METABOLIC ALTERATIONS OF FREE AND PROTEIN-BOUND BIOTIN IN RATS DURING DIETARY BIOTIN MANIPULATION AND ENDOTOXIN EXPOSURE By BRANDON JAMES LEWIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003 ACKNOWLEDGMENTS I would like to express my gratitude toward Drs. Bobbi Langkamp-Henken and Robert McMahon for their continuous guidance, advice, and support throughout the course of my Ph.D. work. My great appreciation also goes to my committee, Drs. Robert Cousins, Jesse Gregory III, and Susan Frost, for constantly providing valuable comments and suggestions during my Ph.D. experience. I would also like to express my gratitude towards my wife, Darci, and my parents, Pete and Sharon Lewis, for their support and undying faith that this would eventually be finished. Finally, I would like to thank Sara Rathman for all her help and especially her reliability in the lab and Amy Mackey for her support and enthusiasm in answering my many questions. ii TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. ii ABSTRACT....................................................................................................................... vi CHAPTER 1 LITERATURE REVIEW ................................................................................................1 Biotin............................................................................................................................... 1 Structure and Function ............................................................................................. 1 Sources ..................................................................................................................... 5 Absorption................................................................................................................ 5 Transport .................................................................................................................. 7 Deficiency ................................................................................................................ 7 Toxicity .................................................................................................................. 10 Biotin Status........................................................................................................... 10 Role in Immune Function ...................................................................................... 11 Lipopolysaccharide....................................................................................................... 12 Structure ................................................................................................................. 12 Binding................................................................................................................... 13 Elicited Response................................................................................................... 15 Rationale and Hypotheses............................................................................................. 16 Dietary Modulation of Biotin Metabolism............................................................. 16 Modulation of Biotin Metabolism by Endotoxin................................................... 17 2 GENERAL MATERIALS AND METHODS...............................................................21 Preparation of Radiolabeled and Nonradiolabeled Bisnorbiotin and Biotin Sulfoxide Standards........................................................................................................... 21 Bisnorbiotin............................................................................................................ 21 Biotin Sulfoxide ..................................................................................................... 22 Biotin, Bisnorbiotin, Biotin Sulfoxide, and Biocytin Separation by High Performance Liquid Chromatography.................................................................................... 22 Preparation of Biotinylated Bovine Serum Albumin.................................................... 23 Competitive Assay of Biotin......................................................................................... 24 Preparation of Avidin AlexaFluor 430 ......................................................................... 25 Markwell Assay for Protein Concentration .................................................................. 25 Carboxylase Separation by SDS-PAGE ....................................................................... 26 iii Acetyl CoA Carboxylase ....................................................................................... 26 Pyruvate Carboxylase ............................................................................................ 26 Methylcrotonyl-CoA Carboxylase, and Propionyl-CoA Carboxylase................... 26 Detection of Carboxylases After SDS-PAGE............................................................... 26 Activity Assays of Pyruvate Carboxylase and Methylcrotonyl CoA Carboxylase ...... 27 Pyruvate Carboxylase ............................................................................................ 27 Methylcrotonyl CoA Carboxylase ......................................................................... 28 Isolation of P61 by Monomeric Avidin Column .......................................................... 28 3 DIETARY BIOTIN INTAKE MODULATES THE POOL OF FREE AND PROTEIN- BOUND BIOTIN IN RAT LIVER ...............................................................................30 Introduction................................................................................................................... 30 Methods.................................................................................................................. 32 Animals and Dietary Treatments ........................................................................... 32 Sample Preparation and Analysis........................................................................... 33 Measurement of Free Biotin................................................................................... 34 Measurement of Protein-Bound Biotin .................................................................. 34 Statistical Analysis................................................................................................. 37 Results........................................................................................................................... 37 Effect of Dietary Biotin Intake on Rat Growth and Food Intake........................... 37 Effect of Dietary Biotin Intake on Biotin Status.................................................... 38 Effect of Dietary Biotin Intake on the Relative Abundance of Biotinylated Carboxylases .............................................................................................. 40 Discussion..................................................................................................................... 41 4 LIPOPOLYSACCHARIDE INDUCED INFLAMMATORY RESPONSE ALTERS CIRCULATING BIOTIN AND THE RELATIVE ABUNDANCE OF THE BIOTIN- DEPENDENT CARBOXYLASES...............................................................................47 Introduction................................................................................................................... 47 Methods......................................................................................................................... 49 Materials................................................................................................................. 49 Animals and Dietary Treatments ........................................................................... 50 Dose-response experiments .............................................................................50 Time-dependent and dietary status experiments..............................................51 Sample Preparation ................................................................................................ 51 Dose-response experiments .............................................................................51 Time-dependent and dietary status experiments..............................................51 Competitive Binding Assay of Biotin.................................................................... 52 Detection and Quantification of Biotinylated Proteins .......................................... 52 Serum Zn and Serum Glucose................................................................................ 52 PC and MCC Activity............................................................................................ 52 Statistical Analysis................................................................................................. 53 Dose-response experiments .............................................................................53 iv Time-dependent and dietary status experiments..............................................53 Results........................................................................................................................... 53 Dose Dependent Effects of an LPS Induced Inflammatory Response................... 53 Serum zinc and glucose ...................................................................................53 Urine, serum, and tissue free biotin .................................................................54