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

THE ERYTHROCYTE MEMBRANE • • • • • • • • • • • • • • • • •

A. Normal erythrocytes • • • • • • • • • • • • • • • • • • se B. Abnormal erythrocytes • • • • • • • • • • • • • • • • 60

1. G6PD deficient erythrocytes • • • • • • • • • • • • • • • • • • • • 60 2. Drug-induced hemolytic anemia in non-G6PD deficient subjects •••••••••••••••••••••••••••••• 61

). Other acquired hemolytic anemias • • • • • • • • • • • • • • • 72

Hereditary spherocytosis • • • • • • • • • • • • • • • • • • • • • • • 75

5. Other conditions • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 77 6. Splenic sequestration of SH deficient red cells •••••••••••••••••••••••••••••••••••••• 77

PART III: GENERAL DISCUSSION: • • • • • • • • • • • • • • • • • • • • • • 1. The role of SH groups in the function of the red cell membrane •••••••••••••••••••••••••• 79 2. A brief review of the transmembrane cation transport in erythrocytes •••••••••••••••••••••• e1 ). The analogy of muscle and red cell ATPase: a hypothesis for the role of membrane SH groups in the function of the erythrocyte membrane ATPase •••••••••••••••••••••••••••••••• g4 viii

Page

PART III: (Cont'd) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 4. The leakage of potassium from erythrocytes exposed to oxidative compounds •••••••••••••••• 86 5. The leakage of potassium from PMB incubated erythrocytes •••••••••••••••••••••••• 88 6. A proposed mechanism for the participation of SH groups in the active transport of cations in erythrocytes ••••••••••••••••••••••• 90 7. The significance of the findings of reduced membrane sulfhydryl content in certain hemo1ytic conditions •••••••••••••••••••••••••• 92

SUMMARY AND CONCLUSION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 94 TABLES I - XVII • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 98

FIGURES 1 - 12 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 117 BIBLIOGRAPHY •••••••••••••••••••••••••••••••••••••••••• 129 LIST OF FIGURES

Figure 1 - The Reactions of Para-chloromercuribenzoate 2 - Absorbancy of P-mercuribenzoate and Its Mercaptide with Glutathione 3 - PMB Binding by Erythrocytes 4 - Effect of various Concentrations of PMB on the Erythrocyte GSH Content a. Erythrocyte GSH Levels 5 - Effect of Various Concentrations of PMB on the Erythrocyte GSH Content b. Per Gent Decrease of GSH Level 6 - Time Course of PMB Uptake by Erythrocytes 7 - Time Course of PMB Uptake by Erythrocytes in Acquired Hemolytic Anemia 8 - Time Course of PMB Uptake by Erythrocytes from Patient with Congenital Spherocytosis 9 - Absorbancy of Red Cell Hemolysate Dilutions at Various Wave Lengths 10 - Losa of Potassium from Red Cella Incubated with various Concentrations of PMB 11 - Losa of Potassium from PMB Incubated Red Cells: Comparison of PMB Effect on Normal and Defective Erythrocytes 12 - The Mean and Range of Erythrocyte Membrane Sulf­ hydryl Content in Various Conditions - x -

LIST OF ABBREVIATIONS

ACD acid citrate dextrose (blood preservative) APH acetylphenylhydrazine ATP adenosine triphosphate ATPase adenosine triphosphatase BUN blood-urea-nitrogen ca++ calcium DPN diphosphopyridine nucleotide G6PD glucose-6-phosphate dehydrogenase GSH reduced glutathione GSSG oxidized glutathione GSSG-R glutathione reductase H.A. hemolytic anemia HGB hemoglobin Hct hematocrit Hg++ mercury ~ potassium Mg++ magnesium MCV mean corpuscular volume Na sodium NEM N-ethyl maleimide PMB p-mercuri (chloro) benzoate Pr:N - packed cell volume Retics reticulocyte count RBC erythrocyte (red blood cell}

SH suli'hydryl WBC leukocyte (white blood cell)

TPN triphosphopyridine nucleotide - 1 -

INTRODUCTION

The history of medicine in the twentieth century may be characterized more by the discovery and increasing application of chemical compounds in the treatment of disease than by any other event. The upsurge of new drugs inevitably carried the risk of unwanted and often unfore­ seeable aide effects. Indeed~ the recognition of drug-induced 11 blood dyscrasias11 --toxic drug effects on the blood and blood­ forming organs-- has demanded increasing alertness on the part of the clinician and has received increasing attention in the medical literature. One of the most puzzling drug-induced blood diseases is the hemolytic anemia produced by certain compounds. Several drugs have been observed to induce hemolytic anemia quite frequently~ but unpredictably, in certain individ­ uals but not in others. This has led to a long series of investigations resulting in an increasing insight into the mechanism of drug-induced hemolysis, and has focused consider­ able attention on the not too well known metabolic processes in the erythrocyte. It has been known for over a hundred years that direct exposure to various chemicals with potent oxidant action~ auch as anilin~ nitrobenzene, phenylhydrazine, etc., will result in the development of intracellular inclusions~ referred to as - 2 -

Heinz bodies~ in the erythrocyte~ and in eventual hemolytic red-cell destruction. Strong oxidant compounds~ or excessive amounts of weaker ones~ are capable of producing hemolysis uniformly in every exposed subject. It was noted~ however, in the 1940 1 s, that therapeutic doses of certain commonly used drugs, such as primaquine, or the sulfonamides~ may produce a similar type of hemolysis in susceptible individuals. The cause of susceptibility was later identified as an intrinsic red-cell abnormality: the deficiency of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) (2~3~4,5~6,7). This inborn error of metabolism resulta in deficiency and/or instability of reduced glutathione (GSH) in the erythrocytes, because the pentose-phosphate pathway of glucose metabolism which is responsible for the conversion of oxidized glutathione (GSSG) to the reduced form in red cells depends on the activity and availability of its rate-limiting enzyme, G6PD. The inadequate maintenance of GSH supply interferes with the efficiency of the self-regenerating electron transport system that protects the hemoglobin and other internal cell constituants from oxidation. Thus, ingestion of oxidative compounds by such an individual will result in oxidation of the hemoglobin Heinz body formation and in subsequent splenic sequestration and hemolysis of the affected erythrocytes (6). This discovery called attention to the importance of sulfhydryl compounds in the protecting mechanism of the red cell. - 3 -

Another entirely different intrinsic red-cell defect involving the hemoglobin has been described recently as being responsible for sensitivity to oxidative compounds. Patients with 11 hemoglobin Zurich" may develop acute severe hemolytic anemia if treated with sulfonamides (8). Why the replacement of histidine by arginine on the sixty-third place of the beta chain of this defective hemoglobin makes the red cel1s susceptible to oxidative drug action is not yet understood (9). The occurrence of drug-dependent, auto-immune hemo1ytic anemia a1so has been reported (10,11,12) where readministration of the offending agent will precipitate an antigen-antibody reaction resu1ting in hemo1ysis of the antigen-coated red cells. This mechanism, however, is very rare and it can be identified by the presence of anti-drug antibodies demonstrable with positive indirect Coombs 1 test in the presence of the offending drug (11). That other factors may play an important role in the deve1opment of drug-induced hemolysis was demonstrated by the finding that typical Heinz-body hemolytic anemia can be induced by therapeutic doses of sulfonamides or nitrofurantoin in subjects not deficient in erythrocyte G6PD (1). This suggested that deficiency or instability of intracellular erythrocyte GSH is not the only mechanism by which therapeutic - 4 - doses of drugs produce oxidative hemolysis. Whether the cause of this phenomenon is related to extra-corpuscular factors, or whether any other intrinsic defect is involved, is not known. This has given the impetus to undertake the present investigation, one aspect of which, the study of erythrocyte membrane sulfhydryl groups, is the subject of this thesis. - 5 - .

CHAPTER I

HISTORICAL BACKGROUND

PART I SALIENT FEATURES OF SULFHYDRYL GROUPS IN BIOLOGICAL MATERIAL

The biological importance of sulfhydryl compounds in various tissues has been known for long as evidenced by the extensive literature on this subject. The nature and bio­ logical function of sulfhydryl (SH) groups and their association with enzymatic processes is well summarized by Work and Work (13) and by Boyer {14). A detailed study of SH groups is not within the scope of this thesis, but a brief review of sorne basic factors may be pertinent to the understanding of this study.

1. The Role of SH Groups in Protein Structure and FUnction Many enzymes reqUire SH groups ror their activity. These radicals often are located in the active center of the enzyme protein and are, in fact, a part of the active core. SH groups adjacent to the active center may be essential for the proper function of the enzyme and determine enzyme activity. Other more peripheral SH groups may be important for the stability of the enzyme protein. SH groups often participate in the formation of the enzyme-substrate complex during the enzymatic - 6 - reactions. A substance which interferes with the reactivit.y of an essential group in the active center of the enzyme may inhibit its activity by rendering it incapable of combining with its substrate. SH groups are also important in the maintenance of tertiary structures of the enzyme or other protein molecules by formation of S-S bridges or other intra- or inter-molecular bonds. An enzyme that loses its activity when sorne, certain, or all of its SH groups undergo chemical modification is usually called a ~ulfhydryl enzyme (14). Oxidation or blockage of certain SH groups in such enzymes cause complete loss of its activity. This may occur by three different mechanisms: (1) Blockage of an SH group which has a primary role in the catalytic activity of the enzyme itself. (2) Blockage of an SH group may cause structural changes in the enzyme protein proper. (3) Blockage of an SH group may affect other groups attached to the -s of the -SH group.

Sorne enzymes contain s-s linkages in their resting1 inactive forms, but the S-S bridge is very labile, and SH groups can be formed when enzyme activity is required (13). Others may regenerate their SH groups through non-enzymatic reduction by GSH while the resultant GSSG can be reconverted enzymatically to GSH: glutathione reductase GSSG + 2TPNH + ~ ------~ 2GSH + 2TPN+ - 7 -

This enzymatic regeneration or reduced glutathione takes place in erythvocytes during the first step of the pentose phosphate pathway.

2. The Reactivity of the SH Groups The activity of the reactive SH groups is more important than the total SH content of a tissue or biological material (14). For the description of the degrees of reactivity, various terms are used: (1) Reactive, free, or rapidly reacting SH groups. (2) Intermediate, slowly reacting, or nsluggishu SH groups. (3) Unreactive, unavailable, or blocked SH group. The latter is a relative term since, by various methods, these groups also can be brought into reaction. The difference in reactivity could be presumably due to various factors: (a) Hydrogen bonding of sorne SH groups may occur by the formation of intra- or intercellular hydrogen bonds. (b) covalent bond formation with SH groups is one possibility,

(c) Steric blocking of sorne SH groups by the tertiary molecular structural arrangements (ring formation, certain helix formations, etc.). This is probably the most likely cause of the differences in reactivity. Consequently, changes in the steric arrangement of molecular structure may produce changes in the reactivity of the SH groups - 8 -

involved. For example, urea may increase reactivity of sorne SH groups by unfolding of seme tertiary molecular structures. (d) Oxidation or chemical blockage may render the SH groups totally unreactive. This can be irreversible, but it may represent a transient or reversible state. For example, S-S bridges may break and reduced forms may regenerate, thus regaining their reactivity under certain circumstances, or a blocking agent may become detached from the SH group. Cyanides, for example, may detach p-chloromercuribenzoate from SH groups.

3. Chemical Reactions and Inhibition of SH Groups a. Formation of thiel ester bonds between enzyme SH groups and the substrate of the enzyme is an example of the variety of chemical reactions that SH groups may enter. Sorne reactions may result in the inhibition of the reactivity of SH groups. b. Several compounds are known to inhibit the SH groups. Heavy metals, particularly mercury, have a high affinity to SH groups. Mercury, for example, may act as a Hg++ ion, or as a complex mercury compound, forming mercury-mercaptide linkages (14). - 9 -

s /S-Hg-Q-coon

Enzyme/t or Enzyme ~1s ~-Hg--Q-COOH

c. Reaction of SH groups with oxidative compounds results in the formation of S-S bridges between SH groups with consequent loss of their reactivity. This may take place by direct electron transfer or by mediated electron trans fer:

2 GSH + H202 -----~GSSG + 2 H20

2 GSH + 2 TPN-----~ GSSG + 2 TPNH S-S bridges may be formed between neighboring SM groups of a protein molecule by the effect of strong oxidants:

exidant++ ------>

That certain drugs may act by blocking SH groups and hence interfere with metabolic processes of the living cell~ was suggested by the early observation that compounds which oxidize the surface SH groups of certain bacteria are bacterio­ cidal~ ahd~that Atebrin and Quinine, which may interfere with the reactivity of certain SH groups, inhibit the utilization of ATP by the malaria parasite (13). - 10 -

PART II RELATION OF SH GROUPS TO STRUCTURAL AND FUNCTIONAL COMPETENCE OF ERYTHROCYTES

1. Correlation of Red Cell Integrity and Intracellular SH Groups The correlation of erythrocyte integrity and SH groups was demonstrated in 1954 by Benesch and Benesch (15). Studying the effect of mercurial compounds on the hurnan erythro­ cyte in vitro, they concluded that SH groups are essential for the maintenance of the intact erythrocyte structure. They showed that the hemolytic effect of p-chloromercuribenzoate (PMB) and other organic mercurials is not due to denaturation of the hemoglobin, but to blockage of essential cellular thiols such as glutathione. The role of thiols in oxidative drug action on the erythro­ cytes was studied by Allen0.and Jandl who suggested that SH groups are involved in the physiological activity of hemoglobin and in the maintenance of cellular integrity (16). They postulated that the prirnary site of oxidative drug action is the intracellular thiol groups, and that GSH, acting as a hydrogen donor, may facilitate the enzyrnatic destruction of peroxides, generated by oxidative compounds such as acetyl­ phenylhydrazine, protecting all other cellular constituents from the danger of peroxidation. Thus intracellular GSH may protect not only the SH groups of native hemoglobin from - 11 - oxidation, but it may protect the membrane SH groups as well by a similar mechanism. They also demonstrated that PMB, which acts directly on the membrane SH groups, causes spherocytosis, increased erythrocyte fragility and osmotic hemolysis, depending on the concentration of the PMB and the length of incubation. Several other studies have been published on the role of intracellular thiols (17,18,19) and their function in the glycolytic enzymes of the erythrocytes (20,21). The discovery of certain enzymes in the red cell stroma focused more attention on the function and structure of the erythrocyte membrane.

