuminuria is associated with cardiovascular disease, kidney dysfunction and DM [151 153] Urinary NGAL expression is linked to nephritis and urinary tract infections [105, 154] Creatinine is a general marker of myolysis and kidney disease [155, 156] Myoglobin is another marker of myolysis; it was considered the gold standard of cardiac markers prior to the characterization of troponin. There were no significant gender based results in the analysis of IL6, microalbumin, NGAL or myoglobin. There was a statistically significant increase in myoglobin demonstrated in male cocaine positive urine specimens as compared to male control urine.

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105 Oxidative stress has been implicated in a variety of inflammatory processes. For example, i ncre ased vascular oxidative stress leads to endothelial dysfunction and hypertension. VEGF, a marker of angiogenesis, may also be elevated by endothelial dysfunction [157] Microalb umin expression is enhanced by many of the same pathophysiological conditions as VEGF. It is linked to increased cardiovascular and renal risk factors. Neither microalbumin nor VEGF were statistically significant in any analysis. Increased oxidative stress is implicated in the activation of the RAS and subsequent elevations in aldosterone. Aldosterone is a renal hormone in the RAS secreted in response to hypotension. Aldosterone was higher in female control urine, when compared to male control urine, by a statistically significant amount. There was also a statistically significant increase in aldosterone in male cocaine positive urine when compared to male control urine specimens. Increased cardiac concentrations have been linked t o heart failure. It has been observed that inhibition of the RAS results in a decline in ROS production [65, 66] proANP is a hypotensive peptide that counteracts the RAS by inhibiting the release of hormones such as aldosterone and renin [158] Increased ANP levels are detected in adult congestive heart failure, chronic renal failure and in severe essential hypertension [158, 159] No statistically significant results were seen in this study in any analysis of proANP. While there are noted gender based differences in the etiology of cardiac and vascular disease, particularly in the area of comorbid conditions, underlying blood pressure and resultant mortality, the basis of the gender dimorphism is unclear. Clearly hormones may play an impor tant role as cardiac muscle and vascular tissue are influenced by hormones such as estrogen and testosterone [1, 9, 10, 12, 59 61] The

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106 relationship between inflammatory cytokines and gender has not been clearly elucidated, although some differences in serum levels of risk markers, including lipids and acute phase proteins have been noted [6, 27, 29, 30, 80 82] Studies suggest there may be gender related differences in the regulation of IL 1 so it is possible that the interleukins serve different functions depending on gender [160, 161] were significantly higher in the control urine of women than in men. IL 6 has both pro and anti inflammatory roles depending on the target cell. It has contraction and has been found to act synergistically with both IL 1 subunits [162, 163] Elevations in IL 6 frequently precede increases in CRP [164] It may be possible that alterations in urinary IL 6 expression were transitory in our study. This could explain t he failure to detect a significant difference in IL6 between control and metabolite positive urine specimens. W omen tend to have slightly higher serum CRP than men with the same BMI and age; however, these differences are not pronounced enough to warrant gender dependent reference ranges [135, 165] The role of gender in predisposition to oxidative stress has yet to be determined. The clinical and prognostic relevance of these differences requires further research. CRP is an acute phase inflammatory marker considered to be a reliable prognostic indicator of atherosclerosis and acute myocardial infarction (AMI) [61, 82, 86] Urina ry levels peak approximately 24 hours following the inflammatory event and are undetectable within 13 to 16 hours. The differences between male and female CRP means in control urines in this study were just outside of statistical significance

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107 ( p=0.0662 ) Cocaine metabolites can remain in the urine for 48 to 72 hours following acute use, whereas CRP in urine quickly w anes. The results from this study suggest that CRP levels in urine may not be a reliable prognostic indicator for these pathophysiological pro cesses at least under the conditions of this study. As a measure of leukocyte infiltration, the concentration of MPO may increase significantly in an acute infection such as those of the urinary tract Elevated MPO is linked to adverse outcomes in acute co ronary syndromes and can be used to identify patients at risk for cardiac events in the absence of cardiac necrosis [54, 87] Elevated MPO levels have been shown to independently predict increased risk of major adverse cardiac events including MI at 30 days and 6 months [166] Th e predictive value of this marker has not been fully characterized; however, it is associated with long term adverse cardiac events as well. The differences between male and female MPO means in control urines in this study were just outside of stat istical significance ( p=0.0562 ) Cocaine is well absorbed mucosally and metabolized almost immediately by serum cholinesterase [56] The serum half life of cocaine is less than an hour. The pharmocodynamics do not change substantia lly for different routes of entry despite differences in onset of action, peak absorbance and duration of effect [167] BE and EME are the primary metabolites; up to 50% of cocaine is transformed to BE and 30 to 40% is metabolized to EME. Although neither metabolite is pharmacologically active, both BE and EME been shown to elevate blood pressure [43] The intense cardiotoxic interaction between ethanol and cocaine was the primary reason for excluding ethanol

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108 positive urine specimens from analysis. CE has a s ignificantly longer half life than both cocaine and BE; inclusion of specimens that could potentially contain CE could affect the expression of biomarkers such as myoglobin, CRP and MPO. Cocaine increases intracellular oxygen demand, elevates heart rate and arterial blood pressure [35, 168] Cocaine use has been linked to hypertension and tachycardia, although the effects on heart rate are variable [42, 169] Increases in myocardial oxygen demand are associated with the induction of oxidative stress. Cocaine use has been further shown to enhance endotheli al permeability, which may be linked to subclinical cardiovascular disease, vascular dysfunction, kidney disease, hypertension, nephrotic syndrome, glomerulonephritis, interstitial nephritis, and rhabdomyolysis [34, 49, 58, 115] Atherosclerosis, an endothelial cell dysfunction, is characterized by the development of abnormal vascular respon ses and the expression of proinflammatory and prothrombotic factors [50] MPO is expressed as a result of atherosclerotic lesions such as those seen in chronic cocaine use. Females had a significantly higher mean concentration than did males. Estradiol has been suggested as a potential endogenous substrate for MPO in plasma [88] As with CRP, the differences between male and female MPO means in control urines were just outside statistical significance ( p=0.0562 ) Examination of male and female cocaine positive urine specimens yielded no female mean (p=.0650), this result is not statistically significant. It should be noted that

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109 se veral markers that exhibited significant differences in control urines were no longer significant in cocaine positive urine. In cocaine positive urine, the mean male aldosterone was significantly higher than the male control mean (p=0.0095). The female coc aine positive aldosterone mean was somewhat lower than the control mean although the difference was not significant. Mean concentrations of aldosterone were significantly higher in the control urine of women than in men. Increased vascular oxidative str ess may lead to alterations in endothelial function and may be related to hypertension [26] The hypertens ive effects of cocaine use are well established [36, 56] Cocaine has been shown to activate the renin angiotensin system (RAS) which contributes to its cardiotoxicity. ProANP is secreted from cardiac atria in response to myo cyte strain and ischemia; increased levels of proANP have been observed in heart failure, left ventricular dysfunction, coronary artery disease, and renal failure [170] The ANP pathway decreases blood pressure in response to hypertension [171] Elevated proANP is considered predictive in renal failure, AMI and heart failure [172, 173] ProANP is cleared from the circulation in 60 to 120 minutes; it seems likely that there would not be a detectable amount in urine unless the sp ecimen was collected within 8 to 12 hours of the pathological insult. There were no significant differences in any analysis of proANP. While it well known that cocaine affects the thermoregulating centers of the brain, it is unclear if this effect is exe rted directly or via endogenous cytokines such as interleukins. IL capable of resetting the hypothalamus thermoregulatory center to change body

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110 temperature [91, 174] Our results suggest that cocaine may induce hyperexia through the interleu kins. The cellular consequences of heat and ischemic stress are quite similar [175] If cocaine exerted a significant thermogenic effect in our study following usage, IL anticipated to be elevated in urine specimens of users. However, there were no significant differences between the urine of the control group versus the cocaine positive. Unfortunately, no information was available at the time of urine collection as to do nor body temperature. Clearly, this is an important parameter that needs to be investigated further in a prospective study. NGAL exists primarily as a monomer in neutrophils and urine; it also occurs as a complex with matrix metalloproteinase 9 (MMP 9). NGAL is expressed at a low level in other tissues including the kidney, prostate and epithelia of the respiratory and alimenta ry tracts. It is presently being investigated for validation as a biomarker of urinary tract infections [176] NGAL is upregulated in processes that include apoptosis, ischemia, organogenesis and inflammation. Increased levels of NGAL from acute renal i njury can be detected in both urine and blood within two hours of the insult [57] Taken in conjunction with its presence in urine and association with ischemia, NGAL may be a significant marker of cardiac dysfunction stemming from cocaine use [177 179] Most cocaine induced chest pains do not progress to AMI. The chest pains due to cocaine are induced by myocardial ischemia via vasospasm. Given the role of NGAL in oxidative and inflammatory processes, we were surprised not to see a difference in mean values of NGAL expression. The lack of statistically significant differences did allow us to tentatively rule out UTIs as the basis

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111 for higher mean NGAL concentrations seen in female urine specimens. Coupled with the unlikelihood that the entire female cohort suffered from UTIs, the fact that male specimens had uniformly lower NGAL concentrations than did the females appears to suggest gender based expression differences. Myoglobin lacks cardiospecificity; el evated levels are considered strongly suggestive of AMI particularly in the presence of other symptoms. Elevated levels of myoglobin are also associated with rhabdomyolysis, renal failure and trauma [58] Myoglobin is released within 2 to 4 hours of significant myocardial and/or skeletal muscle injury. Levels peak 6 to 12 hours following the pathophysiological event; returning to prenecrotic levels within 36 hour s. An estimated 24% of cocaine users develop rhabdomyolysis [58] When male control urine specimens were compared to cocaine positive specimens, there was a statis tically significant difference. The results of this study suggest there are gender specific and cocaine based differences in oxidative and inflammatory cytokine expression in urine. Males and females exhibit different morbidity and mortality across a wide range of conditions which suggests basic pathophysiology may control on the incidence of multiple disease processes.

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112 Chapter 6.0 Conclusions The purpose of this study was to investigate whether or not gender differences may be present in the expression of a number of urinary proteins which may serve as markers of inflammation and oxidative stress. It was concluded that t he urinary expression of aldosterone, IL appear s to be influenced by gender. Mean significantly higher in female control urine when c ompared to male control urine. The results for hsCRP (p=.0662) and MPO (p=.0562) were also higher in female control urine when compared to male control urine; however, they did not reach statistical significance. The pathophysiological mechanisms associated with gender based differences in marker expression will require additional prospective study with expanded demographic information. The urinary expression of aldost erone and myoglobin in males is influenced by the use of cocaine. There were statistically significant increases in myoglobin (p=.0095) and aldosterone (p=.0332) expression in the cocaine positive urine of males. Oxidative metabolites of cocaine generate significant ROS; much of the pathophysiological damage

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113 associated with cocaine use stems from oxidative stress and electron transfer [32] Cocaine use affects almost every organ system; myocardial infarction, arrhythm ias, renal failure, hypertension, atherosclerosis, and rhabdomyolysis are all in the spectrum of acute and chronic cocaine toxicity [114, 115]

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114 References 1. Wizemann, T. and M. Pardue, eds. Exploring the biological contributions to human health: does sex matter? J Womens Health Gend Based Med. 2001. 288. 2. Berkley, K.J., S.S. Zalcman, and V.R. Simon, Sex and gender differences in pain and inflammation: a rapidly maturing field. Am J Physiol Regul Integr Comp Physiol, 2006. 291 (2): p. R241 4. 3. Arain, F.A., et al., Sex/gender medicine. The biological basis for personalized care in cardiovascular medicine. Circ J, 2009. 73 (10): p. 1774 82. 4. Hutchens, M.P., et al., Renal ischemia: does sex matter? Anesth Analg, 2008. 107 (1): p. 239 49. 5. McCarthy, M., The "gender gap" in autoimmune disease. Lancet, 2000. 356 (9235): p. 1088. 6. Olafsdottir, I.S., et al., Gender differences in th e association between C reactive protein, lung function impairment, and COPD. Int J Chron Obstruct Pulmon Dis, 2007. 2 (4): p. 635 42. 7. Perucca, J., et al., Sex difference in urine concentration across differing ages, sodium intake, and level of kidney di sease. Am J Physiol Regul Integr Comp Physiol, 2007. 292 (2): p. R700 5. 8. Spitzer, J.A., Gender differences in some host defense mechanisms. Lupus, 1999. 8 (5): p. 380 3. 9. Murphy, E. and C. Steenbergen, Gender based differences in mechanisms of protection in myocardial ischemia reperfusion injury. Cardiovasc Res, 2007. 75 (3): p. 478 86. 10. Ostadal, B., et al., Gender differences in cardiac ischemic injury and protection -experimental aspects. Exp Biol Med (Maywood), 2009. 234 (9): p. 1011 9. 11. Liu, P.Y., A.K. Death, and D.J. Handelsman, Androgens and cardiovascular disease. Endocr Rev, 2003. 24 (3): p. 313 40. 12. Quyyumi, A.A., Women and ischemic heart disease: pathophysiologic implications from the Women's Ischemia Syndrome Evaluation (WISE) St udy and future research steps. J Am Coll Cardiol, 2006. 47 (3 Suppl): p. S66 71. 13. Devarajan, P., Emerging urinary biomarkers in the diagnosis of acute kidney injury. Expert Opin Med Diagn, 2008. 2 (4): p. 387 398. 14. Pontremoli, R., et al., Microalbuminu ria, cardiovascular, and renal risk in primary hypertension. J Am Soc Nephrol, 2002. 13 Suppl 3 : p. S169 72. 15. Kang, A.K. and J.A. Miller, Impact of gender on renal disease: the role of the renin angiotensin system. Clin Invest Med, 2003. 26 (1): p. 38 44

