HEMOLYTIC ANEMIA AND THE REACTIVE SULFHYDRYL GROUPS OF THE ERYTHROCYTE MEMBRANE by Erwin Peter GABOR, M.D., C.M. A thesis presented to the Faculty of Graduate Studies and Research ~n partial fulfillment of the requirements for 1 the degree of Master of Science in Experimental Medicine. McGill University Clinic and Royal Victoria Hospital, Montreal, Quebec. April, 1964. HEMOLITIC ANEMIA AND THE REACTIVE SULFHYDR!L GROUPS OF THE ER'YTHROCITE MEMBRANE by Erwin Peter Gabor ( Abstract ) Membrane sul.fhydryl ( SH) groups have been reported to be important for the maintenance of red cell integrity E, ~ ( Jacob and Jandl, 1962 ). A technique has been developed for the determination of reactive membrane sulfhydryl content in intact erythrocytes, utilizing sub­ hemolytic concentrations of p-chloromercuribenzoate (PMB). The erythrocyte membrane of 52 healthy subjects contained 2.50 - 2.85 x lo-16 moles of reactive SH groups ( mean 2.50 ·~ 0.20 ) per erythrocyte, when determined by this method. A 27-56% reduction of erythrocyte membrane SH content was observed in various conditions characterized by accelerated red cell destruction, including glucose- 6-phosphate dehydrogenase ( G6PD ) deficiency, drug-induced, auto- immune and other acquired hemolytic anemias and congenital spherocytosis. Normal membrane sulfhydryl content was found in iron deficiency anemia, pernicious anemia in relapse, and in other miscellaneous hematological conditions. Inhibition of membrane SH groups with PMB caused marked potassium leakage from the otherwise intact cells. The possible role of membrane suli'hydryl groups in the development of certain types of hemolytic anemias,and in the maintenance of active transmembrane cation transport in the erythrocyte is discussed. - i - PREFACE AND ACKNOWLEDGE:MENTS The investigations described in this thesis represent a part of a larger project initiated to study the mechanism of hemolytic anemia in man. The first phase of this research was undertaken to investigate possible molecular changes occurring in the erythrocyte membrane in hemolytic anemia. The investigation was prompted by the observation of de Leeuw et al. (1) that drug-induced hemolysis, hitherto regarded as an inborn error of metabolism,-deficiency of a red cell enzyme, glucose-6-phosphate dehydrogenase (G6PD), may frequently occur in patients with no such deficiency. The investigation of this phenomenon has led to the study of the erythrocyte membrane, and specifically of the reactive sulfhydryl radicals in the erythrocyte membrane. In the course of this study, a technique has been developed for the quantitative titration of these important membrane constituents. Since early resulta have suggested alterations in the membrane sulfhydryl content of erythrocytes in certain conditions accompanied by increased red cell destruction, the scope of the investigation has been widened to a variety of associated hematological disorders. It is the development of this technique and the resulta obtained by its application that forma the subject of this thesis. The place of the presented findings in the long-range - ii - study of red cell hemolysis will be projected in the discussion of the findings. The more strictly clinical investigation of the patient material which was carried out simultaneously with this study will be omitted from the thesis. During the two years in which the work presented in this thesis was carried out, the writer was a Clinical Fellow of the Hematology Division, Department of Medicine, Royal Victoria Hospital. Throughout this period, advice and encouragement was readily offered by each and every member of the Division and enthusiastic cooperation was always forthcoming on the part of the hematology technicians. The author feels particularly indebted to Dr. Louis Lowenstein, whose guidance and direction in the philosophy and nature of medical research will far outlive the scope of this thesis, and to Dr. N.K.M. de Leeuw, whose help assisted the author in every phase of this study. Grateful thanks are also due to Dr. Bernard A. Cooper, who was always available with excellent ideas and assistance to solve the nunsolvable11 problems as they arose. The useful suggestions of Dr. Rhoda Blostein in the later phase of this study and in the preparation of the thesis is also gratefully acknow­ ledged. - iii - The technical assistance of Mrs. Ghislaine Houle in the laboratory work, Miss Barbara Heward in the preparation of the illustrations, and Miss Sylvia Sentheim in the typing of this thesis is acknowledged with deep appreciation. The research project was supported by a Grant from the Medical Research Council of Canada. iv TABLE OF CONTENTS Page PREFACE AND ACKNOWLEDGEMENTS • • • • • • • • • • • • • • • • • • • • • • i TABLE OF CONTENTS ••••••••••••••••••••••••••••••••• ii LIST OF FIGURES •••••••••·••••••••••••••••••••••••· ix LIST OF ABBREVIATIONS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • x INTRODUCTION •.• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 CHAPTER I: HISTORICAL BACKGROUND • • • • • • • • • • • • • • • • • 5 PART I: SALIENT FEATURES OF SH GROUPS IN BIOLOGICAL MATERIAL • • • • • • • • • • • • • • • • • • • 5 1. The· .ro1e of SH groups in protein structure and function •••••••••••••••• 5 2. The reactivity of SH groups 1 • • • • • • • • • • • 7 3. Chemical reactions and inhibitions of .. SH groups •.•.•..••..•.............• PART II: RELATION OF SH GROUPS TO STRUCTURAL AND FUNCTIONAL COMPETENCE OF ERYTHROCYTES ••• 10 1. Correlation of red ce11 integrity and intrace1lular SH groups ••••••••••• 10 2. Correlation of red ce11 integrity and membrane SH groups •••••••••••••••••••• 11 a. The site of action of heavy metals •• 11 b. The hemolytic action of SH reagents. 12 c. The role of membrane SH groups in cellular integrity ••••••••••••••••• 13 d. The site of action of oxidative drugs . • • . • • • • . • • . • • . • . • . • 14 e. The role of membrane constituants in intracellular metabolism ••••••• 15 v Page CHAPTER II: EXPERIMENTAL • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 18 PART I: MA TERIALS AND METHODS • • • • • • • • • • • • • • • • • • • • là 1. Introduction to the methods of sulfhydryl determination ••••••••••••••••• là 2. Materials • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 20 A. Reagents • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 20 B. Substrate • • • • • • • • • • • • • • • • • • • • • • • • • • • • 22 3. P.reliminary experimenta for the application of Boyer's method of SH titration to intact erythrocytes •••••••••••••••••••••• 25 a) Titration of excess P.MB • • • • • • • • • • • • • • 26 b) Selection of PMB concentration and incubaJ-ion time •••••••••••••••••• 27 i. Incuba~on,of erythrocytes with various' concentrations of PMB • • • • • 2$ ii. The time course of PMB uptake by erythrocytes •••••••••••••••••• 29 4. Determination of intracellular GSH content in erythrocytes incubated with PMB •••••••• 30 5. The effect of leukocyte and reticulocyte content of red cell preparations on the PMB uptake of erythrocytes ••••••••••••••• 33 6. Resume of the technique used for the determination of erythrocyte membrane sulfhydryl content ••••••••••••••••••••••• 34 7. Other methods employed • • • • • • • • • • • • • • • • • • • 37 PART II: RESULTS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 38 A. The reactive membrane sulfhydryl content of normal erythrocytes •••••••• 3à 1. The range and mean of normal membrane sulfhydryl content ...•....••••..............•..•.....•...• 38 2. Studies on ACD preserved blood • • • • • • • • • • • • • • • • • 39 vi Page PART II: (Cont'd) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • B. Determination of erythrocyte membrane sulfhydry1 content in patients ••••••• 39 1. Erythrocyte membrane SR content in drug- induced hemo1ytic anemia •••••••••••••••••••••• 40 2. Membrane SR groups of G6PD-deficient erythrocytes •••••••••••••••••••············••• 42 3. The reactive membrane SH content in acquired hemo1ytic anemia •••••••••••••••••••••••••••••• 42 4. Erythrocyte membrane su1fhydry1 groups in hereditary spherocytosis •••••••••••••••••••••• 45 5. Erythrocyte membrane sulfhydry1 content in misce11aneous conditions with possible hemo1ytic component ···•·•••••••••••••••••••••• 45 6. Erythrocyte membrane su1fhydry1 content in iron deficiency anemia •••••••••••••••••••••••• 45 7. Erythrocyte membrane su1fhydry1 content in pernicious anemia ••••••••••••••••••••••••••••• 46 8. Erythrocyte membrane su1fhydry1 content in misce1laneous hemato1ogical conditions ••••••••• 46 9. The effect of azotemia on the erythrocyte membrane SH content ••••••••••••••••••••••••••• 47 10. The effect of su1fonamides on the erythrocyte membrane SH content ••••••••••••••••••·•······· 47 C. The time course of P.MB uptake in hemolytic anemia ••••••••••••••••• 48 D. The effect of FMB on the erythrocyte GSR 1eve1 ••••••••••••••••••••••••••• 49 E. The effect of PMB on cation transport in erythrocytes • • • • • • • • • • • 50 vii Page CHAPTER III: DISCUSSION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • PART I: DISCUSSION OF THE TECHNIQUE OF ERYTHROCYTE MEMBRANE SULFHYDRYL GROUP DETERMINATION • • • • • • • • • • • • • • • • • • • • • • • • • • • • 51 1. The choice of intact cells as opposed to red cell ghosts •••••••••••••••••••••••••••••••• 51 2. The choice of reagent, concentration, and incubation time •••••••••••••••••••••••••••••••• 54 PART II: DISCUSSION OF THE RESULTS: THE REACTIVE SULFHYDRYL GROUPS OF