2. Correlation of Red Cell Integrity and Membrane SH Groups In 1956 Herbert demonstrated the phosphatase activity of isolated red cell stroma and of intact cell membranes (22). He observed that mercury and other heavy metals inhibit the erythrocyte stromal apyrase (pyro-phosphatase) activity. a. Site of Action of Heavy Metals Rothstein, investigating the toxic effect of heavy metals on tne living cells, has emphasized that tne primary site of action of heavy metals is in the cell membrane (23). He has shown in several experiments that while physiologically inert substances in the cell, such as GSH,_may combine with heavy metals to protect the active, functional SH sites in the cell - 12 - by "soaking up 11 the heavy metal ions, the most important protection is afforded by the marked physical resistance of cell membranes to heavy metals. Thus, while intracellular enzymes are well protected, enzymes in the cell membrane are the first target and most probable site of damage caused by several toxic substances. Heavy metals for example, may produce profound changes in the membrane structure by inter­ acting with important chemical ligands. b. Hemolytj_c Action of SH Reagents Tsen and Collier who studied the protective action of tocopherol against hemolysis of rat erythrocytes in vivo, reported in 1960 that tocopherol protects erythrocytes from oxidative hemolysis by virtue of its anti-oxidant action (25). Sufficient doses of the vitamin protected the membrane SH groups of rat erythrocytes from oxidation by inhibiting the dialuric acid induced peroxide formation in the unsaturated lipid portion of the cell membrane. They demonstrated that intracellular GSH cannot afford protection against the oxidative action of lipid peroKide generating compounds, and that susceptibility to peroxide-induced hemolysis is not related to intracellular GSH content. Similarly, they have presented evidence that lower concentration of PMB can cause hemolysis by direct effect upon the erythrocyte membrane with relatively little decrease in intracellular GSH content, while - 13 - lower concentration of ethyl maleirnide (NEM) caused only little hemolysis in spite of the rapid fall in GSH content produced by this alkylating agent. Thus the relative independence of hemolysis from intrauellular GSH content was first demonstrated. Tsen and Collier concluded that the differing hemolytic actions of various SH-reagents can be attributed to their varying effects on the stromal proteins rather than upon the free intracellular GSH. Heavy metal SH reagents probably cause hemolysis through disruption of cell membrane structure by combining with SH groups and perhaps other important groups of the cell surface {25). c. The Role of Membrane SH Groups in Cellular Integrity The important role of membrane SH sitœin the maintenance of cellular integrity and regulation of erythrocyte life span was demonstrated by Jandl and Jacob in 1962 (26,27). They showed that higher concentration of NEM which readily penetrates into the red cell,rapidly binding all SH sites, caused osmotic swelling and hemolysis of the cells after paralyzing intra­ cellular glucose metabolism, disrupting cation gradients and causing precipitous fall in intracellular GSH content. Similar disruption of cation gradients with subsequent osmotic hemolysis was produced by PMB which, in lower concentration, does not penetrate into the red cella and reacts only with membrane SH groups without interfering with intracellular glucose metabolism - 14 -

or GSH content. Thus they have presented evidence that the shape and viability of human erythrocytes depend primarily on membrane and not upon intracellular SH activity. Inhibition of membrane SH sttes by agents acting directly on the membrane, or oxidation of membrane SH groups secondary to oxidative consumption of intracellular thiols, will result in osmotic hemolysis in vitro (26). In vivo studies demonstrated the rapid and complete splenic sequestration of erythrocytes previously exposed to sub-hemolytic doses of membrane SH inhibitors in vitro upon their re-injection into human subjects. The membrane SH-inhibited cells were removed from the circulation in spite of their normal osmotic tragility, normal intracellular GSH level, and normal glycolytic activity (27). Jandl and Jacob also emphasized the close association of membrane SH inhibition with disturbed cation control and diminished cell viability.

On the basis of their experiments, they postulated that the loss of membrane sulfhydryl activity may be the decisive hemo­ lytic event common to a number of hemolytic processes, including Heinz body anemias. d. The Site of Action of Oxidative Drugs That oxidative compounds like heavy metals may inflict direct damage on the cell membrane was suggested by the observation of Weed, Eber and Rothstein (28) who demonstrated that relatively small amounts of oxidant compounds, such as - 15 - primaquine, cause membrane injury with leakage of potassium

{~) and excessive entry of sodium (Na+) into the exposed red cells. The disruption of cation gradients by low concentrations of primaquine without interference with intra­ cellular metabolic processes, was observed in both G6PD deficient and normal red cells. This observation suggested that primaquine, at least in low concentrations, acts primarily on the erythrocyte membrane, exerting its oxidative effect which eventually leads to oxidation of intracellular thiols and hemolysis in the case of G6PD deficient erythro­ cytes. The effect on the cation gradients also suggested that oxidative drugs may interfere with active cation transport across the cell membrane. e. The Role of Membrane Constituants in Intracellular Metabolism Several other observations supported the possible role of erythrocyte membrane in the intracellular metabolism. Ramot et al. suggested in 1961 that deficient activity of G6PD is due to lack of a stromal activator in primaquine sensitive eryth~ocytes (29). Brunetti et al. proposed, in 1962, that stromal SH groups may play a role in the activation of certain intracellular enzymes such as G6PD. He suggested also that hemolytic drugs act primarily on the stromal SH groups and produce hemolysis by altering the sulfhydryl structure - 16 - of the red cell membrane (30). The theory of stromal activation of G6PD, however, has not been substantiated satisfactorily. Other findings supporting the role of erythrocyte membrane in active cation transport and intra­ cellular metabolism of the red cell will be discussed later. In summary, there seems to be good evidence that SH blocking agents affect the erythrocytes primarily by their interaction with membrane SH sites. Qxidative compounds may profoundly affect the intracellular SH pool, but also act on the cell membrane producing a membrane injury similar to that caused by heavy metals. It seems obvious that any drug or reagent must come into physical contact first with the cell membrane, and therefore, may in sorne degree, inter­ act with its constituents. Their action on membrane vs. intracellular groups depends on their ability to penetrate into the intracellular phase, i.e. on the resistance offered by the cell membrane against their entry into the cell. On the basis of the presented findings, it can be presumed that sulfonamides and similar oxidative drugs may effect primarily, the reactive membrane SH groups of the erythro­ cytes in vivo. Since·.only a small percentage of non-G6PD deficient patients treated with such drugs develop Heinz body­ hemolytic anemia, it was thought that pre-existing or induced dit'ferences in the reactive SH content of the erythrocyte membrane would provide an explanation for this selective hemolysis. - 17 -

Because of this presumption, and because of the obvious importance of membrane SH sites, the determination of membrane SH-content in the red cells of healthy individuals and of patients with various hemolytic and non-hemolytic anemias was thought to be desirable. Thus a study has been undertaken, and a technique has been developed for the determination of the reactive SH content of the intact, non-hemolyzed erythrocyte membrane. - 18 -

CHAPTER II

EXPERIMENTAL

PART I MATERIALS AND METHODS

1. Introduction to the Methods of Sulfhydryl Determination Several methods have been described for the determination of SH content of various biological materials (31-35), however, no attempts have been made to adapt these methods to the quantitative determination of SH content in intact cells, particularly erythrocytes. For this purpose, a method had to be developed which permitted the use of intact cells, rather than hemolyzed cell stromata, the so-called red cell

11 11 ghosts • The process of preparing erythrocyte ghosts may introduce relatively great variables which may prevent accurate determination of, and reliable comparison between the SH content of normal, abnormal and presumably abnormal red cells (see DISCUSSION). Since the cell membrane SH content is only a fraction of the total cellular thiol pool, a SH reagent with a marked selectivity for membrane SH groups was required which, however, would not destroy the ~ed dells. Therefore, p-chloromercuribenzoate (p-mercuribenzoate, PMB) was selected as the reagent of choice for the determination of the reactive SH content of intact erythrocyte membranes. - 19 -

PMB is an organic mercury compound which reacts readily and rather specifically with SH groups by forming mercury­ mercaptide linkages between the S of the SH groups and the Hg of the PMB molecule in a stoichiometric fashion (14,36,37) (figure 1). Large, positively charged ions are transported very slowly across the cell membrane (36) and this pre­ determines the site and mode of their action on the cell (23). Tsen and Collier and Jacob and Jandl demonstrated that in low, non-hemolytic concentrations, the large, positively charged molecules of PMB did not penetrate into the intact erythro­ cytes, but interacted only with the membrane SH groups. Apparently only in concentrations that grossly affected membrane structure and permeability, did PMB enter into the red cells with consequent binding of intracellular GSH and hemolysis of the cells (25,26). In 1954 Boyer described a method for rapid and sensitive spectrophotometric titration of free SH groups of biological materials with PMB, bas.ed on the relatively large increase in ultra-violet light absorption that accompanied mercaptid~' formation (38). In the range of 250-260 m~. the increase in absorption was a linear function of the amount of SH groups added to a buffered solution of PMB. Kaper and Houwing round this method applicable for studying the SH content of turnip yellow mosaic viruses and their shells (39). They reacted PMB with the SH groups of the virus, and titrated the excess PMB - 20 - after the virus was removed from the medium. A similar application of Boyer's method was developed for the titration of membrane SH groups of intact erythrocytes. The materials used and the development of the method will be briefly reviewed before a summarized description of the technique developed for the determination of erythrocyte membrane SH content is given.

2. Materials A. Reagents 1. p-chloromercuribenzoate Before the experimenta, PMB was freshly dissolved in small amounts of dilute NaOH since the solubilization of this reagent requires the presence of OH- ions (38). The dissolved reagent was diluted to the appropriate concentration with the same buffer solution that was used for the suspension or the red cells. The pH was set at 7.4 by the addition of minute amounts of HCl. Stock solutions or PMB were prepared in lo-3 M. concentration, and their O.D. was checked on each occasion. 2. Glutathione GSH was dissolved, just prior to use, in double distilled, ion-.free water. 3. Buffer The preparation of a suitable buffer solution, to assure constant PH and osmolarity during the incubation of the erythro- - 21 - cytes, was an important step in the development of the technique of SH titration. various buffer solutions were tested, and a 0.1 M phosphate buf1'er prepared from a mixture of mono- and di-hydro-sodium phosphate (Na2HP04:NaH2P04) without the addition of hydrochloric acid was round most suitable. The Na2H:NaH2 ratio was calculated from the equation of Henderson and Hasselbach so as to produce a pH of 7.4 when dissolved in double distilled deionized water. This buffer contained 184 mE/Lof Na+. When the presence of K+ ions were required, 10 mM of NaH2P04 was replaced with the same amount of KH2P04 to produce a final ionie concentration of 174 mE of Na+ and 10 mE of ~/L. Potassium was present in the suspending buffer when the effect of PMB on the cation exchange was studied. Incubation of erythrocytes with PMB up to 60 minutes in this buffer solution, caused no or only negligible changes of pH. The maximum change of pH observed occasionally was a slight depression, not more than pH 0.02. 4. Osmolarity The 0.1 M pH 7.4 phosphate buffer was round hypo-osmotic when its osmolarity was determined by r'reezing point determination on a suitable osmometer. various osmotically active substances were tested to make the osmolarity of the buffer iso-osmotic wi th the red cells. Ini ti ally isotonie NaCl solutions,· .buffered - 22 - with sodium phosphate, were used as described by Dacie (41}, but the presence of large amounts of Cl- ions in the buffer was found undesirable. various polysaccharides were tested and finally a simple carbohydrate, sucrose vms chosen. Sucrose is osmotically active, and does not penetrate the red cell membrane. The addition of 3% sucrose to the phos­ phate buffer raised the osmolarity to 310-312 milli-osmols. 200 mg. % glucose was also added to the final mixture as a nutrient.

B. Substrate 1. Source Human blood was collected by venipuncture from healthy adult male and female donors and from patients with various hematological conditions. In few instances ACD stored bloods were used. 2. Anticoagulant Various anticoagulants were tested to prevent clot form­ ation. Heparin (10,000 units/ml.) was round most suitable. Slight heparinization of the syringe used for drawing the blood was enough to prevent blood clotting. 3. Separation and Washing of the Cella Erythrocytes were separated from heparinized fresh blood by removal of the plasma, buffy coat, and the top layer of red cella after 10 minutes centrifugation at 4°c. with 1000 g. - 23 -

The red cells were washed by re-suspension in 0.9% saline eight times the volume of the cells. The O.D. of an aliquot of the washing fluid was determined at 257 rn~. (absorption peak of PMB) after each subsequent washing. The absorbancy of the washing fluids produced by plasma proteins and other materials adsorbed to, or trapped between the red cells, decreased rapidly after each washing. The O.D. of the fourth washing fluid was found invariably zero. Red cells, prepared for determination of their membrane SH groups, therefore, were washed three times before suspension in nutrient buffer solution. 4. Red Cell Suspension The thrice washed erythrocytes were suspended in a volume of 0.1 M. isotonie pH 7.4 cold phosphate buffer, to make a 33% cell suspension. A sample of this suspension was preserved for determination of red cell count, white cell count, hematocrit and mean corpuscular volume before it was distributed into the various incubation containers. 5. Preparation of Glassware and the Handling of Material A major problem durj_ng the early experimenta was the occurrence of hemolysis. This was noted frequently, but not a1ways, during incubation and subsequent rapid sedimentation of the cells, and was about the same in control samples containing no PMB. This presented a serious hinderance which - 24 - was overcome by the application of the following measures: i. Avoiding mechanical damage to the cells by very careful handling during the procedures. ii. Decreased speed of red cell sedimentation: 10 minutes centrifugation at 4°c. with the force of 1000 g. was found adequate to separate the cells from the suspension. The centrifuga! force was calculated from the equation of relative centrifuga! force (RCF): RCF = R x 1.118 x lo-5 x /RPM/2-in g-s where R is the radius of the centrifuge in cm., RPM is the number of revolutions per minute, and 1.118 x lo-5 represents the unit mass. iii. The decisive measure was the introduction of siliconization. All glasswears, tubes, pipets, syringes, etc., which have come into contact with the cells were siliconized with a water-soluble resistant silicon preparation ( 1 Siliclad1 , Clay-Adams). The use of siliconized glassware not only prevented hemolysis, but increased the accuracy of determinations and minimized the need for anticoagulant by delaying the coagulation. Once siliconization was introduced, hemolysis presented no more problem. The hemoglobin content of the post­ incubation supernatants was about, or less than 3 mg.% during the experimenta to be described, and often it was zero when measured by the modified benzidine method of Crosby and Firth (42). - 25 -

Higher concentrations of PMB or longer periods of incubation used in the preliminary experiments to be described, still produced visible hemolysis up to about 30 mg. % hemoglobin in sorne of the samples. Since the hemoglobin concentration of the red cell suspensions was about 2 - 3 gm. %, this represented not more than maximum 1-2% hemolysis.