PAGE 132

115 16. Maric, C. and S. Sullivan, Estrogens and the diabetic kidney. Gend Med, 2008. 5 Suppl A : p. S103 13. 17. Sandberg, K., Mechanisms underlying sex differences in progressive renal disease. Gend Med, 2008. 5 (1): p. 10 23. 18. Seliger, S.L., C. Davis, an d C. Stehman Breen, Gender and the progression of renal disease. Curr Opin Nephrol Hypertens, 2001. 10 (2): p. 219 25. 19. Silbiger, S.R. and J. Neugarten, The impact of gender on the progression of chronic renal disease. Am J Kidney Dis, 1995. 25 (4): p. 51 5 33. 20. Lansang, M.C. and N.K. Hollenberg, Renal perfusion and the renal hemodynamic response to blocking the renin system in diabetes: are the forces leading to vasodilation and vasoconstriction linked? Diabetes, 2002. 51 (7): p. 2025 8. 21. Itoh, Y., et al., 17beta estradiol induces IL 1alpha gene expression in rheumatoid fibroblast like synovial cells through estrogen receptor alpha (ERalpha) and augmentation of transcriptional activity of Sp1 by dissociating histone deacetylase 2 from ERalpha. J Immuno l, 2007. 178 (5): p. 3059 66. 22. Nanda, N., Z. Bobby, and A. Hamide, Oxidative stress and protein glycation in primary hypothyroidism. Male/female difference. Clin Exp Med, 2008. 8 (2): p. 101 8. 23. Kerksick, C., et al., Gender related differences in muscle injury, oxidative stress, and apoptosis. Med Sci Sports Exerc, 2008. 40 (10): p. 1772 80. 24. Nguyen, V.H. and M.A. McLaughlin, Coronary artery disease in women: a review of emerging cardiovascular risk factors. Mt Sinai J Med, 2002. 69 (5): p. 338 49 25. Fearon, I.M. and S.P. Faux, Oxidative stress and cardiovascular disease: novel tools give (free) radical insight. J Mol Cell Cardiol, 2009. 47 (3): p. 372 81. 26. Heitzer, T., et al., Endothelial dysfunction, oxidative stress, and risk of cardiovascul ar events in patients with coronary artery disease. Circulation, 2001. 104 (22): p. 2673 8. 27. Kaysen, G.A. and J.P. Eiserich, The role of oxidative stress altered lipoprotein structure and function and microinflammation on cardiovascular risk in patients with minor renal dysfunction. J Am Soc Nephrol, 2004. 15 (3): p. 538 48. 28. Loscalzo, J., Oxidative stress in endothelial cell dysfunction and thrombosis. Pathophysiol Haemost Thromb, 2002. 32 (5 6): p. 359 60. 29. Miller, A.A., et al., Effect of gender and sex hormones on vascular oxidative stress. Clin Exp Pharmacol Physiol, 2007. 34 (10): p. 1037 43. 30. Surekha, R.H., et al., Oxidative stress and total antioxidant status in myocardial infarction. Singapore Med J, 2007. 48 (2): p. 137 42. 31. Cooper, C.E., et al., Exercise, free radicals and oxidative stress. Biochem Soc Trans, 2002. 30 (2): p. 280 5. 32. Kovacic, P., Role of oxidative metabolites of cocaine in toxicity and addiction: oxidative stress and electron transfer. Med Hypotheses, 2005. 64 (2): p. 350 6. 33. Plotnikov, E.Y., et al., Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney's mitochondria. Biochim Biophys Acta, 2009. 1792 (8): p. 796 803.

PAGE 133

116 34. Jaffe, J.A. and P.L. Kimmel, Chronic nephropathies of cocaine and h eroin abuse: a critical review. Clin J Am Soc Nephrol, 2006. 1 (4): p. 655 67. 35. Qureshi, A.I., et al., Cocaine use and the likelihood of nonfatal myocardial infarction and stroke: data from the Third National Health and Nutrition Examination Survey. Circulation, 2001. 103 (4): p. 502 6. 36. Restrepo, C.S., et al., Cardiovascular complications of cocaine: imaging findings. Emerg Radiol, 2009. 16 (1): p. 11 9. 37. Middleton, P.M., Cerebrovascular Effects Of Cocaine The Internet Journal of Emergency Medic ine, 2004. 2 (1). 38. Toth, A.R. and T. Varga, Myocardium and striated muscle damage caused by licit or illicit drugs. Leg Med (Tokyo), 2009. 11 Suppl 1 : p. S484 7. 39. Pozner, C.N., M. Levine, and R. Zane, The cardiovascular effects of cocaine. J Emerg Me d, 2005. 29 (2): p. 173 8. 40. Keller, K.B. and L. Lemberg, The cocaine abused heart. Am J Crit Care, 2003. 12 (6): p. 562 6. 41. Kloner, R.A., et al., The effects of acute and chronic cocaine use on the heart. Circulation, 1992. 85 (2): p. 407 19. 42. Kloner R.A. and S.H. Rezkalla, Cocaine and the heart. N Engl J Med, 2003. 348 (6): p. 487 8. 43. Karch, S.B., Karch's Pathology of Drug Abuse, Fourth Edition 2009, Boca Raton: CRC/Taylor & Francis. 44. Boess, F., et al., Effects of cocaine and its oxidative met abolites on mitochondrial respiration and generation of reactive oxygen species. Biochem Pharmacol, 2000. 60 (5): p. 615 23. 45. Labib, R., R. Turkall, and M.S. Abdel Rahman, Inhibition of cocaine oxidative metabolism attenuates endotoxin potentiation of cocaine mediated hepatotoxicity. Toxicology, 2002. 179 (1 2): p. 9 19. 46. Wilbert Lampen, U., et al., Cocaine increases the endothelial release of immunoreactive endothelin and its concentrations in human plasma and urine: reversal by coincubation with sig ma receptor antagonists. Circulation, 1998. 98 (5): p. 385 90. 47. Kasahara, Y., et al., [Trend in the study of cocaine addiction]. Nippon Rinsho. 68 (8): p. 1479 85. 48. Nicholls, S.J. and S.L. Hazen, Myeloperoxidase and cardiovascular disease. Arterioscler Thromb Vasc Biol, 2005. 25 (6): p. 1102 11. 49. Chen, Y., et al., Cocaine and catecholamines enhance inflammatory cell retention in the coronary circulation of mice by upregulation of adhesion molecules. Am J Physiol Heart Circ Physiol, 2005. 288 (5): p. H2 323 31. 50. Lin, C.C., et al., The relation of metabolic syndrome according to five definitions to cardiovascular risk factors -a population based study. BMC Public Health, 2009. 9 : p. 484. 51. Vroegop, M.P., et al., The emergency care of cocaine intoxicat ions. Neth J Med, 2009. 67 (4): p. 122 6. 52. Poon, H.F., et al., Cocaine induced oxidative stress precedes cell death in human neuronal progenitor cells. Neurochem Int, 2007. 50 (1): p. 69 73.

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117 53. Fried, L., et al., Inflammatory and prothrombotic markers an d the progression of renal disease in elderly individuals. J Am Soc Nephrol, 2004. 15 (12): p. 3184 91. 54. Klebanoff, S.J., Myeloperoxidase: friend and foe. J Leukoc Biol, 2005. 77 (5): p. 598 625. 55. Matthijsen, R.A., et al., Myeloperoxidase is critically involved in the induction of organ damage after renal ischemia reperfusion. Am J Pathol, 2007. 171 (6): p. 1743 52. 56. Richards, I.S., Health effects of illicit cocaine use. Pulmonary and Critical Care Update American College of Chest Physicians, 1991. 7 (2): p. 1 7. 57. Trof, R.J., et al., Biomarkers of acute renal injury and renal failure. Shock, 2006. 26 (3): p. 245 53. 58. Richards, J.R., Rhabdomyolysis and drugs of abuse. J Emerg Med, 2000. 19 (1): p. 51 6. 59. Reckelhoff, J.F., Gender differences in th e regulation of blood pressure. Hypertension, 2001. 37 (5): p. 1199 208. 60. Sieveking, D.P., R.W. Chow, and M.K. Ng, Androgens, angiogenesis and cardiovascular regeneration. Curr Opin Endocrinol Diabetes Obes, 2010. 61. Vitale, C., et al., Gender differenc es in the cardiovascular effects of sex hormones. Fundam Clin Pharmacol, 2010. 62. Komukai, K., S. Mochizuki, and M. Yoshimura, Gender and the renin angiotensin aldosterone system. Fundam Clin Pharmacol. 63. Sieveking, D.P., R.W. Chow, and M.K. Ng, Androgens, angiogenesis and cardiovascular regeneration. Curr Opin Endocrinol Diabetes Obes. 17 (3): p. 277 83. 64. Kautzky Willer, A. and A. Handisurya, Metabolic diseases and associated complications: sex and gender matter! Eur J Clin Invest, 2009. 39 (8): p. 631 48. 65. Cooper, S.A., et al., Renin angiotensin aldosterone system and oxidative stress in cardiovascular insulin resistance. Am J Physiol Heart Circ Physiol, 2007. 293 (4): p. H2009 23. 66. Leiter, L.A. and R.Z. Lewanczuk, Of the renin angiotensin system and reactive oxygen species Type 2 diabetes and angiotensin II inhibition. Am J Hypertens, 2005. 18 (1): p. 121 8. 67. Coresh, J., et al., Prevalence of chronic kidney disease in the United States. JAMA, 2007. 298 (17): p. 2038 47. 68. Perez Lopez, F.R., et al., Gender differences in cardiovascular disease: hormonal and biochemical influences. Reprod Sci. 17 (6): p. 511 31. 69. Kobayashi, H., et al., Increased prevalence of carotid artery atherosclerosis in rheumatoid arthritis is artery specific. J Rheumatol. 37 (4): p. 730 9. 70. Gameiro, C. and F. Romao, Changes in the immune system during menopause and aging. Front Biosci (Elite Ed). 2 : p. 1299 303. 71. Gu, H.F., et al., SOX2 has gender specific genetic effects on diabetic nephropathy i n samples from patients with type 1 diabetes mellitus in the GoKinD study. Gend Med, 2009. 6 (4): p. 555 64. 72. Rubtsov, A.V., et al., Genetic and hormonal factors in female biased autoimmunity. Autoimmun Rev. 9 (7): p. 494 8.