3. Preliminary Experiments for the Application of Boyer's Method of SH Titration to Intact Erythrocytes The application of the above method to intact cells has not been reported. Since the use of intact cells was desired, the following modification of Boyer's method was developed for the determination of the reactive sulfhydryl content of the erythrocyte membrane. Washed erythrocytes were suspended in an isosmotic nutrient solution buffered at pH 7.4. Various concentrations of PMB, dissolved in the same buffered solution, were added to samples of the erythrocyte suspension, and the mixture was incubated at 37°C. for the designated period of time. At the end of the incubation, the cells were rapidly sedimented by centrifugation in the cold and removed. The excess PMB content of the supernatant solution was determined by its light absorption. - 26 -

a. Titration of Excess PMB The concentration of PMB in the post-incubation supernatant solution was determined by measuring the absorption increment at 257 rn~. in the presence of excess GSH using a Bausch and Lomb 'Spectronic 505' spectrophotometer. Preliminary experi­ menta have shown that in the presence of excess SH compounds, the increase in absorption at 257 rn~. caused by the mercaptide formation between the SH compounds and the available PMB, is a linear function of the PMB concentration in the solution. This is a reversai of Boyer 1 s technique (38), where standard amounts of excess PMB is added to buffered solutions containing the unknown quantity of SH groups. It was also observed during the preliminary studies that the light absorption of buffered PMB solutions alone, at 257 rn~., was linear over a wide range of PMB concentrations. The addition of glucose, sucrose, dextran, NaCl or KCl to the buffer solution did not affect the behaviour of PMB, provided that the pH of the solution was kept constant. Nor was the optical density of the buffered PMB solutions affected by incubation at 37°C., or by rapid cooling and centrifugation, or other procedures. Thus it was possible to determine the PMB concentration of a clear solution from its O.D.257· Figure 2 shows the absorption of various concentrations of PMB at 257 rn~. The increase in O.D. (AO.D.} produced by the mercaptide formation between PMB and GSH is also depicted - 27 - on this figure. These results have been found accurately reproducible whenever a new stock of PMB solution was checked out during the various stages of the experiments. b. The Selection of PMB Concentration and Incubation Ti me The hemolytic effect of PMB depends on the concentration and on the incubation time, during which the red cells are exposed to the action of mercury (17,25,26). Exposure to high concentrations of PMB (10-23 ~ molès per ml. RBC) (25) or prolonged incubation (1-8 hrs.) with lower concentration of the reagent (26) will result in hemolysis. According to Rothstein, this phenomenon is due to specifie changes in the molecular structure of the membrane induced by mercury {23). The formation of mercaptide bridges between membrane SH groups and the mercury compound causes molecular stress, and when this stress reaches a threshold level for a given cell, a generalized breakdown of membrane function ensues, starting with the breakdown of the membrane's permeability barrier and entry of the mercury compound into the cell with simultaneous or subsequent release of cellular constituents (23). Indeed, Tsen and Collier observed that there is no, or very little decrease in the intracellular GSH content of the PMB-exposed red cells until a critical hemolytic level is reached. Then the GSH content gradually falls to zero, paralle~led by nearly complete hemolysis of the cells. - 28 -

These observations suggested that, ror determination or membrane SR groups, a concentration of PMB had to be round, which was in excess of membrane SR groups, yet caused no hemolysis. Similarly, since the reaction between PMB and reactive SR groups in the red cell is not immediate (17), the minimum time for the complete titration of the reactive membrane SR groups had to be determined. i. Incubation of Erythrocytes with Various Conc­ entrations of PMB To 3 ml. aliquots of 33% red cell suspension various concentrations of PMB were added after thermal eqUilibration and the rinal volume was made up to 11 ml. with bufrer. Thus the samples contained 10 ml. suspending medium with the PMB, and 1 ml. (approximately lolO) packed red cells. Figure 3 shows the uptake of PMB by the red cells as plotted against the concentrations of PMB in the incubation solution when washed erythrocytes were incubated for 30 minutes under standard conditions. The concentration of PMB is expressed in ~ moles per milliliter of packed red cell in the incubation mixture. The solid line of the figure representa the mean of 10-20 determinations carried out on erythrocytes from healthy donors. The actual values are given in Table I. When cells were incubated with 1 ~mole of PMB/ml. RBC, the entire amount of PMB was usually taken up by the cells. The uptake increased - 29 - with the increasing PMB concentrations until a plateau was reached at 3 ~ moles/ml RBC. There was no statistically significant difference between mean values of PMB uptake at 3, 4, and 4.5 ~ moles/ml RBC. A further rise occurred in the uptake of PMB when cella were incubated with 5 ~ moles or more PMB/ml RBC. This was interpreted as possibly due to the gradua! changes taking place in the cell membrane structure by which further structural SH groups have become available for interaction with mercury. This interpretation was supported by measurements of intracellular GSH levels, and by the occurrence of slight hemolysis in most, but not all samples incubated with 5 or 6 ~ moles/ml RBC. Expressed hemolysis invariably complicated the determinations when PMB was applied in concentrations of 8, 9, or lü ~ moles/ml RBC. The amount of hemolysis was measured by the absorption of the supernatant at 410 m~ and was found to be very variable, and not necessarily the same when experimenta were repeated on erythrocytes from the same donor. ii. The Time Course of PMB Uptake by Erythrocytes Since the uptake of PMB varies with the time of incubation, the time course of the reaction was followed in order to determine the minimum time required for the complete titration of reactive membrane SH groups. A typical time dourse is depicted in figure 6. Washed erythrocytes were incubated at - 30 -

37°c. with 3-4 ~ moles of PMB/ml RBC for various lengths of time. After an initial rise of PMB uptake, a plateau was reached at 30 minutes time, and further incubation up to 60 minutes caused no, or only negligible increase of PMB uptake. No hemolysis occurred in samples incubated for 60 minutes, when normal erythrocytes were used, however, increasing hemolysis was obvious when erythrocytes of patients with hemolytic disorders were incubated with the same concentration of PMB for 40 minutes or longer. These erythrocytes showed a somewhat different time course of PMB uptake (figures 7 and 8), but a plateau was also reached at 30 minutes. These findings will be discussed later. These results are in accordance with the observations of Sheets et al. who demonstrated that PMB, bound to the cell surface, could be washed off by simple physical means after short periods of incubation, but not if the red cells were incubated for 30 minutes at 37°C. (17).

4. Determination of Intracellular Reduced Glutathione (GSH) Content in Erythrocytes Incubated with PMB The PMB uptake by the incubated erythrocytes may represent the number of membrane SH groups, provided that PMB did not penetrate into the interior of the cell. The number of 'readily available', free, or reactive SH groups •in the erythro­ cyte membrane that reacted with the mercury compound in 30 minutes may, or may not, reflect the total SH content of the - 31 - erythrocyte membrane, but it has been shown by others that physiological importance is attached mostly to the reactive, rather than the total SH content of biological materials (14,37,40). To demonstrate whether PMB has or has not penetrated into the cells under the conditions of the experimenta, the effect of PMB on the intracellular GSH content was determined. The GSH level of erythrocytes was measured before and after incubation, and the GSH content of PMB incubated cella was compared to the GSH content of cells incubated without the addition of PMB. In these experimenta, the 1 Alloxan 305 1 method of Patterson and Lazarow was used for GSH determinations (43) because this method does not require the use of sodium cyanide which splits the Hg-GSH complex at low concentrations of mercury (26,44). Erythrocytes incubated with PMB were washed twice with 0.9% cold saline, then hemolyzed and their GSH content determined. The resulta, expressed in mg. GSH per lOO ml packed red cella, were corrected to a standard number of 1012 RBC's in order to avoid possible errors caused by a relative increase of the packed cell volume due to swelling of the PMB incubated cella. The RBC count was determined in each sample of incubated, rewashed, and resuspended erythrocytes before the determination of their GSH content. One ml of packed cella usua11y contained 0.92-1.15 x 1010 erythrocytes. - 32 -

The erythrocyte GSH content, as determined by this method, has given values similar to those obtained by the method of Grunert and Phillips (45) as modified by Beutler (46). The normal range has varied between 54-75 with a mean of b6 mg. GSH/lo12 RBC's (Table 2). The erythrocyte GSH content of four G6PD deficient subjects, as determined by this method, was between 24-38, with a mean of 30 mg. GSH/1012 RBC (Table 3). Figure 4 shows the GSH content of erythrocytes incubated with various concentrations of PMB for 30 minutes at 37°C. No change or a slight increase of GSH content was observed in cells incubated with PMB concentrations of 3 ~ moles or lower. A slight, gradual decrease of GSH was noted in cells incubated with PMB concentrations of 5 ~ moles/ml RBC or higher. This suggested that the increasing PMB uptake of erythrocytes incubated with 5 ~ moles PMB/ml RBC or more (figure 3), could probably be due in part to seepage of sorne of the PMB molecules through the injured and, therefore, more permeable cell membrane. Figure 5 depicts the per cent decrease of GSH in the PMB incubated cells as compared to the GSH content of cells inàubated in buffer without the addition of PMB. Because of the above findings, PMB was used in the standard procedures for the determination of erythrocyte membrane SH content in the latèr· experimenta in a concentration of 3 ~ moles per ml packed red cells. - 33 -

5. The Effect of Leukocyte and Reticulocyte Content of the Red Cell Preparations on the PMB Uptake of Erythrocytes 1. Leukocytes Erythrocyte preparations always contained sorne leuko­ cytes. In the ?3% RBC suspension containing approximately 6 3,2 - 3,7 x 10 red cells/cu mm, the leukocyte count ranged between 450 - 980/cu mm. A control experiment was carried out with leukocyte rich (8.065) and leukocyte poor (850 WBC per cu mm) preparations of red cells from the same donor. No difference in the average PMB uptake was found between the two preparations. Reticulocytes Sorne erythrocyte preparations from patients with hemolytic anemia may have contained more reticulocytes than others with no reticulocytosis. To establish the effect of reticulocyte content on the average PMB uptake of the erythrocytes, reticulo­ cyte rich (21%) and reticulocyte poor (3%) erythrocyte suspensions were prepared from the blood of a patient with reticulocytosis subsequent to the administration ' of Bl2 for his pernicious anemia in relapse. The values of the average membrane SH content in bath samples were almost identical with each ether, and with values of previous determinations obtained before the appearance of reticulocytic response. This demonstrated that the relative reticulocyte content of the experimental samples had no effect - 34 - on the values of average SH content of erythrocyte membranes as determined by this method. As the result of the described preliminary and control experimenta, the following standard procedure has been established for the titration of reactive membrane sulfhydryl content in intact erythrocytes.

6. Resumé of the Technique Used for the Determination of Erythrocyte Membrane Sulfhydryl Content 1. Incubation Procedure Erythrocytes from freshly drawn heparinized blood are washed three times with eight volumes of cold isotonie (.17 M) NaCl. The top layer is removed after the third washing and discarded; the remaining cells are suspended in an isotonie phosphate-sucrose solution, buffered at pH 7.4 and containing 200 mg% glucose, to make a 33% red cell suspension. PMB, dissolved in the same buffer, is added to 3 ml aliquots of the 33% RBC suspensions after 5 minutes of thermal equilibration at 37°C. to make a final concentration of

3 ~ moles/ml RBC. The final volume in the test tubes is 11 ml, containing 1 ml packed cells and 10 ml of 10 -4 M. buffered PMB solution. Control tubes contain the same volume of buffer with no PMB. The samples are incubated in a Dubnoff metabolic shaking incubator for 30 minutes at 37°C. At the end of incubation the - 35 - red cells are sedimented by 10 minutes centrifugation at 4°C. with 1.000 g in an International Refrigerated Centrifuge and separated from the supernatant immediately. The incubated cells are washed again twice, and their intracellular GSH content is determined by the method of Patterson and Lazarow (43) if it is required. The supernatant fluid is analyzed for reàidual PMB, pH, and Na+-~ concentrations. 2. Cell Counts The red and white cell counts, reticulo- cyte count, micro-hematocrit and mean corpuscular volume (MCV) is determined from aliquots of the initial cell suspension. Determinations of red cell count, hematocrit, and MCV are repeated from aliquots of the incubated samples. 3. Titration of Residual PMB in the Supernatant The PMB content of the supernatant solution is determined by its ultraviolet light absorption at 257 rn~. A Bausch and Lomb

1 Spectronic 505 1 spectrophotometer was used for the readings. Correction for traces of hemoglobin or other proteins or nucleotides that may have-escaped into the solution during the incubation is made from the absorbancy of the samples at 410 m~. The PMB content of the samples gives practically no absorption at this wave length which is most sensitive to hemoglobin. The correction factor is read from a standard curve prepared from dilutions of red cell hemolysates (figure 9). - 36 -

The result is checked by adding equal volumes of excess concentration of GSH in water to aliquots of the supernatant, and determining the O.D.257 of the mixture. The àO.D. (2 x the O.D. of the diluted sample minus the O.D. of the undiluted sample) produced by the mercaptide formation is a function of the PMB content of the solution, and is unaffected by other factors. Therefore, the AO.D. of the control samples containing no PMB should be zero. The difference of the initial and post-incubation concentrations of PMB in the suspending solution is divided by the number of erythrocytes in the suspension which gives the average PMB binding capacity, hence the reactive SH content of the cell membranes. 4. Determination of the pH of the Supernatant Solution Determinations were made on a Beckman expanded scale pH­ meter. There was no, or negligible decrease of pH during the incubation of the red cells. 5. Sodium and Potassium Concentrations of the Supernatant

The Na+ and ~ content of the supernatant solutions were determined from appropriate aliquots of the samples, incubated with or without the addition of PMB on a Baird K-Y type flame­ photometer. - 37-

7. Other Methods Employed During the Course of various Studies 1. The hemoglobin content of supernatant solutions was determined by the modified benzidine method of Crosby and Firth (42). 2. For the determination of GSH content in PMB-incubated erythrocytes, the 'Alloxan 305' method of Patterson and Lazarow was used (43). 3. The nitroprusside method of Grunert and Phillips was used for routine laboratory determinations of erythrocyte GSH levels (45) as modified by Beutler for the determination of GSH stability (46). 4. Erythrocyte G6PD activity was determined by the method of Zinkham (47). 5. Routine hematological techniques for the examination of peripheral blood samples were used as described by Wintrobe (48).

6. Osmotic ~ragility tests, Coombs tests and Heinz body preparations were made according to the description by Dacie (49). 7. Blood sulfonamide determinations were carried out according to the method of Brutton and Marshall {50) as modified by Rieder {51). 8. Cell counts were performed by use of a Model B Coulter electronic blood cell counter (52). - 38 -

PART II RESULTS

A. THE REACTIVE MEMBRANE SULFHYDRYL CONTENT OF NORMAL ERYTHRO­ CYTES 1. The Range and Mean of Normal Membrane SH Content Using the described technique, a large number of deter­ minations were carried out on erythrocytes obtained from healthy donors (technicians, secretaries, medical students, physicians, etc.). The range of normal erythrocyte membrane SH content was established on the basis of repeated determinations carried out on the bloods of 20 healthy donors. Results of the repeated determinations on blood samples obtained at different times from the same donor checked very closely, or were identical, with each other. Each of these determinations was carried out either in duplicate, triplicate or quadruplicate. The results of these multiple determinations were practically, or nearly identical. The values of normal membrane SH groups, given in Table IV, represent the average of 2 or 3 determinations on each individual of the first 20 donors (donors no. 1-20). Erythrocytes obtained from healthy donors contained 2.30 - 2.85 x lo-16 moles of reactive membrane SH groups per red blood cell. The mean value of the determinations was 2.51 x lo-16 moles/RBC with a standard deviation of ± 0.21 x lo-16. - 39 -

During the various stages of experimental studies, the erythrocyte membrane SH content was determined on the bloods of an additional 12 healthy donors (Table IV, donors 21-32). The values obtained were within the established normal range with a mean of 2.47 x lo-16. The mean value of determinations on all 32 healthy donors is 2.50 x 1o-l6 moles of reactive membrane SH groups per erythrocyte, with a standard deviation of ~ 0.20. This would represent an average of 1.51 x 108 reactive sulfhydryl sites in the erythrocyte membrane. 2. Studies on ACD - Preserved Bloods The erythrocyte membrane SH content was determined in three samples of stored bloods. The values obtained were within the normal range: 2.6, 2.35, and 2.41 x lo-16 moles of SH groups/RBC on 1, 21 and 23 day-old ACD-stored bloods respectively (Table XVI). The investigation of membrane SH content of stored erythrocytes was not pursued further.