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118 73. Brahmachari, S. and K. Pah an, Gender specific expression of beta1 integrin of VLA 4 in myelin basic protein primed T cells: implications for gender bias in multiple sclerosis. J Immunol. 184 (11): p. 6103 13. 74. Dinesh, R.K., B.H. Hahn, and R.P. Singh, PD 1, gender, and autoimmunit y. Autoimmun Rev. 9 (8): p. 583 7. 75. Gao, H.X., et al., Sex and autoantibody titers determine the development of neuropsychiatric manifestations in lupus prone mice. J Neuroimmunol. 76. Maioli, M., et al., Number of autoantibodies and HLA genotype, more t han high titers of glutamic acid decarboxylase autoantibodies, predict insulin dependence in latent autoimmune diabetes of adults. Eur J Endocrinol. 163 (4): p. 541 549. 77. Shen, H., et al., Gender dependent Expression of Murine Irf5 Gene: Implications for Sex Bias in Autoimmunity. J Mol Cell Biol. 78. Khan, N., et al., Effects of age, gender, and immunosuppressive agents on in vivo toll like receptor pathway responses. Hum Immunol. 71 (4): p. 372 6. 79. Li, X., et al., 17beta estradiol enhances the response of plasmacytoid dendritic cell to CpG. PLoS One, 2009. 4 (12): p. e8412. 80. Jankord, R., et al., Sex difference in link between interleukin 6 and stress. Endocrinology, 2007. 148 (8): p. 3758 64. 81. Lee, C.E., A. McArdle, and R.D. Griffiths, The role of h ormones, cytokines and heat shock proteins during age related muscle loss. Clin Nutr, 2007. 26 (5): p. 524 34. 82. Yeun, J.Y. and G.A. Kaysen, C reactive protein, oxidative stress, homocysteine, and troponin as inflammatory and metabolic predictors of ather osclerosis in ESRD. Curr Opin Nephrol Hypertens, 2000. 9 (6): p. 621 30. 83. Libby, P., P.M. Ridker, and A. Maseri, Inflammation and atherosclerosis. Circulation, 2002. 105 (9): p. 1135 43. 84. Libby, P., P.M. Ridker, and G.K. Hansson, Inflammation in athero sclerosis: from pathophysiology to practice. J Am Coll Cardiol, 2009. 54 (23): p. 2129 38. 85. Ridker, P.M., et al., C reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med, 2000. 342 (12): p. 8 36 43. 86. Sander, K., et al., High sensitivity C reactive protein is independently associated with early carotid artery progression in women but not in men: the INVADE Study. Stroke, 2007. 38 (11): p. 2881 6. 87. Wu, A.H., Novel biomarkers of cardiovascula r disease: myeloperoxidase for acute and/or chronic heart failure? Clin Chem, 2009. 55 (1): p. 12 4. 88. Zhang, C., et al., Interaction of myeloperoxidase with vascular NAD(P)H oxidase derived reactive oxygen species in vasculature: implications for vascula r diseases. Am J Physiol Heart Circ Physiol, 2003. 285 (6): p. H2563 72. 89. Jarvie, J.L. and J.M. Foody, Recognizing and Improving Health Care Disparities in the Prevention of Cardiovascular Disease in Women. Curr Cardiol Rep. 12 (6): p. 488 496. 90. Dowd, J.B., A. Zajacova, and A.E. Aiello, Predictors of inflammation in u.s. Children aged 3 16 years. Am J Prev Med. 39 (4): p. 314 20.

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119 91. Ferrero Miliani, L., et al., Chronic inflammation: importance of NOD2 and NALP3 in interleukin 1beta generation. Clin Exp Immunol, 2007. 147 (2): p. 227 35. 92. Pottratz, S.T., et al., 17 beta Estradiol inhibits expression of human interleukin 6 promoter reporter constructs by a receptor dependent mechanism. J Clin Invest, 1994. 93 (3): p. 944 50. 93. Corcoran, M.P., et al., Sex hormone modulation of proinflammatory cytokine and C reactive protein expression in macrophages from older men and postmenopausal women. J Endocrinol. 206 (2): p. 217 24. 94. Gilbert, K.C. and N.J. Brown, Aldosterone and inflammation. Curr Opin Endocrinol Diabetes Obes. 17 (3): p. 199 204. 95. Jeong, Y., et al., Aldosterone activates endothelial exocytosis. Proc Natl Acad Sci U S A, 2009. 106 (10): p. 3782 7. 96. Colgan, S.P. and C.T. Taylor, Hypoxia: an alarm signal during intestinal inflammation. Nat Rev Gastroenterol Hepatol. 7 (5): p. 281 7. 97. Asselbergs, F.W., et al., Vascular endothelial growth factor: the link between cardiovascular risk factors and microalbuminuria? Int J Cardiol, 2004. 93 (2 3): p. 211 5. 98. Aviv, A., The reactive oxidativ e species renin angiotensin system link. J Hypertens, 2002. 20 (12): p. 2357 8. 99. Wu, C.C., et al., Myeloperoxidase serves as a marker of oxidative stress during single haemodialysis session using two different biocompatible dialysis membranes. Nephrol Di al Transplant, 2005. 20 (6): p. 1134 9. 100. Carbone, A., et al., Human Harvey ras is biochemically different from Kirsten or N ras. Oncogene, 1991. 6 (5): p. 731 7. 101. Maggio, M., et al., The relationship between testosterone and molecular markers of inf lammation in older men. J Endocrinol Invest, 2005. 28 (11 Suppl Proceedings): p. 116 9. 102. Flogel, U., et al., Role of myoglobin in the antioxidant defense of the heart. FASEB J, 2004. 18 (10): p. 1156 8. 103. Brancaccio, P., G. Lippi, and N. Maffulli, Bio chemical markers of muscular damage. Clin Chem Lab Med. 48 (6): p. 757 67. 104. Brown, N.J., Aldosterone and vascular inflammation. Hypertension, 2008. 51 (2): p. 161 7. 105. Bennett, M., et al., Urine NGAL predicts severity of acute kidney injury after card iac surgery: a prospective study. Clin J Am Soc Nephrol, 2008. 3 (3): p. 665 73. 106. De Vito, P., et al., Atrial natriuretic peptide and oxidative stress. Peptides. 31 (7): p. 1412 9. 107. Videla, L.A., Hormetic responses of thyroid hormone calorigenesis in the liver: Association with oxidative stress. IUBMB Life. 62 (6): p. 460 6. 108. Wilson, L.D., et al., Cocaine, ethanol, and cocaethylene cardiotoxity in an animal model of cocaine and ethanol abuse. Acad Emerg Med, 2001. 8 (3): p. 211 22. 109. Wiener, S.E. et al., Patients with detectable cocaethylene are more likely to require intensive care unit admission after trauma. Am J Emerg Med, 2009.

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120 110. Ndikum Moffor, F.M., T.R. Schoeb, and S.M. Roberts, Liver toxicity from norcocaine nitroxide, an N oxidative m etabolite of cocaine. J Pharmacol Exp Ther, 1998. 284 (1): p. 413 9. 111. Ferris, M.J., et al., Cocaine Insensitive Dopamine Transporters with Intact Substrate Transport Produced by Self Administration. Biol Psychiatry. 112. Lloyd, S.A., et al., Cocaine sel ectively increases proliferation in the adult murine hippocampus. Neurosci Lett. 113. Zayara, A.E., et al., Blockade of nucleus accumbens 5 HT(2A) and 5 HT (2C) receptors prevents the expression of cocaine induced behavioral and neurochemical sensitization in rats. Psychopharmacology (Berl). 114. Su, J., et al., Cocaine induces apoptosis in primary cultured rat aortic vascular smooth muscle cells: possible relationship to aortic dissection, atherosclerosis, and hypertension. Int J Toxicol, 2004. 23 (4): p. 233 7. 115. van der Woude, F.J., Cocaine use and kidney damage. Nephrol Dial Transplant, 2000. 15 (3): p. 299 301. 116. Baumann, B.M., et al., Cardiac and hemodynamic assessment of patients with cocaine associated chest pain syndromes. J Toxicol Clin Toxicol, 2000. 38 (3): p. 283 90. 117. Kiyatkin, E.A., Brain hyperthermia as physiological and pathological phenomena. Brain Res Brain Res Rev, 2005. 50 (1): p. 27 56. 118. Halpern, J.H., et al., Diminished interleukin 6 response to proinflam matory challenge in men and women after intravenous cocaine administration. J Clin Endocrinol Metab, 2003. 88 (3): p. 1188 93. 119. Ershler, W.B., Interleukin 6: a cytokine for gerontologists. J Am Geriatr Soc, 1993. 41 (2): p. 176 81. 120. McCance Katz, E.F ., et al., Gender effects following repeated administration of cocaine and alcohol in humans. Subst Use Misuse, 2005. 40 (4): p. 511 28. 121. Galankin, T., E. Shekunova, and E. Zvartau, Estradiol lowers intracranial self stimulation thresholds and enhances cocaine facilitation of intracranial self stimulation in rats. Horm Behav. 122. Sinha, R., et al., Sex steroid hormones, stress response, and drug craving in cocaine dependent women: implications for relapse susceptibility. Exp Clin Psychopharmacol, 2007. 15 (5): p. 445 52. 123. Minerly, A.E., et al., Testosterone differentially alters cocaine induced ambulatory and rearing behavioral responses in adult and adolescent rats. Pharmacol Biochem Behav. 94 (3): p. 404 9. 124. Festa, E.D., et al., Cocaine induced s ex differences in D1 dopamine receptor mRNA levels after acute cocaine administration. Ethn Dis. 20 (1 Suppl 1): p. S1 24 7. 125. Sun, W.L., et al., Sex differences in dopamine D2 like receptor mediated G protein activation in the medial prefrontal cortex a fter cocaine. Ethn Dis. 20 (1 Suppl 1): p. S1 88 91. 126. Kilts, C.D., et al., The neural correlates of cue induced craving in cocaine dependent women. Am J Psychiatry, 2004. 161 (2): p. 233 41.

PAGE 138

121 127. Carrero, J.J., et al., Prevalence and clinical implication s of testosterone deficiency in men with end stage renal disease. Nephrol Dial Transplant. 128. Ciambrone, G. and J.C. Kaski, Gender differences in the treatment of chronic ischemic heart disease: prognostic implications. Fundam Clin Pharmacol, 2009. 129. Doering, L.V., et al., Gender specific characteristics of individuals with depressive symptoms and coronary heart disease. Heart Lung. 130. Goetzl, E.J., et al., Gender specificity of altered human immune cytokine profiles in aging. FASEB J. 24 (9): p. 3580 9. 131. Groleger, U. and V. Novak Grubic, Gender, psychosis and psychotropic drugs: differences and similarities. Psychiatr Danub. 22 (2): p. 338 42. 132. Hung, M.Y., et al., Interactions among gender, age, hypertension and C reactive protein in coronary v asospasm. Eur J Clin Invest. 133. Jarvie, J.L. and J.M. Foody, Recognizing and Improving Health Care Disparities in the Prevention of Cardiovascular Disease in Women. Curr Cardiol Rep. 134. Jimenez Navarro, M.F., et al., Influence of gender on long term pr ognosis of patients with chronic heart failure seen in heart failure clinics. Clin Cardiol. 33 (3): p. E13 8. 135. Khera, A., et al., Race and gender differences in C reactive protein levels. J Am Coll Cardiol, 2005. 46 (3): p. 464 9. 136. Legato, M.J., et a l., Gender specific care of the patient with diabetes: review and recommendations. Gend Med, 2006. 3 (2): p. 131 58. 137. Leuzzi, C. and M.G. Modena, Coronary artery disease: clinical presentation, diagnosis and prognosis in women. Nutr Metab Cardiovasc Dis 20 (6): p. 426 35. 138. Minh, H.V., et al., Gender differences in prevalence and socioeconomic determinants of hypertension: findings from the WHO STEPs survey in a rural community of Vietnam. J Hum Hypertens, 2006. 20 (2): p. 109 15. 139. Puustinen, P.J., et al., Gender specific association of psychological distress with cardiovascular risk scores. Scand J Prim Health Care. 28 (1): p. 36 40. 140. Tan, Y.Y., G.C. Gast, and Y.T. van der Schouw, Gender differences in risk factors for coronary h eart disease. Maturitas. 65 (2): p. 149 60. 141. Vitale, C., et al., Gender differences in the cardiovascular effects of sex hormones. Fundam Clin Pharmacol. 142. Xu, R., et al., Gender differences in age related decline in glomerular filtration rates in he althy people and chronic kidney disease patients. BMC Nephrol. 11 : p. 20. 143. Goo, Y.A., et al., Urinary proteomics evaluation in interstitial cystitis/painful bladder syndrome: a pilot study. Int Braz J Urol. 36 (4): p. 464 78; discussion 478 9, 479. 144. Adachi, J., Kumar, C., Zhang, Y., Olsen, J.V., & Mann, M., The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biology, 2006. 7 (9): p. R80. 145. Pejcic, M., S. Stojnev, and V. Stefanovic, Urinary proteomics -a tool for biomarker discovery. Ren Fail. 32 (2): p. 259 68. 146. Fliser, D., et al., Advances in urinary proteome analysis and biomarker discovery. J Am Soc Nephrol, 2007. 18 (4): p. 1057 71.