B. DETERMINATION OF ERYTHROCYTE MEMBRANE SULFHYDRYL CONTENT IN PATIENTS In addition to the above, 69 membrane SH determinations were carried out on the erythrocytes of 43 patients with various hematological conditions. All cases were thoroughly studied clinically and laboratory determinations were carried out when indicated. These included determinations of hemoglobin, hematocrit, reticulocyte count, Heinz-body preparations, study - 40 - of the morphology of the peripheral blood smear, Coobms• test, and blood-urea-nitrogen (BUN) determinations. The G6PD activity, GSH content and GSH stability of the red cells were deterrnined whenever it was possible. Osmotic fragility tests, serum bilirubin determinations and blood sulfonamide level determinations were undertaken when it was indicated. Red cell survival studies using Cr51 labelling technique, were carried out on 6 patients. The following groups of cases have been studied: 1. Drug-induced hemolytic anemia 2. G6PD deficient erythrocytes 3. Auto-immune and other acquired hemolytic anemias 4. Congenital spherocytosis 5. Pernicious anemia 6. Iron deficiency anemia 7. Other types of anemia 8. The effect of azoternia 9. The effect of prolonged sulfonarnide therapy on the erythro­ cyte membrane SH content was also studied. A résumé of the findings is given in Table XVI. 1. Erythrocyte Membrane SH Content in Drug-Induced Hemolytic Anemia Membrane SH content has been deterrnined in eight cases of drug-induced Heinz-body hemolytic anemia (Tables V and VI). - 41 -

Reduced membrane SH content was round in 7 patients, ranging from 1.27 - 2.0 x lo-16 moles/RBC, and normal membrane SH groups in one {2.48) who had a very mild degree of Heinz-body hemolytic anemia, presumably induced by a moderate phenacetine intake. The offending drugs are listed in Table V. The relationship of the known oxidative compounds in cases 1-6 to the development of hemolytic anemia is clear. Case number 7 was treated with tolbutamide and colcemid when the determinations were performed. This patient had marked hemolysis, and Heinz­ body stains were strongly positive. Although colcemid is a kno~m cytotoxic agent and tolbutamide is a sulfonamide derivative, neither of these compounds were known to cause Heinz-body anemia. Besides the known oxidative compound, sulfamethizole, patient number 1 also received an antibody-suppressive agent, azo-thio­ purine (Imuran*) which has been reported to affect sulfhydryl groups {52), and could have played a role in the development of the drug-induced hemolysis. Heinz bodies were identified in the erythrocytes of all cases included in this group. All patients had normal erythro­ cyte GSH levels. The degree of anemia and reticulocytosis is shown in Table VI. Five patients had renal disease, and out of the eight cases, sorne degree of azotemia was present in four. Case number 4 had a long history of hemolytic anemia and red cell survival studies, with Cr51 tagging, showed a T 1/2 life of 10 days (normal: 24-35 days). - 42 -

The mean value of erythrocyte membrane SH content determinations in this group of patients was 1. 81 x 10 -16 moles/RBC which represents a significant reduction from the normal. 2. Membrane SH Groups of G6PD Deficient Erythrocytes Since drug-induced hemolytic anemia is a usual complication of the administration of oxidative compounds to G6PD deficient subjects, the membrane SH content of G6PD deficient erythro­ cytes was also studied. Reduced membrane SH content was found in the erythrocytes of four G6PD deficient subjects studied. The lowest value was 1.5, and the highest was 2.0 with a mean value of 1.79 x 1o-l6 moles/RBC, which represents a significant reduction from the normal mean. The erythrocytes of these four patients had zero G6PD activity, and markedly reduced and unstable erythrocyte GSH content as shown in Table VII. Cases number 11 and 12 had increased reticulocyte counts suggesting a possible compensated chronic hemolytic process. Cr51 red cell survival study, under­ taken in case number 11, showed impa±red red cell survival. The T 1/2 was 19 days (normal: 24-35 days). None of these four patients had taken oxidative drugs or any other medications, and no Heinz bodies were round in their red cells. 3. The Reactive Membrane SH Content in Acquired Hemolytic Anemia Membrane SH content has been determined in three cases of Coombs' positive auto-Ïmmune hemolytic anemia and in two other - 43 - cases of acquired hemolytic anemia (Tables VIII and IX). Four other cases where the presence of hemolytic anemia was suspected but was not sufficiently confirmed, are reviewed in the MISCELLANEOUS group. The hemolytic anemia was due to infectious hepatitis in case number 13, and presumably to renal disease in case number 14. The latter patient, however, received nitrofurantoin and Imuran treatment, and peripheral blood smears were suggestive of drug-induced hemolysis inj_tially. Shortly after the diagnosis this patient was transfused, and repeated Heinz- body preparations failed to confirm this suspicion. This patient also developed megaloblastic blood changes wnile on Imuran, seven weeks after the initial SH group determination. The membrane SH content remained low, and the accelerated red cell destruction persisted. The latter was confirmed by the markedly shortened Cr51 erythrocyte survival time: the T 1/2 was 14 days. Erythrocyte membrane SH group content ranged between 1.75 - 2.10 x lo-16 moles/RBC, giving a mean of 1.91. This representa a marked decrease compared to that of normal red cells. The lowest values for membrane SH content were round in the three cases of auto-immune hemolytic anemia (cases 15,16,17; Tables VIII and IX) where it ranged between 1.33 - 1.85 x 1o-l6 moles/RBC with a mean value of 1.61, representing a 36% reduction - 44 - from the normal mean. All three cases had positive Coombs' test and increased osmotic fragility. The auto-immune hemolysis was due to Systemic Lupus Erythematosus in two cases (cases 16 and 17) and cold agglutinins were round in one (case 15). All patients in this group had normal GSH levels, and all but one had normal blood-urea-nitrogen levels. Case 17 was repeatedly studied because of the very low membrane SH content in this patient. This young woman suffered from recurrent bouts of hemolytic episodes as well as thrombocytopenia which necessitated splenéctomy. The membrane SH groups were determined twice before splenectomy, during the operation and five weeks post-operatively. The results are shown in Table X. A slight difference was noted between the membrane SH content of erythrocytes from the arterial and the venous splenic blood obtained during the operation. The membrane SH content remained well below normal up to five weeks after the splenectomy, however, a marked improvement in all post-operative values, including the membrane SH groups was apparent. The patient had not received transfusions at any time. Cr51 survival studies showed accelerated red cell destruction, resulting in a 17-day, half life of the tagged cells. - 45 -

4. Erythrocyte Membrane Sulfhydryl Groups in Hereditary Spherocytosis One patient was studied in whom the hemolytic anemia was due to congenital spherocytosis. The erythrocyte membrane SH content was 1.66 x 1o-l6 moles/RBC during the acute hemolytic crisis which necessitated splenectomy. The SH values had risen to 1.95 three weeks after splenectomy

(Table x~ patient b and Tables VIII and IX, case number 18), however, this patient required blood transfusions before and during the operations. Determinations showed significant decrease in the membrane SH content of this patient. 5. Erythrocyte Membrane SH Content in Miscellaneous

Conditions with P0 ssible Hemolytic Component Decreased erythrocyte membrane SH content was found in four cases where increased red cell destruction was suspected but not confirmed adequately (Tables VIII and IX, cases number 19-22). All four patients had renal disease with sorne degree of azotemia; one was markedly uremie. The clinical picture was complicated in these cases by infection, bleeding, iron deficiency or other factors. 6. Erythrocyte Membrane SH Content in Iron Deficiency Anemia The SH content of the erythrocyte membrane had been round within the normal range in four cases of iron deficiency anemia. The values are given in Tables XI and XVI. - 46 -

7. Erythrocyte Membrane SH Content in Pernicious Anemia The membrane SH content was studied in two cases of pernicious anemia in relapse and was found normal in both cases. Determinations were done before and after administration of intramuscular Bl2 and during the reticulocytic response. No significant differences were seen in the membrane SH content (Table XII). 8. Erythrocyte Membrane SH Content in Miscellaneous Hematological Conditions Normal membrane SH content was round in ether cases. The diagnosis and the membrane SH content are shown in Table XIII. Membrane SH groups were studied in case number 32 because of the reticulocytosis, slight splenomegaly and sorne sphero­ cytosis observed in this patient. Subsequent studies, however, did not confirm the suspicion of spherocytic hemolytic anemia in this patient. 9. The Effect of Azotemia on the Erythrocyte Membrane SH Groups Since azotemia had been observed frequently in non-G6PD deficient patients who developed drug-induced hemolytic anemia, the erythrocyte merrilirane SH content was studied in patients who suffered from severe azotemia. The results are presented in Table XIV. The membrane SH content was slightly decreased in one case. Normal membrane SH groups were found in four, - 47 - giving a mean value of 2.40 x lo-16 moles/RBC, not significantly different from the normal mean. Increased red cell destruction was present in two of these patients (cases number 36 and 37) evidenced by shortened life span of the Cr51 tagged erythro­ cytes (T 1/2 = 16 and 17 days respectively). The effect of azotemia on normal erythrocytes could be studied particularly well in patient number 38 who developed acute renal failure and severe azotemia after post-operative complications. Because of the development of very severe anemia and hypovolewia, this patient received massive trans­ fusions two weeks prior to the experiment. Thus it could be presumed that a greater part of the circulating cells at the time of the experiment were those transfused two weeks before and exposed to the effects of azotemia since. In spite of the persisting azotemia, the erythrocyte membrane SH content was normal (2.50) in this patient. 10. The Effect of Sulfonamides on the Erythrocyte Membrane SH Groups Since, in the majority of previously studied cases of drug-induced hemolytic anemia in non-G6PD deficient patients (1), sulfonamides were found to be the offending drug, the effect of prolonged sulfonawide therapy on the membrane SH groups was studied in three patients. Normal membrane SH content was tound in two, slightly decreased in one as shown in Table xv. - 48 -

Unfortunately none of these patients had membrane SH deter­ minations done prior to the onset of sulfonamide therapy. The mean value of the three determinations is 2.33 x 10-16 moles/SH, not significantly different from the normal mean. The above presented findings are summarized in Table XVI, and depicted in figure 12. Significant reduction in the mean SH content of the erythrocyte membrane was round in 16 cases of drug-induced, auto-immune, and ether acquired hemolytic anemias in four G6PD deficient subjects, and in one case of hereditary spherocytosis, while normal erythrocyte membrane SH groups were round in pernicious and iron-deficiency anemias and in other cases.

C. THE TIME COURSE OF PMB UPTAKE IN HEMOLYTIC ANEMIA The time course of PMB uptake has been studied in one patient 'l.rith auto-immune hemolytic anemia (case number 17), in one patient with hereditary spherocytosis (case number 18), in the mother of this patient (case number ), and in a case of iron deficiency anemia (case number 26), and uremia (case number 38). The rate of PMB uptake by the erythrocytes of the patient with auto-immune hemolytic anemia was different from that of normal red cells as shown in figure 7. Figure 8 shows the markedly different rate of PMB uptake by the erythrocytes of the patient with hereditary spherocytosis, as determined before - 49 - and after splenectomy (twin black circles), as well as the uptake by the erythrocytes of the patient's mother. The course of PMB uptake by the erythrocytes of the iron-deficient patient, and of the patient with renal failure and severe uremia was similar to that in normal erythrocytes.

D. THE EFFECT OF PMB ON THE ERYTHROCYTE GSH LEVEL The effect of various concentrations of PMB on the erythrocyte GSH level is shown in figures 4 and 5 and has been discussed in the development of the technique of membrane SH group determination (see METHODS). Incubation of the red cells with PMB up to 4 1-L moles/ml,RBC caused no, or negligible decrease of intracellular GSH, while PMB concentrations above this level caused a slight and gradual decrease of GSH content (Table II). The effect of various concentrations of PMB on the GSH level of erythrocytes obtained from various patients was similar to that in normal erythrocytes with the exception of G6PD deficient patients. The mean values of the GSH content of PMB incubated erythrocytes obtained from healthy donors, from G6PD deficient subjects, and from patients with various hemolytic conditions, are shown in Table III. The initial decline in erythrocyte GSH content was observed at a slightly lower concentration of PMB (4 1-L moles/ml RBC) in patients with hemolytic anemia than in cells of healthy donors

(5 J..L moles/ml RBC). - 50 -

The GSH content of G6PD deficient erythrocytes was not only markedly reduced as expedted, but the incubation of these cells with PMB affected them somewhat differently from that observed in any other case: 30-minutes incubation of G6PD deficient erythrocytes with 2 and 3 ~ moles of PMB/ml RBC caused an itial slight increase of GSH levels of the incubated cells as if low concentrations of PMB stimulated GSH production in these cells (figure 4, Table III).

E. THE EFFECT OF PMB ON CATION TRANSPORT 'IN ERYTHROCYTES The blockage of membrane SH groups by PMB caused a gradual leakage of potassium from the incubated erythrocytes (figure 10).

Sorne leakage of ~ has occurred in every case from erythrocytes incubated With a minimum of 2 ~ moles of PMB/ml RBC. The range and mean of potassium loss from the erythrocytes of 10-20 healthy donors are given in Table XVII. Greater potassium loss at lower concentration of PMB was observed in G6PD deficient erythrocytes and in red cells of patients with Coombs 1 positive auto-immune hemolytic anemia (two cases of each) (Table XVII and figure 11). The loss of potassium from erythrocytes of patients with other diseases (Coombs' negative hemolytic anemias, pernicious and iron-deficiency anemias, etc.) was within the range of potassium loss from normal erythrocytes. - 51 -

CHAPTER III

D I S C U S S I 0 N

PART I DISCUSSION OF THE TECHNIQUE OF ERYTHROCYTE MEMBRANE SULFHYDRYL GROUP DETERMINATION

1. The Choice of Intact Cells as Opposed to Red Cell Ghosts In order to determine the erythrocyte membrane SH content under standard physiological conditions, it was necessary to develop a technique that permitted the use of intact, non­ hemolyzed red cells for this purpose. It was realized that accepted methods of separating the erythrocyte membrane from the rest of the cell content may introduce numerous variables affecting the accurate determination and reliable comparison of the membrane SH content of normal and abnormal red cells. The question of the extent to which hemoglobin constitutes a structural part or the erythrocyte membrane 1s not settled, and the concept that hemoglobin is an essential structural component of the red cell membrane has been challenged recently (54,55,56). This problem is of great importance from the point of SH titration, since every hemoglobin molecule contains 6-8 SH groups (57,58,59) at least 2-4 of which are "readily reactive" {58,59). Efforts to npurify" the red cell - 52 - membrane by removal or the hemoglobin are complicated by the losa of essential membrane constituents in spite of the retention of basic membrane functions. Removal of erythrocyte contents by hemolysis or other methods for isolation of erythrocyte membranes,yields discoid bodies referred to as "red cell ghosts" or post­ hemolytic residues. various methods of ghost preparation have been reviewed by Ponder (60) and more recently by others (56). The most accepted methods employ hemolysis in hypotonie solutions for removal of hemoglobin. The hemoglobin content of various ghost preparations by these methods varied from 30% of the dry weight ( 61) to 1. 4% of the dry weight in the relatively hemoglobin-free ghosts prepared by the method of Danon et al. (54). Ponder, Prankerd and Harris considered hemoglobin molecules as essential structural components of the functioning cell membrane, for.ming a part of the membrane ultrastructure itself (62,63,64,65). Weed and Dodge and their assooiates, however, demonstrated that hemoglobin oan be removed almost completely from the post-hemolytic residue of the red cella, and olaimed that hemoglobin is not an integral structural part of the erythro­ cyte membrane {55,56). Nevertheless, the newer methods of preparation of "hemoglobin free" ghosts introduced another variable by the possible losa of essential membrane constituants - 53 - during the process of 11 leachingn the membranes re lati vely free of hemoglobin. Substantial loss of lipid phosphorus was reported by Anderson and Turner ( 66), but only "minimal11 losses were round by Dodge et al. (55). Analysis of the latter ghost preparate revealed, however, substantial losa of non-hemoglobin nitrogen containing substances, most likely mucoproteins, from the oell membranes. Weed et al. claimed the absence of significant losses of membrane lipids or 11 obvious11 losses of stroma! proteine from their 11 hemoglobin­ free11 red cell ghosts (56). They pointed out that it is impossible to define red cell ghosts except in terme of their preparation, s~nce their composition varies widely with the method of preparation. The profound influence of numerous variables auch as pH, ionie strength, and other properties of the hemolyzing solution was demonstrated by ethers (55). These reports raised doubts whether certain observed charact­ eristics of red cell ghosts are truly confined to the membrane or represent the activity of some residual intracellular component. In the opinion of the author, any process used for dis­ ruption of the continuity of the intact cell membrane for removal of the cell content and for "purification.. of the membrane preparate, may result in the losa of essential SH­ containing membrane constituents and may influence the reactivity - 54 - of SH groups remaining in the cell membrane. Even if a highly reproducible standard method is selected for the preparation of red cell ghosts from normal eryth~ocytes, it is very questionable whether these processes will have the same effect on the damaged or more vulnerable cell membranes of Coombs 1 positive, or other abnormal erythrocytes susceptible to hemolysis, as on the membranes of normal red cella. Because of these considerations, it was decided that intact, non-hemolyzed red cella should be used for the quantitative determination of the erythrocyte membrane SH content. 2. The Choice of Reagent, Concentration and Incubation Time These questions have been discussed already in the previous chapter describing the development of the technique of SH titration in intact cell membranes. PMB was selected as the most sUitable reagent because of its high specificity for SH groups (14,37,38) and apparent inability to penetrate into the red cell in low, non-hemolytic concentration, and hence its interaction with only the membrane SH groups in intact cella (36,25,26). A concentration of this reagent was selected which was in excess of the SH content of the red cell membranes, but which did not penetrate into the cella. Erythrocytes were incubated with various concentrations of the reagent and their - 55 -

PMB uptake was determined (figure 3). A plateau was reached at a PMB concentration of 3 ~ moles/ml RBC. The PMB uptake did not increase appreciably with 4 and 4.5 ~ moles of PMB/ml RBC, indidating that the readily reacting membrane SH groups were saturated with PMB at a minimum concentration of 3 ~ moles per ml RBC. The time course of PMB uptake was also studied (figure 6) and a plateau was observed after 30 minutes of incubation which seemed to be the minimum time required for the completion of the reaction of PMB with membrane SH groups. This period was selected as the incubation time. The affect of various concentrations of PMB on the intracellular GSH content was studied using various samples of abnormal as we~l as normal erythrocytes in order to ascertain that PMB had not penetrated into the cell at the given concettration (figures 4,5). Intact cells were then incubated with the selected concentration of PMB (3 ~ moles/ml RBC) and the excess PMB content of the suspending medium was determined following rernoval of the cells after the incubation. If the reagent had not penetrated into the interior of the cells, then the uptake of PMB measured the readily available, free SH groups of the erythrocyte membrane which reacted with the mercury compound. This number may or may not reflect the total SH content of the erythrocyte membrane, but physiological - 56 - importance is attached primarily to the reactive rather than to the total SH content or a biological material (14,37,40).

The observation that PMB in a concentration or 3 ~ moles/ml RBC did not errect the GSH content or the incubated red cells in any or these experimenta, confirmed that it did not penetrate into the cells and interacted only with the membrane constituents. The claim by Weed and associates that mercury prererentially combines with the SH groups or the hemoglobin to "protect the biochemically important GSH which does not combine with mercury until approximately 70 per cent or the total cellular sulfhydryl sites have reacted" {40), has been contradic&ed by others {26) and was subsequently withdrawn by the authors (44). Since it had been reported that the cyanide used in the nitroprusside method or GSH determination splits the mercury-GSH complex (26),

Weed ~al. reinvestigated their findings and observed that while red cell stroma has the greatest affinity for mercury, the mercury that enters the intracellular phase reacts with GSH first, and no interaction with the SH groups or the hemoglobin takes place until the GSH content or the cell is saturated with mercury (44). This indicates that the determination or intra­ cellular GSH content or the PMB-incubated erythrocytes is a good measure or the possible entry or PMB into the cells. - 57 -

The rurther rise in the PMB uptake or erythrocytes

1ncubated with 5 ~ moles or more PMB/ml RBC after a plateau was already reached (rigure 3) was accompanied by a decline in the GSH content of these cells (figure 4,5). Inhibition of the reactive membrane SH groups with PMB may have changed the native state of the membrane by causing alterations in the steric configurations of the molecules

in the membrane. ~s unfording of molecular structures

couilid, in turn, make •masked 1 SH groups available for inter­ action with the reagent, thus resulting in the breakdown of the barrier function of the cell membrane responsible for opposing the entry of 1 noxious agents• into the cell. A seepage of PMB molecules into the red cell may begin, thus causing the decline in the tntracellular GSH level. Indeed, Weed and associates demonstrated recently by the application of Hg203-

labelled HgC12 that amounts of mercury taken up in excess of 5 ~ moles/ml RBC by the cells, began to react with intra­ cellular reduced glutathione. In summary, it was concluded that the PMB uptake of cells incubated with the reagent as described in the Resumé of the Technique, measured the true reactive SH content of the erythro­ cyte membranes. Furthermore, this technique was found to be relatively easy to perform and it yielded resulta which were highly reproducible. Multiple and repeated determinations on - 58 - bloods obtained from the same donor checked very closely With each other (see paragraph no. 1 of RESULTS). The values obtained are discussed in the fol1owing section.

PART II DISCUSSION OF THE RESULTS: THE REACTIVE SULF­ HYDRYL GROUPS OF THE ERYTHROCYTE MEMBRANE

A. Normal Erythrocytes Although the values of membrane SH determinations on red cella obtained from hea1thy donors varied between 2.30 - 2.85 x 1o-16 moles/RBC, most of the values were close to the mean of 2.50 x 1o-l6 moles/RBC, giving a re1ative1y sma11 standard error (S.D.) of± 0.20. A stil1 narrower range was obtained with the erythrocytes of the last 12 of the 32 healthy donors (Table IV) where the S.D. was on1y ± 0.14. This would represent 1.505 x 108 reactive SH groups per erythrocyte as the average number of freely avai1ab1e SH radicals, or reactive SH sites, capable of interacting with PMB in the native state of the erythrocyte membrane. This value is in good agreement with those reported by Weed and associates after our experimenta were initiated (67,68). 20 They used Hg 3-1abel1ed HgC12 for the titration of membrane 11 11 SH sites on hemog1obin-poor stroma • By using cèrtain extra­ polations they calculated the amount of mercury bound by stromal - 59 - proteins, as opposed to the mercury binding of the stromal hemoglobin. The result of these calculations was reported initially in a comprehensive study ( 40) in which the ••small but critically important number of stroma.l SH groupstt were defined as 5 per cent of the total potential cellular SH -16 content. This would represent 2.1 x 10 moles of free SH groups per erythrocyte. Subsequently Weed and Reed reported that the stroma of healthy red cella contain 1.5 - 2.5 x 10 -16 moles SH groups/RBC, determined by the labelled HgC12 titration technique (67). In their most recent communication, Weed and Berg reported the normal value to be 1.8 x lo-16 moles/RBC using the same technique (68). A much smaller number of stromal SH groups were identified by the same workers when they used a Hg203-labelled organic mercurial, chlormerodrin, or c14-labelled n-ethyl maleimide (NEM) for the titration of the reactive SH content of so-called 1'hemoglobin free red cell ghosts" (4 x lo-17, 1.5 x lo-17 respectively) (68). The technique of quantitative titration of membrane SH groups in intact erythrocytes developed during the present study has yielded constant and reproducible values. Thus it bas the advantage of being adaptable to comparative studies or normal and abnormal cella. - 60 -

B. Abnormal Erythrocytes The membrane SH content of abnormal erythrocytes has not been reported up to date. The only relevant reference has been that of Weed and Reèd (67) stating that "normal membrane SH content was round in sickle cell disease11 and umarked reduction of membrane SH content was round in G6PD- deficiencyIl • No values were given.

1. G6PD Deficient Erythrocytes Reduced membrane SH content was round in G6PD deficient erythrocytes. The mean value of determinations on·'.the red cells obtained from four G6PD deficient donors {1.79 x lo-16) represented a 28% decrease from that of the normal mean. Total lack of G6PD activity and marked reduction of erythrocyte GSH content was recorded on all four patients. Apart from minor ailments in one, all four subjects were generally healthy, had carefully avoided taking any oxidative drug since their deficiency was discovered, and all smears for Heinz bodies were persistently negative. The four patients were Caucasians, two of them sephardic, and one an Ashkenazy Jew. Two of the patients had no apparent ailments that could have been associated with their red cell defect, two gave history of possibly hemolytic episodes with jaundice. The latter two patients, although otherwise in good health, showed slight but prolonged reticulocytosis - 61 - over a period of severa! months. The membrane SH content determined on these patients when the reticulocytosis subsided, showed no significant change. Erythrocyte survival time determined in one of these pat~ents with Cr51-tagging of the red cells showed a hamf-lif:e of 23 days. Spontaneous, low-grade, clinically compensated, chronic hemolysis and shortened erythrocyte life span in Caucasian and Negro G6PD deficient subjects have been reported earlier (6,69,90). Since GSH is considered the major source of free SH radicals in erythrocytes (70) 1 it is possible that the decreased membrane SH content in these subjects waw secondary to an intrinsic red cell defect, the markedly reduced erythro­ cyte GSH level. Although earlier reports that G6PD deficiency is possibly due to the lack of stroma! activation by SH or other groups in the red cell membrane (29,30) have not been substantiated, the possibility that membrane SH deficiency has a primary role in the development of hemolytic anemia in primaquine sensitive erythrocytes exposed to oxidative drugs cannot be ruled out. 2. Drug-Induced Hemolytic Anemia in Non-G6PD Deficient Subjects The occurrence of this phenomenon was discussed in the INTRODUCTION and the literature was quoted on this subject. Eight cases of drug-induced hemolytic anemia, diagnosed over a period of five months, were subjects for the determination of - 62 - membrane SH content. Reduced membrane SH content was round in seven patients and normal membrane SH content was observed in one patient for whom the diagnosis or drug-induced hemolytic anemia was not tirmly established. A moderate anemia and slight reticulocytosis was discovered in this patient (case number 8), the etiology or which is still being investigated. Since occasional Heinz bodies were discovered in the patient•s erythrocytes, and she admitted occasional self-medication with a phenacetine containing compound, drug-induced etiology was thought to be possible. Phenacetine has been reported to cause hemolytic anemia if taken in excessive doses. However, contrary to the usual course, the Heinz bodies did not disappear atter the presumed otfending drug was discontinued. Thus it is possible that this patient's anemia was due to other yet undetermined ractors. In contrast, another patient (case number 2), a young woman with markedly elevated erythrocyte GSH level and G6PD activity who developed typical drug-induced hemolytic anemia with severe hemolysis, methemoglobinemia and sulfhemoglobinemia due to selr-induced phenacetine intoxication, showed 20 per cent reduction in the erythrocyte membrane SH content. Thirty to 50 per cent reduction or membrane SH content was observed in the other six cases where the hemolysis was induced by hydralazine in two, and by sultamethizole, pyridium, - 63 - nitrofurantoin, and colcemid in one of each case respectively. The mean value of determinations in all eight patients showed an average 27 per cent reduction or the membrane SH content, or 30 per cent reduction with the exclusion of case number 8. It is of interest that the majority of the 27 previously observed non-G6PD deficient drug-induced hemolytic anemia cases were due to sulfonamide or nitrofurantoin (1, and unpublished). Colcemid has not been reported to produce Heinz body anemia, although it is a known cytotoxic agent. Pyridium was reported to produce methemoglobinemia {71,72), but pyridium-induced Heinz body hemolytic anemia has not been reported previously. When G6PD deficient erythrocytes were incubated with pyridium in vitro, it produced a marked fa11 of the GSH level, proving the oxidative potential of this drug. Hemolytic anemia was produced with high doses of hydralazine in dogs (73) and mild anemia has been reported occurring during hydralazine therapy in man (74), but no cases of Heinz body hemolytic anemia have been reported in G6PD deficient, or non­ deficient patients where hydra1azine was the only known offending agent, as in the two cases observed. De Leeuw, et al. reported a case where hydralazine contributed to the severity of the sulfonamide induced hemolysis (1). They showed that hydralazine was as effective as acetylphenylhydrazine in reducing - 64 -

the GSH content of G6PD-deficient red cells, and predicted

that hydralazine alone ~y produce hemolytic anemia (1). rt should be noted that renal disease was present in five patients, causing marked azotemia in four. Three patients had no apparent renal impairment. The clinical picture was cornplicated by chronic myeloid leukemia and myeloid metapalsia in one case. Heinz bodies were present in all eight cases ranging from 0.8% in case number 8 to 32% in case number 1. The necessity of repeated blood transfusions or hemo­ dialysis interferred with the proper follow up of most of these cases. Whether the membrane SH content was reduced in the erythrocytes of these patients before the development of the drug-induced hemolytic anemia is not known. In two cases, however, where complete follow up was possible, the erythrocyte membrane SH content remained low even arter the discontinuation of the offending oxidative compound, and disappearance of the Heinz bodies. Since the mechanism of non-G6PD-deficient drug-induced hemolytic anemia is not known, the possible relationship of diminished membrane SH content to the development of this condition is of considerable importance. The"~question remains as to whether this biochemical defect of the red cell membrane is the cause or the effect of oxidative hemolysis in these cases; i.e. Does the hemolytic anemia develop when these patients - 65 - are challenged by usual therapeutic doses or oxidative drugs because or this derect, or because of possible other intra­ corpuscular defects (apart rrom G6PD deficiency and GSH content which were normal), or do extracorpuscular ractors, other than the drug play a role in the development or drug-induced hemolysis? In 1959 Desrorges and co-workers reported a single case or hemolytic anemia induced by accidenta! overdosage or sulroxone in a patient who had no G6PD deficiency, and they concluded that drug-induced hemolytic anemia may occur without demonstrable enzymatic derect or the erythrocytes (75). De Leeuw and associates reported ,15. thoroughly studied cases or drug-induced hemolytic anemia in non-G6PD dericient subjects with normal red cell GSH content. They concluded that the mechanism or the development of drug-induced hemolytic anemia in non-G6PD dericient patients is obscure, and requires rurther investigation (1).