PAGE 139

122 147. Schnabel, R.B., et al., Multiple marker approach to risk stratification in patients with stable coronary artery disease. Eur Heart J. 148. von Kanel, R., et al., Heart rate variability and biomarkers of systemic inflammation in patients with stable coronary heart disease: findings from the Hear t and Soul Study. Clin Res Cardiol. 149. Michowitz, Y., et al., Usefulness of serum myeloperoxidase in prediction of mortality in patients with severe heart failure. Isr Med Assoc J, 2008. 10 (12): p. 884 8. 150. Cannon, J.G., et al., Interleukin 1 beta, in terleukin 1 receptor antagonist, and soluble interleukin 1 receptor type II secretion in chronic fatigue syndrome. J Clin Immunol, 1997. 17 (3): p. 253 61. 151. Aguilar, M.I., et al., Albuminuria and the risk of incident stroke and stroke types in older adu lts. Neurology. 152. Cottone, S., et al., Microalbuminuria and early endothelial activation in essential hypertension. J Hum Hypertens, 2007. 21 (2): p. 167 72. 153. Riaz, S., et al., Proteomic Identification of Human Urinary Biomarkers in Diabetes Mellitus Type 2. Diabetes Technol Ther. 154. Wu, Y., et al., Pathological Significance of a Panel of Urinary Biomarkers in Patients with Drug Induced Tubulointerstitial Nephritis. Clin J Am Soc Nephrol. 155. Bagshaw, S.M. and R. Bellomo, Cystatin C in acute kidney injury. Curr Opin Crit Care. 156. Smith, G.L., et al., Renal impairment and outcomes in heart failure: systematic review and meta analysis. J Am Coll Cardiol, 2006. 47 (10): p. 1987 96. 157. Machalinska, A., et al., Different populations of circulating end othelial cells in patients with the exudative form of age related macular degeneration: A novel insight into pathogenesis. Invest Ophthalmol Vis Sci. 158. Ruskoaho, H., Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol Rev, 1992. 44 (4): p. 479 602. 159. Yandle, T.G., Biochemistry of natriuretic peptides. J Intern Med, 1994. 235 (6): p. 561 76. 160. Bry, K., et al., Interleukin 1 receptor antagonist in the fetomaternal compartment. Acta Paediatr, 1995. 84 (3): p. 233 6. 161. Lynch, E.A. C.A. Dinarello, and J.G. Cannon, Gender differences in IL 1 alpha, IL 1 beta, and IL 1 receptor antagonist secretion from mononuclear cells and urinary excretion. J Immunol, 1994. 153 (1): p. 300 6. 162. Kleemann, R., S. Zadelaar, and T. Kooistra, Cytokin es and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc Res, 2008. 79 (3): p. 360 76. 163. Renes, J., et al., Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4 hydroxynonenal. Bio chem J, 2000. 350 Pt 2 : p. 555 61. 164. Thakore, A.H., et al., Association of multiple inflammatory markers with carotid intimal medial thickness and stenosis (from the Framingham Heart Study). Am J Cardiol, 2007. 99 (11): p. 1598 602.

PAGE 140

123 165. Orri, J.C., S.R. Carter, and E.B. Howington, Gender comparison of C reactive protein and cardiovascular disease risk in college students and intercollegiate athletes. J Sports Med Phys Fitness. 50 (1): p. 72 8. 166. Baldus, S., et al., Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation, 2003. 108 (12): p. 1440 5. 167. Lange, R.A., J.E. Cigarroa, and L.D. Hillis, Theodore E. Woodward award: cardiovascular complications of cocaine abuse. Trans Am Clin Climatol Assoc 2004. 115 : p. 99 111; discussion 112 4. 168. Tuncel, M., et al., Mechanism of the blood pressure -raising effect of cocaine in humans. Circulation, 2002. 105 (9): p. 1054 9. 169. Reed, S.C., et al., Cardiovascular and subjective effects of repeated smoked cocaine administration in experienced cocaine users. Drug Alcohol Depend, 2009. 102 (1 3): p. 102 7. 170. Bocek, T., et al., Use of pro atrial natriuretic peptide in the detection of myocardial ischaemia. Eur J Clin Invest, 2005. 35 (7): p. 450 6. 171. Dolci, A. and M. Panteghini, The exciting story of cardiac biomarkers: from retrospective detection to gold diagnostic standard for acute myocardial infarction and more. Clin Chim Acta, 2006. 369 (2): p. 179 87. 172. Franz, M., W. Woloszczuk, and W.H. Horl, Plasma concentration and urinary excretion of N terminal proatrial natriuretic peptides in patients with kidney diseases. Kidney Int, 2001. 59 (5): p. 1928 34. 173. Gardner, T.J. and T.R. Kosten, Therapeutic options and challenges for substances of abuse. Dialogues Clin Neurosci, 2007. 9 (4): p. 431 45. 174. Jackson, A.M., et al., Changes in urinary cytokines and soluble intercellular adhesion molecule 1 (ICAM 1) in bladder cancer patients after bacillus Calmette Guerin (BCG) immunotherapy. Clin Exp Immunol, 1995. 99 (3): p. 369 75. 175. Hayase, T., Y. Yamamoto, and K. Yamamoto, Toxic cocaine and convulsant induced modification of forced swimming behaviors and their interaction with ethanol: comparison with immobilization stress. BMC Pharmacol, 2002. 2 : p. 19 176. Yilmaz, A., et al., Early prediction of urinary tract infection with urinary neutrophil gelatinase associated lipocalin. Pediatr Nephrol, 2009. 24 (12): p. 2387 92. 177. Pisitkun, T., R. Johnstone, and M.A. Knepper, Discovery of urinary biomarkers. M ol Cell Proteomics, 2006. 5 (10): p. 1760 71. 178. Sirera, R., et al., Quantification of proinflammatory cytokines in the urine of congestive heart failure patients. Its relationship with plasma levels. Eur J Heart Fail, 2003. 5 (1): p. 27 31. 179. Wagener, G., et al., Urinary neutrophil gelatinase associated lipocalin and acute kidney injury after cardiac surgery. Am J Kidney Dis, 2008. 52 (3): p. 425 33.

PAGE 141

124 Bibliography 1. Afonso, L., T. Mohammad, and D. Thatai, Crack whips the heart: a review of the cardiovascular toxicity of cocaine. Am J Cardiol, 2007. 100 (6): p. 1040 3. 2. Alayash, A.I., R.P. Patel, and R.E. Cashon, Redox reactions of hemoglobin and myoglobin: biological and toxicological implications. Antioxid Redox Signal, 2001. 3 (2): p. 313 27. 3. Alkerwi, A., et al., First nationwide survey on cardiovascular risk factors in Grand Duchy of Luxembourg (ORISCAV LUX). BMC Public Health. 10 : p. 468. 4. Almkvist, O. and B. Winblad, Early diagnosis of Alzheimer dementia based on clinical and biological factors. Eur Arch Psychiatry Clin Neurosci, 1999. 249 Suppl 3 : p. 3 9. 5. Apple, F.S., et al., Multiple biomarker use for detection of adverse events in patients presenting with symptoms suggestive of acute coronary syndrome. Clin Chem, 2007. 53 (5): p. 874 81. 6. Apple, F.S., et al., Assessment of left ventricular function using serum cardiac troponin I measurements following myocardial infarction. Clin Chim Acta, 1998. 272 (1): p. 59 67. 7. Apple, F.S., et al., Future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome. Clin Chem, 2005. 51 (5): p. 810 24. 8. Arant, C.B., et al., Multimarker approach predicts adverse cardiovascular events in women evaluated for suspected ischemia: results from the national heart, lung, an d blood institute sponsored women's ischemia syndrome evaluation. Clin Cardiol, 2009. 32 (5): p. 244 50. 9. Aslibekyan, S., E.B. Levitan, and M.A. Mittleman, Prevalent cocaine use and myocardial infarction. Am J Cardiol, 2008. 102 (8): p. 966 9. 10. Asselber gs, F.W., et al., Vascular endothelial growth factor: the link between cardiovascular risk factors and microalbuminuria? Int J Cardiol, 2004. 93 (2 3): p. 211 5. 11. Aviv, A., The reactive oxidative species renin angiotensin system link. J Hypertens, 2002. 20 (12): p. 2357 8. 12. Bahmani, P., et al., Neutrophil gelatinase associated lipocalin induces the expression of heme oxygenase 1 and superoxide dismutase 1, 2. Cell Stress Chaperones. 15 (4): p. 395 403. 13. Basu, S., et al., Microalbuminuria: A novel biom arker of sepsis. Indian J Crit Care Med. 14 (1): p. 22 8.

PAGE 142

125 14. Baumann, B.M., et al., Cardiac and hemodynamic assessment of patients with cocaine associated chest pain syndromes. J Toxicol Clin Toxicol, 2000. 38 (3): p. 283 90. 15. Bazille, C., et al., Brain damage after heat stroke. J Neuropathol Exp Neurol, 2005. 64 (11): p. 970 5. 16. Beasley, K.N., et al., The effect of oral vs. Intravenous rehydration on circulating myoglobin and creatine kinase. J Strength Cond Res. 24 (1): p. 60 7. 17. Bennett, M., et al. Urine NGAL predicts severity of acute kidney injury after cardiac surgery: a prospective study. Clin J Am Soc Nephrol, 2008. 3 (3): p. 665 73. 18. Bernard, T.E., et al., WBGT clothing adjustment factors for four clothing ensembles and the effects of metab olic demands. J Occup Environ Hyg, 2008. 5 (1): p. 1 5; quiz d21 3. 19. Berton, G., et al., Comparison of C reactive protein and albumin excretion as prognostic markers for 10 year mortality after myocardial infarction. Clin Cardiol. 33 (8): p. 508 15. 20. Bhangoo, P., A. Parfitt, and T. Wu, Best evidence topic report. Cocaine induced myocardial ischaemia: nitrates versus benzodiazepines. Emerg Med J, 2006. 23 (7): p. 568 9. 21. Bianchi, S., R. Bigazzi, and V.M. Campese, Antagonists of aldosterone and protein uria in patients with CKD: an uncontrolled pilot study. Am J Kidney Dis, 2005. 46 (1): p. 45 51. 22. Bocek, T., et al., Use of pro atrial natriuretic peptide in the detection of myocardial ischaemia. Eur J Clin Invest, 2005. 35 (7): p. 450 6. 23. Boess, F., et al., Effects of cocaine and its oxidative metabolites on mitochondrial respiration and generation of reactive oxygen species. Biochem Pharmacol, 2000. 60 (5): p. 615 23. 24. Borgeson, E. and C. Godson, Molecular circuits of resolution in renal disease. S cientificWorldJournal. 10 : p. 1370 85. 25. Bortone, A.S., et al., Inflammatory response and angiogenesis after percutaneous transmyocardial laser revascularization. Ann Thorac Surg, 2000. 70 (3): p. 1134 8. 26. Bouchama, A. and J.P. Knochel, Heat stroke. N Engl J Med, 2002. 346 (25): p. 1978 88. 27. Brahmachari, S. and K. Pahan, Gender specific expression of beta1 integrin of VLA 4 in myelin basic protein primed T cells: implications for gender bias in multiple sclerosis. J Immunol. 184 (11): p. 6103 13. 28. B rancaccio, P., G. Lippi, and N. Maffulli, Biochemical markers of muscular damage. Clin Chem Lab Med. 48 (6): p. 757 67. 29. Brevetti, G., et al., Myeloperoxidase, but not C reactive protein, predicts cardiovascular risk in peripheral arterial disease. Eur H eart J, 2008. 29 (2): p. 224 30. 30. Briet, M. and E.L. Schiffrin, Aldosterone: effects on the kidney and cardiovascular system. Nat Rev Nephrol. 6 (5): p. 261 73.

PAGE 143

126 31. Brinton, E.A., Effects of ethanol intake on lipoproteins and atherosclerosis. Curr Opin Li pidol. 21 (4): p. 346 51. 32. Brooks, G.C., M.J. Blaha, and R.S. Blumenthal, Relation of C reactive protein to abdominal adiposity. Am J Cardiol. 106 (1): p. 56 61. 33. Brown, N.J., Aldosterone and vascular inflammation. Hypertension, 2008. 51 (2): p. 161 7. 34. Bruckner, L., et al., Pneumocystis carinii infection sensitizes lung to radiation induced injury after syngeneic marrow transplantation: role of CD4+ T cells. Am J Physiol Lung Cell Mol Physiol, 2006. 290 (6): p. L1087 96. 35. Brueckmann, M., et al., Ma rkers of myocardial damage, tissue healing, and inflammation after radiofrequency catheter ablation of atrial tachyarrhythmias. J Cardiovasc Electrophysiol, 2004. 15 (6): p. 686 91. 36. Budd, G.M., Wet bulb globe temperature (WBGT) -its history and its limi tations. J Sci Med Sport, 2008. 11 (1): p. 20 32. 37. Burnett, L.B. and J. Adler. Toxicity, Cocaine 2008 [cited 2009 Sept 12]; Nov 10, 2008:[Available from: http://emedicine.medscape.com/article/813959 overview. 38. Callaway, C.W. and R.F. Clark, Hyperth ermia in psychostimulant overdose. Ann Emerg Med, 1994. 24 (1): p. 68 76. 39. Cantilena, L.R., et al., Prevalence of abnormal liver associated enzymes in cocaine experienced adults versus healthy volunteers during phase 1 clinical trials. Contemp Clin Trial s, 2007. 28 (6): p. 695 704. 40. Capodanno, D. and D.J. Angiolillo, Impact of race and gender on antithrombotic therapy. Thromb Haemost. 104 (3): p. 471 84. 41. Caravello, V., et al., Apparent evaporative resistance at critical conditions for five clothing e nsembles. Eur J Appl Physiol, 2008. 104 (2): p. 361 7. 42. Carbone, A., et al., Human Harvey ras is biochemically different from Kirsten or N ras. Oncogene, 1991. 6 (5): p. 731 7. 43. Carey, R.M., Aldosterone and cardiovascular disease. Curr Opin Endocrinol Diabetes Obes. 17 (3): p. 194 8. 44. Carrero, J.J., et al., Prevalence and clinical implications of testosterone deficiency in men with end stage renal disease. Nephrol Dial Transplant. 45. Cervellin, G., I. Comelli, and G. Lippi, Rhab domyolysis: historical background, clinical, diagnostic and therapeutic features. Clin Chem Lab Med. 48 (6): p. 749 56. 46. Chang, L., et al., Gender effects on persistent cerebral metabolite changes in the frontal lobes of abstinent cocaine users. Am J Psy chiatry, 1999. 156 (5): p. 716 22. 47. Chaudhary, K., et al., The emerging role of biomarkers in diabetic and hypertensive chronic kidney disease. Curr Diab Rep. 10 (1): p. 37 42. 48. Chen, H.M., et al., Evaluation of Metabolic Risk Marker in Obesity related Glomerulopathy. J Ren Nutr. 49. Chen, Y., et al., Cocaine and catecholamines enhance inflammatory cell retention in the coronary circulation of mice by upregulation of adhesion molecules. Am J Physiol Heart Circ Physiol, 2005. 288 (5): p. H2323 31.