One case or 8-~oquinoline induced hemolytic anemia was reported in 1961 (76) in which susceptibility to hemolysis was presumably due to glutathione reductase (GSSG-R)dericiency in a Caucasian patient with normal G6PD activity. This patient, however, had low red cell GSH content, not a reature in any or the cases studied in our series. Furthermore, the role or GSSG-R in the development or drug-induced hemolysis has not - 66 - been substantiated. Recent investigations by Beutler demonstrated that although GSSG-R activity in human red cells can be maintained with DPNH, instead of TPNH, the DPNH-linked enzyme activity could not be "harnessedu to reduce oxidized glutathione in the intact erythrocyte (77). Therefore, it seems logical that the reduced glutathione content in GSSG-R deficient red cella would markedly decrease when challenged by strong oxidants such as acetylphenylhydrazine (APH) contrary to the findings in the eight cases studied. These facts seem to rule out the possibility of GSSG-R deficiency being primarily responsible for the development of drug-induced hemolysis in these cases. The possible role of an isolated catalaze of peroxidase deficiency in the development of drug-induced hemolytic anemia is negated by the large amounts of catalaze and peroxidase reserves present in human erythrocytes (6,7). The clinical picture of drug-induced hemolytic anemia, and the appearance of inclusion bodies identified in the erythro­ cytes of patients with Hemoglobin Zurich (8,9) is different from that observed in the cases studied. Moreover, this hemoglobino­ pathy is exceedingly rare, having been identified in only a few familias. The course or the hemolytic anemia, the negative Coombs' test and the presence of typical Heinz bodies in each one of - 67 - the patients described, rules out the possibility that the hemolytic anemia developed on the basis of sensitization by a previous administration of the offending drug in these patients. In two cases tested, no ânui-drug antibodies could be demonstrated (cases number 1 and 3). Although the presence of an oxidative compound in the plasma does not necessarily lead to increased red cell destruction, extracorpuscular factors may have enhanced the hemolytic potential of the oxidative drugs in the described cases. (a) Relative overdosage due to impaired renal clearance or insufficient detoxification of the drug is to be considered. (b) The combination of azotemie plasma and oxidative compounds may have a special delete~ous effect ~ the erythrocytes. (c) Abnorrnal metabolites of the drug may have been produced, which are capabàé of increased peroxide generation in the blood, exceeding the detoxifying capability of the red cells. The role of peroxide intoxication in the development of the primaquine type drug-induced hemolytic anemia has been reported recently (78,79). Peroxide generation has been demonstrated to produce severe hemolysis in rats in ~ and in vitro by peroxidation of the erythrocyte membrane constituents (24,25). The clinical observation, however, that only relatively few of the numerous patients with severe renal disease and azotemia who are treated with sulfonamides, nitrofurantoin, - 68 -

pyridium~ or hydralazine develop drug-induced hemolysis, makes the primary role of the above-listed factors unlikely. Blood analysis in case number 1 and in several other cases (not reported herein) where the hemolytic anemia was induced by sulfonamides~ showed that the blood-sulfonamide level did not exceed the suggested therapeutic range. Furthermore, azotemia~ renal disease or other diseases that could account for abnormal drug-metabolism and detoxification is not a constant feature of drug-induced hemolytic anemia in non-G6PD deficient patients. Thus the probability that extracorpuscular factors alone may increase the susceptibility of intrinsically normal red cells to the hemolytic action of oxidative drugs given in usual doses is rather remote. In conclusion, it is suggested that in the observed cases of Heinz body anemia, the increased red cell destruction was due to the combination of two factors: (a) An extracorpuscular factor - the presence of an oxidative compound in the blood, and (b) an intracorpuscular defect in the erythrocyte, which is responsible for the increased susceptibility of these cella to oxidative hemolysis induced by such compounds. In the absence of G6PD deficiency or other detectable intracellular defect, it is suggested that the decreased erythrocyte membrane sulf­ hydryl content is the most important etiological factor in the individual sensitivity to oxidative drugs. - 69 -

The cause of reduced erythrocyte membrane SH content has to be considered separately. Two possibilities will be considereà: (i) It cannot be excluded that extracorpuscular factors played a role in the depletion of reactive membrane SH groups. The membrane SH content was determined in the erythrocytes of three patients on relatively long-term sulfonamtde treatment, and in five patients with severe azotemia (Tables XIV and XV). The mean values of reactive membrane SH content in these groups of patients did not differ significantly from that of the normal mean. As mentioned previously, the presence of severe azotemia did not affect the SH content of erythrocytes from transfused, ACD-stored blood in patient number 38. Neither of these patients developed drug-induced hemolytic anemia. Since one patient was round in each group whose erythro­ cyte membrane SH content was slightly below the normal range (cases number 35 and 39), the possibility that azotemia, administration of oxidative compounds, or abnormal metabolites of these drugs contributed to the deficiency of membrane SH groups cannot be ruled out. The finding of low membrane SH content in G6PD deficiency and other cases of hemolytic anemia where these extracorpuscular factors could be entirely excluded would suggest that, although these factors may have contributed to the reduction of the membrane SH content, they alone cannot be responsible. - 70 -

(11) Deficiency of membrane SH groups observed in drug­ induced hemolytic anemia may represent a congenttal red cell defect, resulting in decreased resistance to oxidative injury. Since it was not possible to predict which patient will develop drug-induced hemolytic anemia, no studies could have been undertaken on patients prior to the onset of hemolysis to test this concept. Follow-up studies in two cases showed that, although the membrane SH content increased slightly after the recovery from the acute hemolytic attack, it remained significantly below the normal range. The mechanism by which the decreased membrane SH content causes increased susceptibility to oxidative hemolysis could be two-fold: (a) Increased permeability of SH deficient erythrocyte membranes to oxidative drugs. Previous studies on the distribution of various sulfonamides in human and canine blood demonstrated that most of the drug remains in the plasma, primarily adsorbed to serum proteins, and very little, if any, penetrates into the red cell (80-83). It 1s conceivable that membrane SH groups are important for the function of the membrane as a protective barrier against the entry of oxidative drugs or other harmful substances into the red cell. SH groups thus may forma 'first line of defence' against oxidative agents by preventing their entry into the cell. Deficiency of - 71 - these groups may result, thererore, in decreased resistance or the cell membrane to the penetration or oxidative compounds into the cell where they may inactivate vulnerable glycolytic or other enzymes. The observation that the onset or decrease or intracellular GSH level in PMB incubated erythrocytes of drug-induced hemolytic anemia patients occurred at a slightly lower concentration or PMB than in normal red cella (Table III), seems to support this possibility. (b) Decreased resistance and impaired function of SH deficient erythrocyte membranes. Membrane SH groups may be required ror the function of the red cell membrane in maintaining the characteristic shape, integrity and stability or the erythrocyte through the active trans-membrane cation transport and other enzymatic processes (see GENERAL DISCUSSION). Recently Desforges demonstrated that oxidative agents auch as acetylphenylhydrazine (APH) may act directly on the red cell membrane, causing profound membrane injury with resultant potassium leak, accelerated breàk• down and depletion of adenosine triphosphate leading to red cell destruction (84). It is conceivamle that if an abundance of free SH groups is not readily available in the cell membrane to combat and neutralize the offending oxidant, perhaps through an elaborate electron-carrier system securing the regeneration of SH groups in the cell membrane, then the multiplicity of important enzymes located in the erythrocyte membrane {85) and - 72 -

necessary for the maintenance or proper membrane function~ become vulnerable to oxidative or other injuries afflicted by various agents. In summary, it is suggested that congenital or induced deficiency of erythrocyte membrane SH groups is responsible, alone, or in combination with extra-corpuscular factors~ for the development of drug-irlduced hemolytic anemia in non-G6PD deficient patients.

3. Other Acquired Hemolytic Anemias The reactive membrane sulfhydryl content was round to be decreased in the erythrocytes of nine patients where the development of hemolytic anemia was not induced by drugs. These resulta are shown in Tables VIII - IX and details are given in the RESULTS section of Chapter II. (a) The development of hemolytic anemia was due to auto­ immune mechanism in three out of the nine cases of acquired hemolytic anemia. Coombs' anti-human-globuline test was positive in these cases and the osmotic fragility of the red cells was increased (see RESULTS). Repeabed determinations on the red cells of these patients showed very low membrane SH content (1.33 - 1.85 x lo-16 moles/RBC) and the mean membrane SH content (1.61) was one-third (36%) less than that of the normal erythrocytes. - 73 -

Erythrocyte membrane insufficiency was observed in these cases (see the effect of PMB on cation transport in RESULTS) evidenced by increased potassium leak and decreased resistance to hemolysis. The mechanism of membrane injury induced by antibody coating of the erythrocytes has been extensively

1nvestigated1 but it is not known (144). Tishkoff reported recently that the nature of red cell injury in Coombs 1 positive hemolytic anemia may reside in a specifie chemical alteration of certain erythrocyte receptors ( 86) • Others:.~presented evidence of direct membrane injury in antibody-and-camplement induced hemolysis of human erythrocytes, resulting in complete disruption of trans-membrane cation gradients, and rapid escape of hemo­ globin through the darnaged cell membrane (87). It is conceivable that deficiency of the membrane SH groups may represent a specifie biochemical alteration associated with the development of autoirnmune hemolytic anemia: (i) Coating of the red cell surface with abnormal protein factors may induce changes in the reactivity of membrane SH groups. Jandl and Jacob suggested that certain antibodies may act by affecting the membrane SH groups {27). (11) .. Another possibility is that changes in the membrane SH content may produce critical alterations in the red cell surface leading to antibody formation under certain circurn­ stances. It has been suggested tljat antôirnrnune type of antibodies - 74 - may be formed against red cella of the sè!f through alteration of the red cell surface so that in essence, it becomes foreign (144). Such changes might be brought about by faulty protein biosynthesis or by various plasma factors (144).

(b) The Coombs 1 test was negative in the remaining six cases of acqUired hemolytic anemia, rour of which were most likely due to renal disease. Extensive investigations confirmed the presence of marked hemolysis in case 13, a patient with infectious hepatitis, and in case 14, a patient recovering from chronic uremia with the aid of a transplanted kidney. Shortened red cell survival was proven in this case by chromium survival etudies. The possibility of nitrofurantoin induced hemolytic anemia was suspected in this patient, but the absence of Heinz bodies from the patient's erythrocytes did not substantiate this suspicion. Detailed information about this case is given in the RESULTS section of Chapter II. Renal disease and infection complicated the clinical picture in four patients (cases number 19-22). Anemia of renal disease is usually due to the combination of (a) erythropoietic depression and (b) the production of poorly formed red cella with diminished life span, giving a hemolytic component to the anemia. This may have been the case in these patients in whom reticulocytosis, severe anemia and splenomegaly (case number 21), initially increased erythrocyte fragility (case - 75 - number 22), previous history of drug-induced hemolysis (case number 19) or other factors suggested the presence of evert hemolysis. Since the presence of accelerated red cell destruction was not substantiated satisfactorily, these four cases were classified as miscellaneous conditions with "possible.. hemolytic anemia.

4. Hereditary Spherocytosis A 33% reduction of the erythrocyte membrane SH content was found in one patient with hemolytic anemia due to congenital spherocytosis. This four-month pregnant patient had severe anemia, marked reticulocytosis, markedly increased red cell osmotic fragility and negative Coombs' test, apart from the morphological features of spherocytosis (case number 18, Tables VIII -X). Her elder sister is thought to suffer from the same disease, but it was not round in her mother. Her rather was unknown. Apart from the low membrane SH content, an abnormal time course of PMB uptake was also featured by her erythrocytes. The number of SH groups slightly increased in the patient's erythrocytes after splenectomy (Table X), but the time rate of PMB uptake had not changed (figure 8). The time course of PMB uptake studied in one of the cases of Coombs' positive acquired hemolytic anemia (case number 17), was also different from that round in normal erythrocytes (figure 7). The importance of different rates of PMB uptake in these two cases of hemolytic anemia is not apparent. - 76 -

Dunn et al., studying the erythrocyte metabolism in hereditary spherocytosis, could not identify any major metabolic defect and concluded that nan improperly constituted membrane could explain many of the abnormalities reported in hereditary spherocytosis" {88). They observed that glycolysis of the spherocyte is more sensitive to the gl7colytic inhibitor iodoacetate than glycolysis of normal cella. Iodoacetate is also known as a relatively non-specifie SH inhibitor. The predicted membrane abnormality was demonstrated as an increased permeability to cations by Jacob and Jandl (89). In a recent paper they reported a specifie defect in the membrane of spherocytic red cella, resulting in increased passive cation fluxes requiring excessive ATPase activity and active cation transport in expense of increased ATP consumption by the pump in order to balance the increased passive cation fluxes. This compensatory mechanism presumably becomes inadequate at times of metabolic stress, auch as erythrostasis and glucose deprivation in the splenic sinuses, and osmotic swelling and hemolysis then ensues. They also reported a feature, specifie to hereditary spherocytosis cella: a marked increase in auto­ hemolysis in the presence of Ouabain, a known specifie inhibitor of the Na+- K+ dependent membrane ATPase {89). The possible role of SH groups, in the function of the above-named enzyme~ will be considered in the GENERAL DISCUSSION. - 77 -

However, from the above-mentioned data, the possibility of some association between reduced membrane SH content and increased membrane permeability in hereditary spherocytosis is apparent. Binee the finding of reduced membrane SH groups is limited to one case of hereditary spherocytosis, further determinations and investigations are required.

5. Other Conditions Normal erythrocyte membrane SH content was found in pernicious anemia, iron deficiency anemia, and in one case of sickle cell trait, as we!l as in other miscellaneous conditions (Tables XI-XIII). This seems to indicate that reduced reactive membrane SH content is not a necessary pre­ requisite of shortened erythrocyte life span, but it is rather associated with conditions where imparied red cell viability is due to susceptibility to hemolysis. The hemolytic episode usually occurs only when the membrane SH deficient erythrocytes arechhallenged by an extracorpuscular factor auch as oxidative drug, antibody, or metabolic stress in the spleen.

6. Splenic Sequestration of SH Deficient Red Cella There is abundant evidence that the spleen is highly selective toward erythrocytes with various qualitative alter­ ations, auch as spherocytic shape change, incomplete antibody coating, metalloprotein membrane complexes, and membrane SH - 78 - group inhibition. The various reports were reviewed recently by Azeb and Schiller (145). Jacob and Jandl suggested that it is the inhibition of erythrocyte membrane SH groups through which the spleen recognizes and specifically traps the erythro­ cytes affected by various conditions, including natural senescence (27). It was thought that if selective sequestration of membrane SH deficient erythrocytes takes place in the spleen then the average number of membrane SH groups of erythrocytes obtained from the (efferent) splenic vein, should be higher than that obtained from the (afferent) splenic artery. We had an opportunity to obtain fresh heparinized blood from human splenic vein and artery at the time of splenectomy in a patient with autoimmune hemolytic anemia and markedly reduced membrane SH content. The findings of this single experiment (Table X) seemed to be compatible with this possibility. However, it must be recognized that the difference observed between the membrane SH content of venous ( 1.7 4 x 10 -16 ) and arterial splenic blood (1.56 x lo-16) was within the range of standard error of the experimental technique (S.D. ~ ± 0.20 x lo-16).

Further experimenta are reqUired for the studying of ~he splenic sequestration of membrane SH deficient eryhhrocytes. - 79 -

PART III GENERAL DISCUSSION

1. The Role of SH Groups in the Function of the Red Cell Membrane The findings of rrandl and Jacob, who demonstrated the unique role of membrane SH groups in the integri ty and survival of human erythrocytes in vitro and in vivo {26,27), have been discussed previously. References have been made to other reports emphasizing the importance of erythrocyte membrane SH groups {15,25,29,30). However, the exact role of the erythrocyte membrane SH groups is not kn.own. More information is available about the role of SH groups in other tissues, such as in renal cortical cells {91) or in muscle cells. Kielley et !!· and Madsen et al. demonstrated that the SH groups of the muscle cell are closely associated with the enzymatic activity of myosin ATPase, and are essential for muscle phosphorylase activity (92-94). Kiell~ and Bradley identified inhibitor and activator SH groups regulating the activity of myosin ATPase in rabbit muscle; binding these SH groups with appropriate concentrations or PMB caused profound functional changes ~nd finally complete inhibition or the enzyme activity {92). Sekine et al. localized one specifie SH group in the active site of myosin ATPase by radioactive fingerprint analysis or the trypsin digest of myosin (95). - 80 -

Blum demonstrated in 1962 that activator and inhibitor SH groups in myosin ATPase are responsible for the conformation changes of the active site of the enzyme (96}. Applying various concentrations of PMB and other SH inhibitors, he observed that SH groups are not only reqUired for the regulation of the ATP Bydrolyzing activity of myosin ATPase, but that they also play a role in the mechanochemical coupling or energy liberation to the contractile machinery or the muscle cell (97}. In the case of actomyosin, Blum showed that hydrolysis of ATP may still proceed after inhibition of specifie SH groups by PMB, but it will no longer be coupled to the contractile machinery of the cell (97}. Similar observation was made by Kono and Colowick, who round that PMB inhibited the link between ATP and the sugar transport mechanism of the muscle cell in rat diaphragm. They also claimed that all sensitive sites or transport are located in the membrane of the muscle cell (98).

These observations suggested that SH groups may have~~a similar role in the function of the erythrocyte membrane where a great part of the glycolytic energy of the red cell is being utilized. Most of the energy made available by erythrocyte metabolism is expended in the cell membrane for maintaining the cell in a functional state (99). This possible analogy between the function of muscle and red cell membrane SH groups - 81 - is supported by recent observations to be discussed in the following paragraphe. It is suggested that erythrocyte membrane SH groups may play a role in the linking of chemical energy generated in the cell during the process of glycolysis to the utilization of this energy for maintaining the normal intracellular electrolyte environment. The maintenance of high intracellular potassium level with paralled extrusion of sodium from the cell is a common protective mechanism of cells with flexible membranes against the osmotic pressure of their interna! soluble proteins auch as hemoglobin in the red cell (lOO).

2. Brief Review of the Trans-membrane Cation Transport in Erythrocytes The cation exchange across the erythrocyte membrane is the net result of two processes: The 1 downhill 1 process of passive diffusion through aqueous pores of the cell envelope during which Na+ and ~ ions follow the electrochemical concentration gradients~ and the •uphill' process of active, carrier facilitated transport against these gradients~ requiring the expenditure of energy (101). The active cation transport is destined to maintain an intracellular ~ concentration thirty times greater than that of the plasma (150:5 ~L) and Na+ level, ten times srnaller than that of the surrounding environment (14:140 mEVL). These large concentration differences are - 82 - maintained by a nNa+ - K'" pump" pumping the cations against their respective electrochemical gradients, driven by metabolic energy derived from the intracellular glycolysis (102-106). The energy ms provided in the form of high energy bonds of adenosine triphosphate (ATP) and made available for the purpose of "running the pump" through the enzymatic hydrolysis of ATP. Hoffman, Dunham, Whi ttam and Nakao independently demonstrated that ATP is the source of potential energy, and that upon hydrolysis of ATP, energy is liberated and converted to the work of active cation transport (107-111). Inhibition of glycolysis or depletion of ATP will result in potassium loss, sodium and water influx, with consequent swelling, distention and finally osmotic hemolysis of the red cell (112-114). The active transport of Na+ and K+ is linked in a single, tightly bond system which is coupled with hydrolysis of ATP in the cell membrane (115,116). In 1952 Clarkson and Maizels suggested that an ATP hydro­ lyzing enzyme is present in the erythrocyte membrane (117). ATPase activity was shown to be essential for the integrity of mammalian erythrocytes (118) and closely associated with the active trans-membrane cation transport {119). The presence of a cation carrier system in the erythrocyte membrane was suggested by the observation that cardiac glycosides, such as Ouabain, can block the active cation transport without effecting - 83 - intracellular glucose metabolism (120-122). Ouabain may dissociate the carrier system from glycolysis by steric dislocation from the energizing source, or by displacing the cations from the carrier site (99}. It may be recalled that PMB in concentrations which have no effect on intracellular glycolysis, similarly disrupts the cation gradients causing ~ leak and Na+ influx (26,27). Thus it seema conceivable that membrane SH inhibition affects the cation carrier system by a similar uncoupling mechanism. Post et !l· (1960) and Dunham and Glynn (1961) identified and located ATPase in broken red cell membrane as a participant in the membrane transport system (123 1 124). Two different ATP hydrolyzing enzymes were characterized, one of them requiring the presence of both Na+ and~ for its activity (123-126). The separation of the two enzymes was reported recently by

Nakao et al. (127). Ouabain and Oligomycin inhibit the Na+ - ~ dependent membrane ATPase, but have no effect on the other enzyme (123-129). The active participation of Na+ and~ ions in the hydrolysis of ATP was recently demonstrated. Experimenta with P32-labelled ATP showed that an enzyme-substrate complex is formed between ATP and ATPase in the erythrocyte membrane (130), whereby a high energy phosphate bond is transferred from the ATP molecule to the enzyme, which requires the presence of Na+ ions. The dephosphorylation of the enzyme and breaking of the complex takes place only in the presence of~ ~ons (1301 131). - 84 -

Skou demonstrated in a cation-dependent membrane ATPase system obtained from ox brain that SH groups play an essential role in the formation and breakdown of the enzyme-substrate­ cation system (131). Nagano and Nakao showed that the Na+- ~ dependent membrane ATPase functions as the actual cation carrier, or as the key molecule of the carrier system (132), as predicted earlier by Burgen (133).

3. The Analogy of Muscle and Red Cell ATPase: A Hypothesis for the Role of Membrane SH Groups in the Function of the Erythrocyte Membrane ATPase A series of studies undertaken by Nakao et al. and Tatibana et al. suggested that in addition to the active transport of cations, the maintenance of the bi-concave discoidal configuration of the erythrocyte is another principal function of the erythrocyte membrane. Nakao demonstrated in 1960 the absolute dependence of the shape of erythrocyte on its ATP content (134,135). They suggested that an ATP­ dependent contractile protein analogous to that found in muscle may be responsible for the variations in cell size and shape. Tatibana et al. claimed that both the active cation transport and the maintenance of the characteristic shape of the erythro­ cyte are conducted by a common structural element in the cell membrane which involves ATPase activity (136,137). - 85 -

Recently Ohnishi and Kawamura reported the isolation of contractile proteins in the red cell membrane similar to those of actin and myosin in the muscle (138,139). They showed that while the contractile proteins certainly play a role in maintaining the shape of the red cells, in addition, they participate in combination with membrane phospholipide in the active cation transport through the erythrocyte membrane (138). These recent reports seem to give further support to the possible analogy between erythrocyte and muscle ATPase. On the basis of the foregoing it is postulated that erythrocyte membrane SH groups may play an important role in the ATPase facilitated active cation transport across the red cell membrane. The role of SH groups in the function of a similar Na+ - K+ dependent ATPase, obtained from ox brain, has been reported (131), but the relationship of erythrocyte membrane SH activity to the Na+ - K+ dependent erythrocyte membrane ATPase has not been studied. Although the presented hypothesis is based on circumstantial evidence and is partly speculative, it could plausibly explain severa! findings of the present study -- particularly the leakage of potassium from the PMB inhibited erythrocytes and is in agreement with similar observations published by others (26). It is also compatible with the finding that oxidative compounds produce ~ leakage from the exposed erythrocytes {28,84,140). - 86 -

4. The Leakage of Potassium from Erythrocytes Ex.posed to OXidative Compounds Weed et al. and Desforges reported that potent oxidative compounds such as primaquine (28) or acetylphenylhydrazine (84,140) directly damage the red cell membrane, presumably through the generation of hydrogen peroxide {78,79,140). Normal erythrocytes are able to detoxify peroxide with the aid of a mediated electron transfer system, protecting the cell from oxidation to a certain extent. If large doses of oxidative compounds generate excessive amounts of peroxide, then the protective mechanism may become insufficient, and oxidative hemolysis ensues. It was observed that disruption of the cation gradients occurs before the onset of hemolysis, and certain pre-hemolytic concentrations of strong oxidants may produce leakage of ~ and intracellular gain of Na+ without overt hemolysis {28). Less potent oxidants such as sulfonamides or lower doses of potent oxidants are sufficient to produce hemolysis of G6PD-deficient erythrocytes which are poorly equipped to handle the oxidative compounds because of their deficiency of GSH, the chief mediator in the electron transport system. It is proposed similarly, that erythrocytes with normal membrane SH content are able to resist the stress caused by therapeutic doses of oxidative drugs, but if the membrane SH - 87 -

content is decreased below a certain critical level~ then relatively minor oxidative stress will result in the breakdown of membrane functions leading to oxidative hemolysis.

Administration of sulfonamides, for example~ may cause a slight reduction of the membrane SH content (Table XV), but the cell is able to cope with this, presumably by regenerating the membrane SH groups~ and hemolysis will not ensue. However, erythrocytes with deficient membrane SH content or deficient ability to ~egenerate their membrane SH groups under the effect of oxidative or other stress become susceptible to hemolysis. Thus, even therapeutic doses of oxidative compounds will be sufficient to interfere with the proper function of the cation pump, resulting in disruption of the physiological cation gradients, leakage of ~~ influx of Na+, and marked increase of membrane permeability to large molecules, thus leading to hemolysis of the cell. Other extracorpuscular factors may contribute to the fore­ going processes. Poor renal clearance of the oxidative drug in kidney disease may produce retention and high plasma level of the compound or its metabolite. Azotemie plasma itself may contribute to the vulnerability of the exposed erythrocytes. Abnormal metabolism of the offending agent in patients with liver or kidney disease ~ result in increased peroxide generation by the administered drug. Inherited differences - 88 - in drug metabolism in man have been reported (141), and evidence has been presented recently for the genetic control of the metabolism of certain sulfonamides and isoniazid in the rabbit (142,143). The possible role of extracorpuscular factors in the development of hemolytic anemia has been discussed.

5. The Leakage of Potassium from PMB-Incubated Erythrocytes The leakage of potassium from intact healthy erythrocytes, the membrane SH groups of which have been blocked with non­ hemolytic concentrations of PMB has been already reported by Jandl and Jacob (26). The present experimenta demonstrated the relationship between the concentration of PMB and the extent of potassium loss from the incubated red cells (figure 10). Potassium leak from PMB-treated normal erythrocytes occurred before hemolysis could be detected. Maximum or near maximum potassium leak ( 60 uE/lo10 RBC) was noted with concentrations of PMB which seemed to be sufficiently htth to gain access to the intracellular phase and to begin reacting With intracellular reduced glutathione. (Since 1010 RBC representa approximately 1 ml packed cells, the maximum ~ leak observed is about 60 mœ/L RBC, or 40% of the total intracellular potassium content).

Much greater ~ loss was observed in Coombs' posttive, antigen-coated erythrocytes, with obviously damaged, nleakyu - 89 - membranes. Not only was the potassium loss greater in these cella, but maximum ~ leak occurred, accompanied by sorne escape of hemoglobin at a lower leve! of PMB concentration

(4 ~ moles~ml RBC) than in normal cells (figure 11). Incubation of these cells in isotonie pH 7.4 buffer for 30 minutes produced sorne leakage of ~ even in the absence of PMB. Some­ what similar findings in G6PD-deficient red cells (figure 11) suggest sorne structural or functional defects in the membranes of the enzyme deficient erythrocytes as we!l. These defects could be the direct consequence of poor SH content and vulnerable cation pumping system in the membrane of these cella. It is also of note that obvious hemolysis occurred in both G6PD­ deficient cells and Coombs' positive erythrocytes at a concent­ ration of PMB (6-8 ~moles/ml RBC) which did not have this effect on normal erythrocytes (figure 11, Table XVII). The available data do not permit conclusions to be drawn in regard to the potassium leak from the erythrocytes of patients with drug-induced or other hemolytic anemias who had negative Coomb's test and normal G6PD activity. In many of these cases, the possibility of studying the effect of PMB on the red cells was limited to one or two concentrations of the reagent. Although' in sorne of the cases, where the effect of SH inhibition could be studied with a wide range of PMB concentrations, the ~ leakage from PMB inhibited erythrocytes - 90 - was round to exceed that of the normal red cells, the mean of ~ loss was not significantly different from that in normal erythrocytes. It must also be considered that the range of ~ leak from healthy red cella was relatively great at any given concentration of PMB (figure 10). The values were obtained by flamephotometric analysis of the supernatants in which the cella were previously incubated. The detection of less than gross differences between the PMB effect on the cation transport in normal and in certain abnormal cella may reqUire more refined methods. The foregoing observations support the suggestion that the reactive membrane SH groups are involved in the active transport of cations across the erythrocyte membrane.

6. A Proposed Mechanism for the Participation of SH Groups in the Active Transport of Cations in Erythrocytes It is proposed that the presence of reactive sulfhydryl groups is essential for the activity of the Na+ and K+ dependent erythrocyte membrane adenosine triphosphatase. OXidation or blockage of these 'critical' SH groups may disrupt the active cation transport by (a) uncoupling the energy liberation from the cation transport, dislocating the carrier mechanism from its source of energy, or (b) by direct inhibition of the cation­ dependent ATPase activity through the displacement of the cations from the active carrier site, or via prevention of the formation - 91 - of enzyme-substrate complex between ATPase and ATP. Primary or secondary deficiency in the SH content of the red cell membrane will permit the interaction of oxidative or other

SH-reactive compounds with the 1 critical 1 SH sttes of the membrane. This, in turn, will interfere witb the effective work of the 1 pump mechanism 1 -- i.e. the ATPase dependent cation carrier system -- with consequent disruption of the cation gradients across the cell membrane leading to cellular swelling, distention and finally disruption of the cell membrane. Thus, the deficiency of membrane SH content will result in increased susceptibility to hemolysis. Decreased membrane SH content was demonstrated in a variety of conditions characterized by increased susceptibility to hemolysis. These findings could be explained by the above­ described mechanism in the following way: 1. Erythrocytes with normal membrane SH content will be able to maintain their membrane SH groups above a critical level and protect their essential SH sites involved in the physiological function of the membrane when faced with various challenges (auch as oxidants, etc.). 2. Red cella with decreased membrane SH content, having no 'protective reserve•, are vulnerable to any metabolic or other stress encountered in the spleen or in the circulation. - 92 -

3. Erythrocytes with decreased membrane SH groups will be unable to maintain the critical level of membrane SH content essential for the functional state of the membrane when challenged by oxidative compounds or other toxic agents. 4. The critical level of SH content in the cell membrane may represent the number of SH sites essential for the activity of the Na+ - x+ dependent ATPase which, in turn, is responsible for the maintenance of the physiological cation gradients in the cell. To test this concept, it is necessary to study the ATPase activity of SR-deficient or SH-inhibited cell membranes during the future course of this project (see PREFACE).

7. The Significance of the Finding of Reduced Membrane SH Content in Certain Hemolytic Conditions The significance of decreased membrane SH content noticed in certain cases of hemolytic anemia requires further invest­ igation: it has not been proven that it representa a cause, rather than an effect of the hemolytic process. The finding of diminished SH content in the membrane of hemolysis-prone G6PD-deficient cells in the absence of overt hemolysis in four healthy individuals, however, would favor the former possibility. On the basis of the previously discussed observations, it was suggested that the deficiency of the reactive membrane SH sites demonstrated in erythrocytes obtained from patients with various hematological conditions, was a major factor in - 93 - the susceptibility of these patients to hemolysis. Accelerated red cell destruction in these clinically different conditions (drug-induced, auto-immune, and other acquired hemolytic anemias, and hereditary spherocytosis) may have resulted from the interaction of severa! factors, decreased membrane SH sites being only one of them. Reporting on the effect of APH on the erythrocyte membrane, Desforges pointed out recently that oxidative hemolysis, for example, may be the result of not one, but the combination of severa! factors, including disordered SH and ATP metabolism of the cell (140). Deficiency of reactive membrane SH sites may represent a primary, perhaps congenital membrane defect in sorne instances. In other cases, it may be secondary to sorne tntracellular, or to extracorpuscular factors, or both. Marked reduction of the intracellular GSH content in G6PD deficiency may represent such an intracorpuscular defect resulting in low, or easily deplatable membrane SH content. various plasma factors, in turn, may represent an extracorpuscular element in the reduction of the membrane SH content. The data presented in this thesis strongly suggest a definite correlation between the deficiency or loss of the reactive membrane SH groups, and the shortened red cell survival or increased susceptibility to hemolysis in the observed cases. - 94 -