PAGE 144

127 50. Che n, Y., et al., Heat shock paradox and a new role of heat shock proteins and their receptors as anti inflammation targets. Inflamm Allergy Drug Targets, 2007. 6 (2): p. 91 100. 51. Chung, N.K., M. Shabbir, and C.L. Lim, Cytokine levels in patients with previ ous heatstroke under heat stress. Mil Med, 1999. 164 (4): p. 306 10. 52. Ciambrone, G. and J.C. Kaski, Gender differences in the treatment of chronic ischemic heart disease: prognostic implications. Fundam Clin Pharmacol, 2009. 53. Clerico, A. and M. Emdin, Diagnostic accuracy and prognostic relevance of the measurement of cardiac natriuretic peptides: a review. Clin Chem, 2004. 50 (1): p. 33 50. 54. Colgan, S.P. and C.T. Taylor, Hypoxia: an alarm signal during intestinal inflammation. Nat Rev Gastroenterol H epatol. 7 (5): p. 281 7. 55. Cooper, C.E., et al., Exercise, free radicals and oxidative stress. Biochem Soc Trans, 2002. 30 (2): p. 280 5. 56. Cooper, J.K., Preventing heat injury: military versus civilian perspective. Mil Med, 1997. 162 (1): p. 55 8. 57. Co rcoran, M.P., et al., Sex hormone modulation of proinflammatory cytokine and C reactive protein expression in macrophages from older men and postmenopausal women. J Endocrinol. 206 (2): p. 217 24. 58. Coresh, J., et al., Prevalence of chronic kidney disease in the United States. JAMA, 2007. 298 (17): p. 2038 47. 59. Cottone, S., et al., Microalbuminuria and early endothelial activation in essential hypertension. J Hum Hypertens, 2007. 21 (2): p. 167 72. 60. Cui, J., et al., Effects of heat stress on thermoregulatory responses in congestive heart failure patients. Circulation, 2005. 112 (15): p. 2286 92. 61. D'Aiuto, F., et al., Oxidative Stress, Systemic Inflammation, and Severe Periodontitis. J Dent Res. 62. Dandona, P., et al., Insulin Suppresses End otoxin Induced Oxidative, Nitrosative and Inflammatory Stress in Humans. Diabetes Care. 63. Das, A.B., et al., Reactions of superoxide with the myoglobin tyrosyl radical. Free Radic Biol Med. 48 (11): p. 1540 7. 64. De Vito, P., et al., Atrial natriuretic p eptide and oxidative stress. Peptides. 31 (7): p. 1412 9. 65. Di Grande, A., et al., Neutrophil gelatinase associated lipocalin: a novel biomarker for the early diagnosis of acute kidney injury in the emergency department. Eur Rev Med Pharmacol Sci, 2009. 13 (3): p. 197 200. 66. Dinarello, C.A., The interleukin 1 family: 10 years of discovery. FASEB J, 1994. 8 (15): p. 1314 25. 67. Dinesh, R.K., B.H. Hahn, and R.P. Singh, PD 1, gender, and autoimmunity. Autoimmun Rev. 9 (8): p. 583 7. 68. Dixen, U., et al., Ra ised plasma aldosterone and natriuretic peptides in atrial fibrillation. Cardiology, 2007. 108 (1): p. 35 9. 69. Doering, L.V., et al., Gender specific characteristics of individuals with depressive symptoms and coronary heart disease. Heart Lung.

PAGE 145

128 70. Dolci A. and M. Panteghini, The exciting story of cardiac biomarkers: from retrospective detection to gold diagnostic standard for acute myocardial infarction and more. Clin Chim Acta, 2006. 369 (2): p. 179 87. 71. Dotsenko, O., J. Chackathayil, and G.Y. Lip, C ardiac biomarkers: myths, facts and future horizons. Expert Rev Mol Diagn, 2007. 7 (6): p. 693 7. 72. Dowd, J.B., A. Zajacova, and A.E. Aiello, Predictors of inflammation in u.s. Children aged 3 16 years. Am J Prev Med. 39 (4): p. 314 20. 73. Egred, M., G. V iswanathan, and G.K. Davis, Myocardial infarction in young adults. Postgrad Med J, 2005. 81 (962): p. 741 5. 74. El Kebir, D., et al., Myeloperoxidase delays neutrophil apoptosis through CD11b/CD18 integrins and prolongs inflammation. Circ Res, 2008. 103 (4) : p. 352 9. 75. Elmarakby, A.A., et al., Induction of hemeoxygenase 1 attenuates the hypertension and renal inflammation in spontaneously hypertensive rats. Pharmacol Res. 76. El Minshawy, O., R.A. Saber, and A. Osman, 24 hour creatinine clearance reliabil ity for estimation of glomerular filtration rate in different stages of chronic kidney disease. Saudi J Kidney Dis Transpl. 21 (4): p. 686 93. 77. Ershler, W.B., Interleukin 6: a cytokine for gerontologists. J Am Geriatr Soc, 1993. 41 (2): p. 176 81. 78. Fan L., et al., Chronic cocaine induced cardiac oxidative stress and mitogen activated protein kinase activation: the role of Nox2 oxidase. J Pharmacol Exp Ther, 2009. 328 (1): p. 99 106. 79. Fassett, R.G., et al., Comparison of markers of oxidative stress, i nflammation and arterial stiffness between incident hemodialysis and peritoneal dialysis patients -an observational study. BMC Nephrol, 2009. 10 : p. 8. 80. Fattore, L., S. Altea, and W. Fratta, Sex differences in drug addiction: a review of animal and huma n studies. Womens Health (Lond Engl), 2008. 4 : p. 51 65. 81. Fearon, I.M. and S.P. Faux, Oxidative stress and cardiovascular disease: novel tools give (free) radical insight. J Mol Cell Cardiol, 2009. 47 (3): p. 372 81. 82. Ferrero Miliani, L., et al., Chro nic inflammation: importance of NOD2 and NALP3 in interleukin 1beta generation. Clin Exp Immunol, 2007. 147 (2): p. 227 35. 83. Ferris, M.J., et al., Cocaine Insensitive Dopamine Transporters with Intact Substrate Transport Produced by Self Administration. Biol Psychiatry. 84. Ferroni, P., et al., Prognostic significance of interleukin 6 measurement in the diagnosis of acute myocardial infarction in emergency department. Clin Chim Acta, 2007. 381 (2): p. 151 6. 85. Festa, E.D., et al., Cocaine induced sex dif ferences in D1 dopamine receptor mRNA levels after acute cocaine administration. Ethn Dis. 20 (1 Suppl 1): p. S1 24 7. 86. Flogel, U., et al., Role of myoglobin in the antioxidant defense of the heart. FASEB J, 2004. 18 (10): p. 1156 8.

PAGE 146

129 87. Franz, M., W. Woloszczuk, and W.H. Horl, Plasma concentration and urinary excretion of N terminal proatrial natriuretic peptides in patients with kidney diseases. Kidney Int, 2001. 59 (5): p. 1928 34. 88. Fried, L., et al., Inflammatory and prothrombotic ma rkers and the progression of renal disease in elderly individuals. J Am Soc Nephrol, 2004. 15 (12): p. 3184 91. 89. Furumatsu, Y., et al., Effect of renin angiotensin aldosterone system triple blockade on non diabetic renal disease: addition of an aldosterone blocker, spironolactone, to combination treatment with an angiotensin converting enzyme inhibitor and angiotensin II receptor blocker. Hypertens Res, 2008. 31 (1): p. 59 67. 90. Galankin, T., E. Shekunova, and E. Zvartau, Estradiol lowers intrac ranial self stimulation thresholds and enhances cocaine facilitation of intracranial self stimulation in rats. Horm Behav. 91. Gameiro, C. and F. Romao, Changes in the immune system during menopause and aging. Front Biosci (Elite Ed). 2 : p. 1299 303. 92. G ao, H.X., et al., Sex and autoantibody titers determine the development of neuropsychiatric manifestations in lupus prone mice. J Neuroimmunol. 93. Gardner, T.J. and T.R. Kosten, Therapeutic options and challenges for substances of abuse. Dialogues Clin Ne urosci, 2007. 9 (4): p. 431 45. 94. Gerszten, R.E. and T.J. Wang, The search for new cardiovascular biomarkers. Nature, 2008. 451 (7181): p. 949 52. 95. Gilbert, K.C. and N.J. Brown, Aldosterone and inflammation. Curr Opin Endocrinol Diabetes Obes. 17 (3): p. 199 204. 96. Goetzl, E.J., et al., Gender specificity of altered human immune cytokine profiles in aging. FASEB J. 24 (9): p. 3580 9. 97. Goldstein, R.A., C. DesLauriers, and A.M. Burda, Cocaine: history, social implications, and toxicity -a review. Dis Mo n, 2009. 55 (1): p. 6 38. 98. Gourgoutis, G. and G. Das, Gastrointestinal manifestations of cocaine addiction. Int J Clin Pharmacol Ther, 1994. 32 (3): p. 136 41. 99. Groleger, U. and V. Novak Grubic, Gender, psychosis and psychotropic drugs: differences and similarities. Psychiatr Danub. 22 (2): p. 338 42. 100. Grosso, S., et al., Isoprostanes in dystrophinopathy: Evidence of increased oxidative stress. Brain Dev, 2008. 30 (6): p. 391 5. 101. Grover, D.S., et al., Lack of clinical utility of urine myoglobin de tection by microconcentrator ultrafiltration in the diagnosis of rhabdomyolysis. Nephrol Dial Transplant, 2004. 19 (10): p. 2634 8. 102. Gu, H.F., et al., SOX2 has gender specific genetic effects on diabetic nephropathy in samples from patients with type 1 diabetes mellitus in the GoKinD study. Gend Med, 2009. 6 (4): p. 555 64. 103. Guder, W.G. and W. Hofmann, Clinical role of urinary low molecular weight proteins: their diagnostic and prognostic implications. Scand J Clin Lab Invest Suppl, 2008. 241 : p. 95 8 104. Hahn, I.H. and R.S. Hoffman, Cocaine use and acute myocardial infarction. Emerg Med Clin North Am, 2001. 19 (2): p. 493 510.

PAGE 147

130 105. Haller, M.J. and D.A. Schatz, Cytokines and type 1 diabetes complications: casual or causal association? Pediatr Diabete s, 2008. 9 (1): p. 1 2. 106. Halpern, J.H., et al., Diminished interleukin 6 response to proinflammatory challenge in men and women after intravenous cocaine administration. J Clin Endocrinol Metab, 2003. 88 (3): p. 1188 93. 107. Hamdy, R.C. Heat Stroke 20 02 [cited 2008 August 9]; Available from: http://www.medscape.com/viewarticle/444158. 108. Han, J.H., et al., The role of cardiac risk factor burden in diagnosing acute coronary syndromes in the emergency department setting. Ann Emerg Med, 2007. 49 (2): p. 145 52, 152 e1. 109. Hayase, T., Y. Yamamoto, and K. Yamamoto, Toxic cocaine and convulsant induced modification of forced swimming behaviors and their interaction with ethanol: comparison with immobilization stress. BMC Pharmacol, 2002. 2 : p. 19. 110. H ayward, R.M., et al., Post collection, pre measurement variables affecting VEGF levels in urine biospecimens. J Cell Mol Med, 2008. 12 (1): p. 343 50. 111. Heitzer, T., et al., Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation, 2001. 104 (22): p. 2673 8. 112. Hoffman, R.S. and J.E. Hollander, Evaluation of patients with chest pain after cocaine use. Crit Care Clin, 1997. 13 (4): p. 809 28. 113. Hollander, J.E., D.E. Brooks, and S.M. Valentine, Assessment of cocaine use in patients with chest pain syndromes. Arch Intern Med, 1998. 158 (1): p. 62 6. 114. Hollander, J.E., et al., Effect of recent cocaine use on the specificity of cardiac markers for diagnosis of acute myocardial infarction. Am Heart J, 1998. 135 (2 Pt 1): p. 245 52. 115. Houlihan, J.L., J.J. Metzler, and J.S. Blum, HSP90alpha and HSP90beta isoforms selectively modulate MHC class II antigen presentation in B cells. J Immunol, 2009. 182 ( 12): p. 7451 8. 116. Huang, J., et al., Role of endothelial lipase in atherosclerosis. Transl Res. 156 (1): p. 1 6. 117. Hung, M.Y., et al., Interactions among gender, age, hypertension and C reactive protein in coronary vasospasm. Eur J Clin Invest. 118. I shino, M., et al., Risk stratification of chronic heart failure patients by multiple biomarkers: implications of BNP, H FABP, and PTX3. Circ J, 2008. 72 (11): p. 1800 5. 119. Itoh, Y., et al., 17beta estradiol induces IL 1alpha gene expression in rheumatoid fibroblast like synovial cells through estrogen receptor alpha (ERalpha) and augmentation of transcriptional activity of Sp1 by dissociating histone deacetylase 2 from ERalpha. J Immunol, 2007. 178 (5): p. 3059 66. 120. Jackson, A.M., et al., Changes in ur inary cytokines and soluble intercellular adhesion molecule 1 (ICAM 1) in bladder cancer patients after bacillus Calmette Guerin (BCG) immunotherapy. Clin Exp Immunol, 1995. 99 (3): p. 369 75. 121. Jaffe, J.A. and P.L. Kimmel, Chronic nephropathies of cocai ne and heroin abuse: a critical review. Clin J Am Soc Nephrol, 2006. 1 (4): p. 655 67.

PAGE 148

131 122. Jarvie, J.L. and J.M. Foody, Recognizing and Improving Health Care Disparities in the Prevention of Cardiovascular Disease in Women. Curr Cardiol Rep. 123. Jeong, Y. et al., Aldosterone activates endothelial exocytosis. Proc Natl Acad Sci U S A, 2009. 106 (10): p. 3782 7. 124. Jimenez Navarro, M.F., et al., Influence of gender on long term prognosis of patients with chronic heart failure seen in heart failure clinics. Clin Cardiol. 33 (3): p. E13 8. 125. Johnsen, A.K., et al., A broad analysis of IL1 polymorphism and rheumatoid arthritis. Arthritis Rheum, 2008. 58 (7): p. 1947 57. 126. Kacila, M., et al., Inflammatory and metabolic response of the myocardium during aorti c valve surgery on the beating heart. Bosn J Basic Med Sci, 2006. 6 (2): p. 59 62. 127. Kaminski, K.A., et al., Oxidative stress and neutrophil activation -the two keystones of ischemia/reperfusion injury. Int J Cardiol, 2002. 86 (1): p. 41 59. 128. Kapoor, J.R., Effect of cocaine on cardiac biomarkers. Am J Cardiol, 2008. 101 (5): p. 744. 129. Karch, S.B., Karch's Pathology of Drug Abuse, Fourth Edition 2009, Boca Raton: CRC/Taylor & Francis. 130. Karnib, H.H. and F.N. Ziyadeh, The cardiorenal syndrome in di abetes mellitus. Diabetes Res Clin Pract. 89 (3): p. 201 8. 131. Kasahara, Y., et al., [Trend in the study of cocaine addiction]. Nippon Rinsho. 68 (8): p. 1479 85. 132. Kautzky Willer, A. and A. Handisurya, Metabolic diseases and associated complications: s ex and gender matter! Eur J Clin Invest, 2009. 39 (8): p. 631 48. 133. Kaysen, G.A. and J.P. Eiserich, The role of oxidative stress altered lipoprotein structure and function and microinflammation on cardiovascular risk in patients with minor renal dysfunct ion. J Am Soc Nephrol, 2004. 15 (3): p. 538 48. 134. Keller, K.B. and L. Lemberg, The cocaine abused heart. Am J Crit Care, 2003. 12 (6): p. 562 6. 135. Khan, F.Y., Rhabdomyolysis: a review of the literature. Neth J Med, 2009. 67 (9): p. 272 83. 136. Khan, N., et al., Effects of age, gender, and immunosuppressive agents on in vivo toll like receptor pathway responses. Hum Immunol. 71 (4): p. 372 6. 137. Kilts, C.D., et al., The neural correlates of cue induced craving in cocaine dependent women. Am J Ps ychiatry, 2004. 161 (2): p. 233 41. 138. Kiyatkin, E.A., Brain hyperthermia as physiological and pathological phenomena. Brain Res Brain Res Rev, 2005. 50 (1): p. 27 56. 139. Klebanoff, S.J., Myeloperoxidase: friend and foe. J Leukoc Biol, 2005. 77 (5): p. 59 8 625. 140. Kleemann, R., S. Zadelaar, and T. Kooistra, Cytokines and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc Res, 2008. 79 (3): p. 360 76. 141. Kloner, R.A., Natural and unnatural triggers of myocardial infarction. Prog Cardiovasc Dis, 2006. 48 (4): p. 285 300. 142. Kloner, R.A., et al., The effects of acute and chronic cocaine use on the heart. Circulation, 1992. 85 (2): p. 407 19.

PAGE 149

132 143. Kloner, R.A. and S.H. Rezkalla, Cocaine and the heart. N Engl J Med, 2003. 348 (6) : p. 487 8. 144. Kobayashi, H., et al., Increased prevalence of carotid artery atherosclerosis in rheumatoid arthritis is artery specific. J Rheumatol. 37 (4): p. 730 9. 145. Komukai, K., S. Mochizuki, and M. Yoshimura, Gender and the renin angiotensin aldo sterone system. Fundam Clin Pharmacol. 146. Konig, D., et al., Exercise and oxidative stress: significance of antioxidants with reference to inflammatory, muscular, and systemic stress. Exerc Immunol Rev, 2001. 7 : p. 108 33. 147. Korish, A.A., Multiple ant ioxidants and L arginine modulate inflammation and dyslipidemia in chronic renal failure rats. Ren Fail. 32 (2): p. 203 13. 148. Kotani, K., et al., Association between coffee consumption and the estimated glomerular filtration rate in the general Japanese population: preliminary data regarding C reactive protein concentrations. Clin Chem Lab Med. 149. Kovacic, P., Role of oxidative metabolites of cocaine in toxicity and addiction: oxidative stress and electron transfer. Med Hypotheses, 2005. 64 (2): p. 350 6 150. Kuhn, C. and R. Francis, Gender difference in cocaine induced HPA axis activation. Neuropsychopharmacology, 1997. 16 (6): p. 399 407. 151. Labib, R., R. Turkall, and M.S. Abdel Rahman, Oral cocaine produces dose related hepatotoxicity in male mice. T oxicol Lett, 2001. 125 (1 3): p. 29 37. 152. Labib, R., R. Turkall, and M.S. Abdel Rahman, Inhibition of cocaine oxidative metabolism attenuates endotoxin potentiation of cocaine mediated hepatotoxicity. Toxicology, 2002. 179 (1 2): p. 9 19. 153. Labib, R., R. Turkall, and M.S. Abdel Rahman, Endotoxin potentiates cocaine mediated hepatotoxicity by nitric oxide and reactive oxygen species. Int J Toxicol, 2003. 22 (4): p. 305 16. 154. Lainscak, M., S. von Haehling, and S.D. Anker, Natriuretic peptides and other biomarkers in chronic heart failure: from BNP, NT proBNP, and MR proANP to routine biochemical markers. Int J Cardiol, 2009. 132 (3): p. 303 11. 155. Landmesser, U. and D.G. Harrison, Oxidant stress as a marker for cardiovascular events: Ox marks the spot. Circulation, 2001. 104 (22): p. 2638 40. 156. Lange, R.A., J.E. Cigarroa, and L.D. Hillis, Theodore E. Woodward award: cardiovascular complications of cocaine abuse. Trans Am Clin Climatol Assoc, 2004. 115 : p. 99 111; discussion 112 4. 157. Lansang, M.C. an d N.K. Hollenberg, Renal perfusion and the renal hemodynamic response to blocking the renin system in diabetes: are the forces leading to vasodilation and vasoconstriction linked? Diabetes, 2002. 51 (7): p. 2025 8. 158. Latchman, D.S., Heat shock proteins and cardiac protection. Cardiovasc Res, 2001. 51 (4): p. 637 46. 159. Lattanzio, F.A., Jr., et al., Cocaine increases intracellular calcium and reactive oxygen species, depolarizes mitochondria, and activates genes associated with heart failure and remodeli ng. Cardiovasc Toxicol, 2005. 5 (4): p. 377 90. 160. Lee, K.S., et al., Simultaneous measurement of 23 plasma cytokines in late life depression. Neurol Sci, 2009. 30 (5): p. 435 8.

PAGE 150

133 161. Lee, M.O., P.M. Vivier, and D.B. Diercks, Is the self report of recent c ocaine or methamphetamine use reliable in illicit stimulant drug users who present to the Emergency Department with chest pain? J Emerg Med, 2009. 37 (2): p. 237 41. 162. Legato, M.J., et al., Gender specific care of the patient with diabetes: review and re commendations. Gend Med, 2006. 3 (2): p. 131 58. 163. Leuzzi, C. and M.G. Modena, Coronary artery disease: clinical presentation, diagnosis and prognosis in women. Nutr Metab Cardiovasc Dis. 20 (6): p. 426 35. 164. Levin, J.M., et al., Gender differences in cerebral perfusion in cocaine abuse: technetium 99m HMPAO SPECT study of drug abusing women. J Nucl Med, 1994. 35 (12): p. 1902 9. 165. Levis, J.T. and G.M. Garmel, Cocaine associated chest pain. Emerg Med Clin North Am, 2005. 23 (4): p. 1083 103. 166. Li, X ., et al., 17beta estradiol enhances the response of plasmacytoid dendritic cell to CpG. PLoS One, 2009. 4 (12): p. e8412. 167. Libby, P. and P.M. Ridker, Inflammation and atherosclerosis: role of C reactive protein in risk assessment. Am J Med, 2004. 116 Suppl 6A : p. 9S 16S. 168. Libby, P., P.M. Ridker, and G.K. Hansson, Inflammation in atherosclerosis: from pathophysiology to practice. J Am Coll Cardiol, 2009. 54 (23): p. 2129 38. 169. Libby, P., P.M. Ridker, and A. Maseri, Inflammation and atherosclerosis Circulation, 2002. 105 (9): p. 1135 43. 170. Lim, L.S., et al., C reactive protein, body mass index, and diabetic retinopathy. Invest Ophthalmol Vis Sci. 51 (9): p. 4458 63. 171. Lin, C.C., et al., The relation of metabolic syndrome according to five defin itions to cardiovascular risk factors -a population based study. BMC Public Health, 2009. 9 : p. 484. 172. Lloyd, S.A., et al., Cocaine selectively increases proliferation in the adult murine hippocampus. Neurosci Lett. 173. Lock, E.A. and J.V. Bonventre, B iomarkers in translation; past, present and future. Toxicology, 2008. 245 (3): p. 163 6. 174. Loria, V., et al., Markers of acute coronary syndrome in emergency room. Minerva Med, 2008. 99 (5): p. 497 517. 175. Loscalzo, J., Oxidative stress in endothelial c ell dysfunction and thrombosis. Pathophysiol Haemost Thromb, 2002. 32 (5 6): p. 359 60. 176. Lu, Y., E. Ku, and V.M. Campese, Aldosterone in the pathogenesis of chronic kidney disease and proteinuria. Curr Hypertens Rep. 12 (4): p. 303 6. 177. Lucena, J., et al., Cocaine related sudden death: a prospective investigation in south west Spain. Eur Heart J. 31 (3): p. 318 29. 178. Luginbuhl, R., et al. Heat Related Deaths Among Crop Workers -United States, 1992 2006 2008 [cited 2008 September 7]; Available fr om: http://www.medscape.com/viewarticle/576721. 179. Maggio, M., et al., The relationship between testosterone and molecular markers of inflammation in older men. J Endocrinol Invest, 2005. 28 (11 Suppl Proceedings): p. 116 9. 180. Maier, C., et al., Endothelial markers may link kidney function to cardiovascular events in type 2 diabetes. Diabetes Care, 2009. 32 (10): p. 1890 5.

PAGE 151

134 181. Maioli, M., et al., Number of autoantibodies and HLA genotype, more than high titers of glutamic acid decarboxylase autoa ntibodies, predict insulin dependence in latent autoimmune diabetes of adults. Eur J Endocrinol. 163 (4): p. 541 549. 182. Malling, T.H., et al., Differences in associations between markers of antioxidative defense and asthma are sex specific. Gend Med. 7 (2 ): p. 115 24. 183. Malyszko, J., et al., Serum neutrophil gelatinase associated lipocalin as a marker of renal function in non diabetic patients with stage 2 4 chronic kidney disease. Ren Fail, 2008. 30 (6): p. 625 8. 184. Mandl Weber, S., et al., Vascular endothelial growth factor production and regulation in human peritoneal mesothelial cells. Kidney Int, 2002. 61 (2): p. 570 8. 185. Mansur, S.J., F.G. Hage, and S. Oparil, Have the Renin Angiotensin Aldosterone System Perturbations in Cardiovascular Disease Been Exhausted? Curr Cardiol Rep. 12 (6): p. 450 463. 186. Martin Schild, S., et al., Intracerebral hemorrhage in cocaine users. Stroke. 41 (4): p. 680 4. 187. Matthijsen, R.A., et al., Myeloperoxidase is critically involved in the induction of organ damage after renal ischemia reperfusion. Am J Pathol, 2007. 171 (6): p. 1743 52. 188. Mazul Sunko, B., et al., Proatrial natriuretic peptide (1 98), but not cystatin C, is predictive for occurrence of acute renal insufficiency in critically ill septic patients. N ephron Clin Pract, 2004. 97 (3): p. c103 7. 189. McCance Katz, E.F., et al., Gender effects following repeated administration of cocaine and alcohol in humans. Subst Use Misuse, 2005. 40 (4): p. 511 28. 190. Meng, Q., et al., Elevated C reactive protein leve ls are associated with endothelial dysfunction in chronic cocaine users. Int J Cardiol, 2003. 88 (2 3): p. 191 8. 191. Mercuro, G., et al., Gender determinants of cardiovascular risk factors and diseases. J Cardiovasc Med (Hagerstown). 11 (3): p. 207 20. 192 Middleton, P.M., Cerebrovascular Effects Of Cocaine The Internet Journal of Emergency Medicine, 2004 2 (1). 193. Miike, K., et al., Proteome profiling reveals gender differences in the composition of human serum. Proteomics. 10 (14): p. 2678 91. 194. Minerly, A.E., et al., Testosterone differentially alters cocaine induced ambulatory and rearing behavioral responses in adult and adolescent rats. Pharmacol Biochem Behav. 94 (3): p. 404 9. 195. Minh, H.V., et al., Gender differences in prevalence and soci oeconomic determinants of hypertension: findings from the WHO STEPs survey in a rural community of Vietnam. J Hum Hypertens, 2006. 20 (2): p. 109 15. 196. Mishra, J., et al., Identification of neutrophil gelatinase associated lipocalin as a novel early urin ary biomarker for ischemic renal injury. J Am Soc Nephrol, 2003. 14 (10): p. 2534 43. 197. Moe, K.T. and P. Wong, Current trends in diagnostic biomarkers of acute coronary syndrome. Ann Acad Med Singapore. 39 (3): p. 210 5.

PAGE 152

135 198. Moritz, F., et al., Role of r eactive oxygen species in cocaine induced cardiac dysfunction. Cardiovasc Res, 2003. 59 (4): p. 834 43. 199. Mussap, M., et al., Acute kidney injury in critically ill infants: the role of urine Neutrophil Gelatinase Associated Lipocalin (NGAL). J Matern Fet al Neonatal Med. 200. Musunuru, K., et al., The use of high sensitivity assays for C reactive protein in clinical practice. Nat Clin Pract Cardiovasc Med, 2008. 5 (10): p. 621 35. 201. Ndikum Moffor, F.M., T.R. Schoeb, and S.M. Roberts, Liver toxicity from norcocaine nitroxide, an N oxidative metabolite of cocaine. J Pharmacol Exp Ther, 1998. 284 (1): p. 413 9. 202. Neubauer, O., D. Konig, and K.H. Wagner, Recovery after an Ironman triathlon: sustained inflammatory responses and muscular stress. Eur J Appl Physiol, 2008. 104 (3): p. 417 26. 203. Nicholls, S.J. and S.L. Hazen, Myeloperoxidase and cardiovascular disease. Arterioscler Thromb Vasc Biol, 2005. 25 (6): p. 1102 11. 204. Niizeki, T., et al., Relation of serum heat shock protein 60 level to severity and prognosis in chronic heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol, 2008. 102 (5): p. 606 10. 205. Nikolaou, N.I., et al., Brain natriuretic peptide increases in septic patients without severe sepsis or shock. Eur J Intern Med, 2007. 18 (7): p. 535 41. 206. Nishiyama, A. and S. Kim Mitsuyama, New approaches to blockade of the renin angiotensin aldosterone system: overview of regulation of the renin angiotensin aldosterone system. J Pharmacol Sci. 113 (4): p. 289 91. 207. Oztezcan, S., et al., The role of stimulated lipid peroxidation and impaired calcium sequestration in the enhancement of cocaine induced hepatotoxicity by ethanol. Drug Alcohol Depend, 2000. 58 (1 2): p. 77 83. 208. Parry, S.N., et al., Myog lobin induces oxidative stress and decreases endocytosis and monolayer permissiveness in cultured kidney epithelial cells without affecting viability. Kidney Blood Press Res, 2008. 31 (1): p. 16 28. 209. Patel, D.R., R. Gyamfi, and A. Torres, Exertional rha bdomyolysis and acute kidney injury. Phys Sportsmed, 2009. 37 (1): p. 71 9. 210. Peake, J.M., et al., Systemic inflammatory responses to maximal versus submaximal lengthening contractions of the elbow flexors. Exerc Immunol Rev, 2006. 12 : p. 72 85. 211. Pel konen, M., et al., Cocaine increases circulating levels of atrial natriuretic peptide and pro atrial natriuretic peptide N terminal fragment in conscious rats. Eur J Pharmacol, 1996. 304 (1 3): p. 55 62. 212. Pepe, M.S., et al., Pivotal evaluation of the ac curacy of a biomarker used for classification or prediction: standards for study design. J Natl Cancer Inst, 2008. 100 (20): p. 1432 8. 213. Perez Lopez, F.R., et al., Gender differences in cardiovascular disease: hormonal and biochemical influences. Reprod Sci. 17 (6): p. 511 31. 214. Perrone, S., et al., Whole body hypothermia and oxidative stress in babies with hypoxic ischemic brain injury. Pediatr Neurol. 43 (4): p. 236 40.

PAGE 153

136 215. Petersen, M.M., C.P. Mikita, and J. Sheikh. Myeloperoxidase Deficiency 2008 [cited 2009 Oct 8]; Available from: http://emedicine.medscape.com/article/887599 overview. 216. Pikkarainen, S., et al., Endothelin 1 specific activation of B type natriuretic peptide gene via p38 mitogen activated protein kinase and nuclear ETS factors. J Biol Chem, 2003. 278 (6): p. 3969 75. 217. Pisitkun, T., R. Johnstone, and M.A. Knepper, Discovery of urinary biomarkers. Mol Cell Proteomics, 2006. 5 (10): p. 1760 71. 218. Plackett, T.P., R.L. Gamelli, and E.J. Kovacs, Gender based differences in cytoki ne production after burn injury: a role of interleukin 6. J Am Coll Surg. 210 (1): p. 73 8. 219. Plotnikov, E.Y., et al., Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney's mitochondria. Biochim Biophys Acta, 2009. 1792 (8): p. 796 803. 220. Pockley, A.G. and J. Frostegard, Heat shock proteins in cardiovascular disease and the prognostic value of heat shock protein related measurements. Heart, 2005. 91 (9): p. 1124 6. 221. Poon, H.F., et al., Cocaine induced oxidative stress precedes cell death in human neuronal progenitor cells. Neurochem Int, 2007. 50 (1): p. 69 73. 222. Pottratz, S.T., et al., 17 beta Estradiol inhibits expression of human interleukin 6 promoter reporter constructs by a receptor dependent mechanism. J Clin Invest, 1994. 93 (3): p. 944 50. 223. Pozner, C.N., M. Levine, and R. Zane, The cardiovascular effects of cocaine. J Emerg Med, 2005. 29 (2): p. 173 8. 224. Prandota, J., Important role of proinflammatory cytokines/other endogenous substances in drug induced hepatotoxicity: depression of drug metabolism during infections/inflammation states, and genetic polymorphisms of drug metabolizing enzymes/cytokines may markedly contribute to this pathology. Am J Ther, 2005. 12 (3): p. 254 61. 225. Puustinen, P.J., et al., Gender specific association of psychological distress with cardiovascular risk scores. Scand J Prim Health Care. 28 (1): p. 36 40. 226. Qureshi, A.I., et al., Cocaine use and the likelihood of nonfatal myocardial infarction and stroke: data f rom the Third National Health and Nutrition Examination Survey. Circulation, 2001. 103 (4): p. 502 6. 227. Reed, S.C., et al., Cardiovascular and subjective effects of repeated smoked cocaine administration in experienced cocaine users. Drug Alcohol Depend, 2009. 102 (1 3): p. 102 7. 228. Renes, J., et al., Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4 hydroxynonenal. Biochem J, 2000. 350 Pt 2 : p. 555 61. 229. Restrepo, C.S., et al., Cardiovascular c omplications of cocaine: imaging findings. Emerg Radiol, 2009. 16 (1): p. 11 9. 230. Rezkalla, S.H. and R.A. Kloner, Cocaine induced acute myocardial infarction. Clin Med Res, 2007. 5 (3): p. 172 6.

PAGE 154

137 231. Richards, I.S., Health effects of illicit cocaine use. Pulmonary and Critical Care Update American College of Chest Physicians, 1991. 7 (2): p. 1 7. 232. Richards, J.R., Rhabdomyolysis and drugs of abuse. J Emerg Med, 2000. 19 (1): p. 51 6. 233. Ridker, P.M., C reactive protein: eighty years from discovery to emergence as a major risk marker for cardiovascular disease. Clin Chem, 2009. 55 (2): p. 209 15. 234. Ridker, P.M., et al., C reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med, 2000. 342 (12 ): p. 836 43. 235. Rodriguez Capote, K., et al., Utility of urine myoglobin for the prediction of acute renal failure in patients with suspected rhabdomyolysis: a systematic review. Clin Chem, 2009. 55 (12): p. 2190 7. 236. Rosa, J.S., et al., Sustained IL 1alpha, IL 4, and IL 6 elevations following correction of hyperglycemia in children with type 1 diabetes mellitus. Pediatr Diabetes, 2008. 9 (1): p. 9 16. 237. Rosano, G.M. and G. Panina, Oestrogens and the heart. Therapie, 1999. 54 (3): p. 381 5. 238. Roy, A., S. Sen, and A.S. Chakraborti, In vitro nonenzymatic glycation enhances the role of myoglobin as a source of oxidative stress. Free Radic Res, 2004. 38 (2): p. 139 46. 239. Rubtsov, A.V., et al., Genetic and hormonal factors in female biased autoimmunity Autoimmun Rev. 9 (7): p. 494 8. 240. Saenger, A.K. and A.S. Jaffe, The use of biomarkers for the evaluation and treatment of patients with acute coronary syndromes. Med Clin North Am, 2007. 91 (4): p. 657 81; xi. 241. Salminen, W.F., Jr., et al., Heat shoc k protein induction in murine liver after acute treatment with cocaine. Hepatology, 1997. 25 (5): p. 1147 53. 242. Saltzberg, M.T., Secondary and Infiltrative Cardiomyopathies. Curr Treat Options Cardiovasc Med, 2000. 2 (5): p. 373 384. 243. Seeman, M.V., Me chanisms of sex difference: a historical perspective. J Womens Health (Larchmt), 2009. 18 (6): p. 861 6. 244. Segarra, A.C., et al., Estradiol: a key biological substrate mediating the response to cocaine in female rats. Horm Behav. 58 (1): p. 33 43. 245. Se ligman, R., et al., Prognostic value of midregional pro atrial natriuretic peptide in ventilator associated pneumonia. Intensive Care Med, 2008. 34 (11): p. 2084 91. 246. Seto, C.K., D. Way, and N. O'Connor, Environmental illness in athletes. Clin Sports Med, 2005. 24 (3): p. 695 718, x. 247. Shapiro, N.I., et al., The diagnostic accuracy of plasma neutrophil gelatinase associated lipocalin in the prediction of acute kidney injury in emergency department patients with suspected sepsis. Ann Emer g Med. 56 (1): p. 52 59 e1. 248. Shen, H., et al., Gender dependent Expression of Murine Irf5 Gene: Implications for Sex Bias in Autoimmunity. J Mol Cell Biol. 249. Siegel, A.J., et al., Effect of cocaine usage on C reactive protein, von Willebrand factor, and fibrinogen. Am J Cardiol, 2002. 89 (9): p. 1133 5.

PAGE 155

138 250. Sieveking, D.P., R.W. Chow, and M.K. Ng, Androgens, angiogenesis and cardiovascular regeneration. Curr Opin Endocrinol Diabetes Obes. 17 (3): p. 277 83. 251. Silva, L.A., et al., N acetylcysteine su pplementation and oxidative damage and inflammatory response after eccentric exercise. Int J Sport Nutr Exerc Metab, 2008. 18 (4): p. 379 88. 252. Sinha, R., et al., Sex steroid hormones, stress response, and drug craving in cocaine dependent women: implica tions for relapse susceptibility. Exp Clin Psychopharmacol, 2007. 15 (5): p. 445 52. 253. Sirera, R., et al., Quantification of proinflammatory cytokines in the urine of congestive heart failure patients. Its relationship with plasma levels. Eur J Heart Fai l, 2003. 5 (1): p. 27 31. 254. Stehr, C.B., et al., Increased levels of oxidative stress, subclinical inflammation, and myocardial fibrosis markers in primary aldosteronism patients. J Hypertens. 255. Straface, E., et al., Gender specific features of plasma tic and circulating cell alterations as risk factors in cardiovascular disease. Fundam Clin Pharmacol. 256. Su, J., et al., Cocaine induces apoptosis in primary cultured rat aortic vascular smooth muscle cells: possible relationship to aortic dissection, a therosclerosis, and hypertension. Int J Toxicol, 2004. 23 (4): p. 233 7. 257. Sun, W.L., et al., Sex differences in dopamine D2 like receptor mediated G protein activation in the medial prefrontal cortex after cocaine. Ethn Dis. 20 (1 Suppl 1): p. S1 88 91. 258. Surekha, R.H., et al., Oxidative stress and total antioxidant status in myocardial infarction. Singapore Med J, 2007. 48 (2): p. 137 42. 259. Suresh, E., Recent advances in rheumatoid arthritis. Postgrad Med J. 86 (1014): p. 243 50. 260. Suzuki, M., et al., Brain natriuretic peptide as a risk marker for incident hypertensive cardiovascular events. Hypertens Res, 2002. 25 (5): p. 669 76. 261. Swanlund, J.M., K.C. Kregel, and T.D. Oberley, Autophagy following heat stress: the role of aging and protein nitra tion. Autophagy, 2008. 4 (7): p. 936 9. 262. Tan, Y.Y., G.C. Gast, and Y.T. van der Schouw, Gender differences in risk factors for coronary heart disease. Maturitas. 65 (2): p. 149 60. 263. Thakore, A.H., et al., Association of multiple inflammatory markers with carotid intimal medial thickness and stenosis (from the Framingham Heart Study). Am J Cardiol, 2007. 99 (11): p. 1598 602. 264. Tonioni, F., et al., Cocaine use disorders and serum magnesium profile. Neuropsychobiology, 2009. 59 (3): p. 159 64. 265. Tor res, A., A.D. Askari, and C.J. Malemud, Cardiovascular disease complications in systemic lupus erythematosus. Biomark Med, 2009. 3 (3): p. 239 52. 266. Toth, A.R. and T. Varga, Myocardium and striated muscle damage caused by licit or illicit drugs. Leg Med (Tokyo), 2009. 11 Suppl 1 : p. S484 7. 267. Toto, R.D., Aldosterone blockade in chronic kidney disease: can it improve outcome? Curr Opin Nephrol Hypertens. 19 (5): p. 444 9.

PAGE 156

139 268. Trof, R.J., et al., Biomarkers of acute renal injury and renal failure. Shock, 2006. 26 (3): p. 245 53. 269. Tsimikas, S., et al., Oxidation Specific Biomarkers, Lipoprotein(a), and Risk of Fatal and Nonfatal Coronary Events. J Am Coll Cardiol. 56 (12): p. 946 55. 270. Tsukamoto, H. and S.C. Lu, Current concepts in the pathogen esis of alcoholic liver injury. FASEB J, 2001. 15 (8): p. 1335 49. 271. Tuncel, M., et al., Mechanism of the blood pressure -raising effect of cocaine in humans. Circulation, 2002. 105 (9): p. 1054 9. 272. Upadhyay, A., et al., Inflammation, kidney function and albuminuria in the Framingham Offspring cohort. Nephrol Dial Transplant. 273. van der Woude, F.J., Cocaine use and kidney damage. Nephrol Dial Transplant, 2000. 15 (3): p. 299 301. 274. van Helvoort, H.A., et al., Systemic inflammatory response to exhau stive exercise in patients with chronic obstructive pulmonary disease. Respir Med, 2005. 99 (12): p. 1555 67. 275. van Rooij, E., et al., Myocyte enhancer factor 2 and class II histone deacetylases control a gender specific pathway of cardioprotection mediated by the estrogen receptor. Circ Res. 106 (1): p. 155 65. 276. Vassallo, J.D., et al., Biomarkers of drug induced skeletal muscle injury in the rat: troponin I and myoglobin. Toxicol Sci, 2009. 111 (2): p. 402 12. 277. Veloza, A., et al., [Inflammator y myopathy with an unusual evolution]. Acta Reumatol Port. 35 (2): p. 254 8. 278. Verma, A., et al., Effect of rosuvastatin on C reactive protein and renal function in patients with chronic kidney disease. Am J Cardiol, 2005. 96 (9): p. 1290 2. 279. Videla, L.A., Hormetic responses of thyroid hormone calorigenesis in the liver: Association with oxidative stress. IUBMB Life. 62 (6): p. 460 6. 280. Visalli, T., R. Turkall, and M.S. Abdel Rahman, Influence of gender on cocaine hepatotoxicity in CF 1 mice. Int J T oxicol, 2005. 24 (1): p. 43 50. 281. Visalli, T., R. Turkall, and M.S. Abdel Rahman, Gender differences in cocaine pharmacokinetics in CF 1 mice. Toxicol Lett, 2005. 155 (1): p. 35 40. 282. Vitale, C., et al., Gender differences in the cardiovascular effects of sex hormones. Fundam Clin Pharmacol. 283. Vitale, C., M.E. Mendelsohn, and G.M. Rosano, Gender differences in the cardiovascular effect of sex hormones. Nat Rev Cardiol, 2009. 6 (8): p. 532 42. 284. Vollaard, N.B., J.P. Shearman, and C.E. Cooper, Exerci se induced oxidative stress:myths, realities and physiological relevance. Sports Med, 2005. 35 (12): p. 1045 62. 285. Vroegop, M.P., et al., The emergency care of cocaine intoxications. Neth J Med, 2009. 67 (4): p. 122 6. 286. Wagener, G., et al., Urinary ne utrophil gelatinase associated lipocalin and acute kidney injury after cardiac surgery. Am J Kidney Dis, 2008. 52 (3): p. 425 33. 287. Wang, T.J., et al., Multiple biomarkers and the risk of incident hypertension. Hypertension, 2007. 49 (3): p. 432 8.

PAGE 157

140 288. W ang, Z. and W.E. Hoy, C reactive protein: an independent predictor of cardiovascular disease in Aboriginal Australians. Aust N Z J Public Health. 34 Suppl 1 : p. S25 9. 289. Weber, J.E., et al., Validation of a brief observation period for patients with coc aine associated chest pain. N Engl J Med, 2003. 348 (6): p. 510 7. 290. Weber, M.A., et al., Myoglobin plasma level related to muscle mass and fiber composition: a clinical marker of muscle wasting? J Mol Med, 2007. 85 (8): p. 887 96. 291. Weinberg, E.O., et al., Identification of serum soluble ST2 receptor as a novel heart failure biomarker. Circulation, 2003. 107 (5): p. 721 6. 292. Weng, C.M., et al., Increased C reactive protein is associated with future development of diabetes mellitus i n essential hypertensive patients. Heart Vessels. 25 (5): p. 386 91. 293. Wiener, S.E., et al., Patients with detectable cocaethylene are more likely to require intensive care unit admission after trauma. Am J Emerg Med, 2009. 294. Wilbert Lampen, U., et al ., Cocaine increases the endothelial release of immunoreactive endothelin and its concentrations in human plasma and urine: reversal by coincubation with sigma receptor antagonists. Circulation, 1998. 98 (5): p. 385 90. 295. Wilson, L.D., et al., Cocaine, e thanol, and cocaethylene cardiotoxity in an animal model of cocaine and ethanol abuse. Acad Emerg Med, 2001. 8 (3): p. 211 22. 296. Wolf, M., et al., Simultaneous detection of C reactive protein and other cardiac markers in human plasma using micromosaic im munoassays and self regulating microfluidic networks. Biosens Bioelectron, 2004. 19 (10): p. 1193 202. 297. Wu, A.H., Novel biomarkers of cardiovascular disease: myeloperoxidase for acute and/or chronic heart failure? Clin Chem, 2009. 55 (1): p. 12 4. 298. W u, C.C., et al., Myeloperoxidase serves as a marker of oxidative stress during single haemodialysis session using two different biocompatible dialysis membranes. Nephrol Dial Transplant, 2005. 20 (6): p. 1134 9. 299. Xu, R., et al., Gender differences in ag e related decline in glomerular filtration rates in healthy people and chronic kidney disease patients. BMC Nephrol. 11 : p. 20. 300. Yamamoto, L.M., et al., Effects of hydration state and resistance exercise on markers of muscle damage. J Strength Cond Res 2008. 22 (5): p. 1387 93. 301. Yan, Y.E., et al., Pathophysiological factors underlying heatstroke. Med Hypotheses, 2006. 67 (3): p. 609 17. 302. Yeun, J.Y. and G.A. Kaysen, C reactive protein, oxidative stress, homocysteine, and troponin as inflammatory and metabolic predictors of atherosclerosis in ESRD. Curr Opin Nephrol Hypertens, 2000. 9 (6): p. 621 30. 303. Yilmaz, A., et al., Early prediction of urinary tract infection with urinary neutrophil gelatinase associated lipocalin. Pediatr Nephrol, 2009. 24 (12): p. 2387 92. 304. Young, W.F., Endocrine Hypertension: Then and Now. Endocr Pract: p. 1 52. 305. Yu, J., et al., Expression and localization of Hsps in the heart and blood vessel of heat stressed broilers. Cell Stress Chaperones, 2008. 13 (3): p. 327 3 5.

PAGE 158

141 306. Zayara, A.E., et al., Blockade of nucleus accumbens 5 HT(2A) and 5 HT (2C) receptors prevents the expression of cocaine induced behavioral and neurochemical sensitization in rats. Psychopharmacology (Berl). 307. Zethelius, B., et al., Use of multip le biomarkers to improve the prediction of death from cardiovascular causes. N Engl J Med, 2008. 358 (20): p. 2107 16. 308. Zhang, J., et al., Collagen targeting vascular endothelial growth factor improves cardiac performance after myocardial infarction. Ci rculation, 2009. 119 (13): p. 1776 84. 309. Zhao, W., D.I. Diz, and M.E. Robbins, Oxidative damage pathways in relation to normal tissue injury. Br J Radiol, 2007. 80 Spec No 1 : p. S23 31. 310. Zlotnik, A. and O. Yoshie, Chemokines: a new classification sys tem and their role in immunity. Immunity, 2000. 12 (2): p. 121 7. 311. Zuliani, G., et al., High interleukin 6 plasma levels are associated with low HDL C levels in community dwelling older adults: the InChianti study. Atherosclerosis, 2007. 192 (2): p. 384 90.

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About the Author Marie Bourgeois is a graduate of University of South Florida with a B.S. in Clinical Chemistry (1991) and an M.P.H. in Toxicology and Risk Assessment (2006) from the University Of South Florida College Of Public Health. In 2007, Mrs. Bourgeois was accepted to the Ph.D. program in Toxicology and Risk Assessment at the University Of South Florida College Of Public Health by the Department of Environmental and Occupational Health. Her degree focused the impact of gender and cocaine use on the urinary expression of biomarkers of oxidative stress and inflammation. She held the position of Graduate Assistant while at the College of Public Health and helped administer online courses. She has been a presenter at national conferences and is the coauthor of several publications and abstracts.