SUMMARY AND CONCLUSION

A technique has been developed for the quantitative determination of reactive membrane sulfhydryl content in intact, non-hemolyzed erythrocytes, utilizing non-penetrating, sub-hemolytic concentrations of p-chloromercuribenzoate. By the application of this technique the normal range of erythro­ cyte membrane SH content has been determined and membrane SH groups have been studied in various hematological disorders. Particular interest was focused on the mechanism of the development of drug-induced hemolytic anemia in certain non­ G6PD deficient patients. Studies have been undertaken in severa! other conditions characterized by impaired red cell survival or other disorders affecting the erythrocyte. It was established that the erythrocyte membrane of 32 healthy subjects contained 2.30 - 2.85 x lo-16 moles of reactive SH groups per erythrocyte when determined by this method. The mean of normal SH content, 2.50 ± 0.20 x lo-16 moles/RBC, representa an average of 1.5 x 108 reactive sulf­ hydryl sites in the intact, normal erythrocyte membrane. The reactive SH content of the erythrocyte membrane was significantly decreased in various hematological conditions characterized by increased susceptibility to hemolysis and/or accelerated red cell destruction. A 27-3~ reduction of - 95 - membrane SH content was observed in the erythrocytes of four G6PD deficient subjects, in seven cases of drug-induced hemolytic anemia in non-G6PD deficient patients, in three cases of autoimmune, and two cases of other acquired hemolytic anemia and in one case of congenital spherocytosis. Normal membrane SH content was round in cold-stored erythrocytes, in iron-deficiency anemia, in pernicious anemia, in one case of sickle cell trait, in one case of possible Heinz body anemia and in other miscellaneous conditions. Inhibition or membrane SH groups with non-hemolytic concentrations of PMB caused marked leakage of potassium from the otherwise intact cells but no decrease or the intracellular

GSH content. Greater degree of potassi~ leak was observed in

Coombs' positive and in G6PD deficient erythrocytes~ which showed decreased resistance to the hemolytic action of PMB. The possible relationship between the readily available membrane SH groups, the active trans-membrane cation transport, the Na+ - K+ dependent membrane ATPase activity, and the integrity or destruction of the red cells was discussed. It was suggested that deficiency or decrease(,_of membrane SH content may play an important role in the development or certain clinically different types of hemolytic anemias. It was postulated that reactive membrane SH groups are essential for the Na+ - K+ dependent membrane adenosine triphosphàtase activity, which in - 96 - turn is responsible for the maintenance of active cation transport across the red cell membrane, and of the characteristic shape of the erythrocytes. According to this postulate, a primary deficiency or secondary reduction of erythrocyte membrane SH content below a critical level, would result in disruption of the physiological cation gradients, osmotic swelling, and subsequent splenic sequestration and hemolysis of the affected cella. Secondary reduction of the membrane SH content could be due to impatred intracellular thiol metabolism, or to the interaction of severa! extracorpuscular factors, auch as (1) azotemia, (2) excessive plasma level of oxidative or other SH blocking compounds due to (a) renal retention, (b) impaired detoxification of these compounds, or (c) production of abnormal metabolites with increased oxidative potential, or (3) any other metabolic stress resulting in oxidation or blockage of the membrane sulfhydryl sites. Increased permeability of the erythrocyte membrane to oxidative or other toxic agents,resulting from changes in the tertiary molecular structure of the red cell membrane induced by oxidation or inhibition of the free SH sites, may also play a role in the development of certain hemolytic anemias. In conclusion, .the experimental data have shown that the erythrocyte membrane SH content is decreased in certain hematological conditions characterized by increased susceptibility to hemolysis and/or accelerated red cell destruction, irrespective - 97 - or intracellular GSH leve! or stability. It is suggested that the development or hemolysis in these conditions 1s related or due to the deficient SH content or the erythrocyte membrane. B8

TABLE I

PMB BINDING OF WASHED ERITHROOlTES

PMB CONCENTRATION IN NO. OF PMB BOUND PER CELL (molesxlo-16) THE INCUBATION MIXTURE DETER-

5 20 2.9 - 5.85 5.45

6 16 5.7 - 4.6 4.10 7 10 5.9 - 5.5 4.70 8 12 4.75 - 6.5 5.51 9 6 4.8 - 7.5 6.26 10 6 6.1 - 8.2 7.00

Incubation : 50 minutes at 5~ C ( pH : 7.4 ) TABΠII

GSH CONTEN.r OF Pr-13 INCUBA.TB:D ERlTHROClTES AS DETERMINED

BY THE ALLOXAN SOS METHOD.

Determinations on erythrocytes of 12 healthy donors.

PMB CONC.IN GSH CONTENr ,mg/101~ PERCENI' LOSS THE INCUBATION OF GSH~ MIXTURE (p. moles/ml RBC) RA:OOE MEAN

BEFORE INCUBATION 0 54- 75 66.0

AFTER INCUBATION 0 54- 78 64.0 0 2 50- 69 63.5 < 1 3 54- 71 63.5 < 1 4 54 - 70 62.5 2

5 52 - 69 56.0 10

6 53 - 68 ss.o 10

8 47 - 63 53.0 14

Incubation time : 50 minutes •rn calculating the percent loss of GSH, the mean GSH content of erythrocytes incubated for 50 minutes without the addition of M , was taken as 100% GSH

content. TABLE III

GSH CONTEN!' OF PMB-nlCUBATEDERYTHROCYTES AS DETERMINEDBY THE ALLOXAN305 METHOD

Mean values or determinations on erythrocytes in healthy donors, in patients with hemolytic anemia and in G-6-PD deficiànt subjects.

PMB CONC.IN ERYTHROCYTEGSH CONrENT (mg per 1012 RED CELLS) THE INCUBATION MIXTURE 12 6 PAT..IJ!il'f.i: ~ 6 PATIENTS WITH 4 DONORSWITH (u moles/ml RBC) HEALTHY WITH HEMOLYTIC DRUG-INDUCED G 6PD DEFIC JEliCY DONORS ANEMIA HEMOLlTIC ANEMIA (acquired or congenital)

!BEFOREmCUBATION 0 66 63 69* 30

!AFTERINCUBATION 0 64 61 70 28 2 63.5 62 68 52

3 65.5 61 69 50 4 62.5 57 61 27

5 56 56 55 21 6 56 55 54 18

8 55 50 51

~--·-·······-

Incubation t:l:m.e: 30 minutes • Increased mean values in this group or patients are due to one patient (ML)with phenacetin induced ~"·C: hemolytic anemia, who had very high erythrocyte GSH content. 0 1.01

TABLE IV

ERYTHROCYTE MEMBRANE SULFHlDRlL GROUPS IN HEALTHY DONORS

NO. DON OR MEMBRANE SH CONTENT NO. DO NOR MEMBRANE SH CONI'EN'I' (xl0-16mo1es/ RBC ) (x10-16moles/RBC )

1 G.H. 2.46 17 N.deL. 2.45 2 s.s. 2.56 18 M.N. 2.36 3 C.J. 2.45 19 N.D. 2.85 4 P.N. 2.61 20 D.P. 2.30 5 T.G. 2.70 21 o.s. 2.48 6 o.sz. 2.45 22 A.H. 2.35 7 J .B. 2.30 23 T.G. 2.56 8 P.G. 2.48 24 s.s. 2.45 9 P.H. 2.46 25 M.S.5 2.54 10 s.Y. 2.56 26 M.s.6 2.52 11 :r-1.5.1 • 2.60 27 M.S.7 2.48 112 M.S.2 2.85 28 M.s.a 2.45 i 13 M.S.3 2.43 1 29 S. Y. 2.56 14 M.S.4 2.32 30 P.G. 2.44

15 H.L. 2.41 51 J .w. 2.50 16 J.J. 2.62 52 D.P. 2.38 i i ' • M.S.= Medical student RANGE: 2.30 - 2.85

MEAN : 2.50 S.D. : ... - 0.20 TABLEV

PATIENTSWITH DRUG-INDUCED HEMOLYTIC ANEMIA a) Clinical data.

CASE PATIENT NO. NA.ME AGE SEI DRUG(S) TAIŒN CLllUCALCONDrriON(S) BUN G6PD GSH GSH mg% units mg/lOOml incubated with /lOOml RBC APH mg/lOOml RBC RBC

1 J.P. 46 F Sulfamethizole pyelonephritis 85 • 81 75 Imuran renal failure - renal transplant 2 M.L. 28 F Phenacetine phenacatin addiction s.s 261 88 124 bleeding ulcer

3 F.B. 79 F Pyridium uri.nary infection 112 121 57 54 4 I.B. 46 F Nitro.furantoin pye1onephritis 16 - 72 75 acidosis ma.labsorption

5 J.B. 37 M Hydralazine chronio nephritis 168 157 42 46 renal faUure h~rtension

6 o.rfc. 41 M Hydralazine chronic nephritis 99 58 78 renal failura - h1J>8rtension 7 M.O• B. 62 F Co1cemid cln-onic myeloid 25.5 - 79 59 Tolbutamide laukemia myeloid metaplasia chronic hemo1ysis ~ .,._.. """" 8 A.K. 62 F ?Phenacetine ? anemia .... 1'1.11\AI... 157 57 82 ~ Normal 4?'-85 : values 10-20 151-214 ~~ - not datermined " TABLE VI

ERYTHROCYTESMEMBRANE SULFHIDRlL GROUPS IN PATIENTSWTI'H DRUG INDUCED HEINZ~BODY HEMOLYTIC ANEMIA

b) Hemato1o~icaldat .NO. NAME AGE SEI DATE OF HGB.gm% RETICS. HEINZ MEMBRANE SH CONTENT:x 10-l.omo1es/ DETERMINATION % BODIES . . RBC % 1. J.P. 46 F 25/10/63 10.4 - 32.0 1.27 29/10/63 9.4 4.2 8.6 1.60 15/11/65 11.1 2.0 0 1.80

2. M.L. 28 F 5/11/63 9.2 2.6 4.2 2.00

5. F.B. 79 F 14/11/65 7.5 15.0 20.0 1.75 18/11/65 7.1 - 14.4 1.65 4. I.B. 46 F 14/11/65 6.9 28.6 9.4 1.85 18/11/65 9.4· 21.4 1.4 1.85

s. J.B. 57 M 10/ 1/64 7.2 2.0 1.4 1.70 20/ 1/64 a.e• 2.0 0 1.85

s. G.Mcc. 41 M 29/ 1/64 6.5 4.8 2.0 1.80 4 7. M.O' B. 62 F 27/ 2/63 7.2 11.2 st.pos. 1.78 e. A.K. 62 F 24/ 2/65 15.5 2.5 o.8 2.48

[.MEAN: 1.81 • Patient was transf'used before this determination was done.

,.. 0 ~ :l04 TABLE VII GSH CONTENT AND MEMBRANE SH GROUPS IN GLUCOSE-S-PHOSPHATE DEHIDROGENASE DEFICIENT ERYTHROCYTES.

CASE N.AME AGE SEI GôPD GSH mg/100 QSH ~l.OO HGB. RETICS. ~Nli: SH NO. ACTIVITY ml RBC ml RBC % % CONTENT x units/100 (APH incub.) 10-16mo1es/ ml RBC RBC

9 A.F. 56 M 0 29.7 3.8 17.8 1~ 2.00

10 H.B. 42 M 0 32.6 4.0 16.5 - 1.60

11 I.B. 42 M 0 22.0 2.5 14.7 3.6 1.71 • - - - 16.4 5.6 1.50 •• 0 29.5 5.55 15.5 1.2 1.95

P-2 I.G. 24 M 0 29.8 5.0 15.1 2.5 2.00

MEAN 1.79

NORMAL RANGE: 151-2~4 45-85 >56 14-18 o.s-1.5 2.50-2.85

• Determination 5 weeks 1atar

•-. Determination 5 months 1ater CiG

·o .....

~

)

INCUBATED

-

68.3

66;.0 62.4

92.2

56.5

mg/lOOml

(APH

GSH

-

52.6

68.5

58.5

55!0

92.0

mg/lOOml

GSH

ANEMIAS

49

~

BUN 18

10

Norm.

Norm.

Norm.

HEMOLITIC

S) OF

-

);tapatitis

lupus

TYPES

lupus

uremia

DISEASE(

VIII

transplant

-

OTHER

VARIOUS TABLE

(pregnant)

Infeotious

Systemic

Systamic

Renal

Chronic

.·

---·----·--·

WITH

dis.

OF

PATIENTS

auto-

auto-

auto-

Renal

ANEMIA

CAUSE

1

AND

immune

immune

:immune

spherocytosis

?Drug-induced.

HEMOL'YTIC

TYPE hepatitis

Acquired.Infectious Congenital,

Acquired. Acquired,

Acquired,

Acquired

F

M

M

F

M

F

SEX

data

54

24 56

42

48

18

AGE

s.e.

F.G. v.T.

A.T.

o.c.

NA.ME

J.n.c.

Clinical

NO.

a) 17

18

~5

~6 ;1.4

15 TABLE VIII continued : a} Clinical data

PATIENTSWITH MISCELLANEOUS CONDITIONS: INCREASEDRED CELL DE3rRUCTIONSUSPEX;TED, BUT NOTPROVEN

~o. NAME AGE SEI TYPEAND CAUSE OF OTHERDISEASE(S) BUN GSH GSH(APHINCUBATED ) HEMOLYTICANEMIA mg% mg/lOOml mg/100 ml

19 H.P. 78 M Acquired. Renal dis.? Chronic pyelo- 55 62.8 55.9 nephritis. Furunculosis Paget's disease 20 o.n. 85 M Acquired. Renal dis.? Pyelonephritis 25 - Benigp prostatic - hypertrophy 21 E.B. 56• F Acquired. Renal dis.? Chronic uremia 160 - - pyelonephritis

22 o.s. 76 M Acquired. Renal dis.? Pyelonephritis 29 Iron deficiency - - Cholecystitis

~--· - Determination repeated l month later .. '"'""'.._, (jJ TABLE IX

ERYTHROCYTEMEMBRANE SH GROUPSIN PATlEN.I'SWITH VARIOUS HEMOLlTIC CONDITIONS B) Hematological data

NO. NAME AGE SEX HGB. RETICS. COOMBS1 OSMOTIC MEMBRANE SH CONTENTxlo-l6moles/RBC gm% % TEST FRAGILITY

~5 F.G. 56 M 11.7 7.4 neg. normal 1.75

~4 v.T. 54 M 13.0 3.8 neg • normal 1.85 tt • 10.7 10.0 - - 2.10 tl ... 10.8 6.0 - - 1.95 15 A.T. 48 F 8.5 8.4 pos. increased 1.90

16 J .n.c. 42 M 5.9 41.6 pos. increased 1.48 17 G.C. 24 F 11.6 5.4 pos. increased 1.53 " 12.7 s.o - - 1.53 n 12.9 4.8 po s. - 1.65 tt • 15.5 2.8 - - 1.85 18 s.e. 18 F 7.4 25.6 neg. increased 1.66 " 12.5 o.8 neg. increased 1.95

MEAN: 1.75

*Determination repeated l month la. ter '...... +• Determination repeated 7 weeks later -~ TABLE IX continued : b) Hematological data

PATIENTS WITH MIJlCELLANEOUSCONDITIONS. I~REASEDRED CELL DEsrRUCTION SUSPECTED

SEX H • I - % FRAGILITY MEMBRANESH CONTENTx lQ-16 moles/RBC M 12.2 3.2 neg. - 2.00 .. • 12.8 1.2 - - 1.95 20 o.n. 83 M 12.9 2.4 -- 2.00 21 E.B. 56 F s.o s.s - - 11.4 ---- 1.95 22 G.s. F 6.6 7.2 neg. increased- 1.94 normal " • 10.2 s.o - normal 2.00

N: 1.97

fi Determination repeated l month later

;...\. • -...... 00 :t09

TABLE X

EFFECT OF SPLENOMEGALY ON ERITHROOYTE MEMBRANE SH GROUPS a) Patient G.C.: Auto-immune hemolytic anemia

DATE SPLEËN' HGB / RETics.l MEMBïïAIŒ SH côllriN'i' gm% % x 10-16 mo1es/RBC 1 1 50/l/64 Before sp1enectomy 11.6 5.4 1.55 5/2/64 Before splenectomy 12.7 6.0 1.55 15/2/64 Blood from aplanie 12.9 4.8 1.74 vain at sp1enectomy Blood from sp1enic - 1.56 artery at sp1enect- - OmJ 26/2/64 5 weeks after 15.5 2.8 1.85 sp1enectomy b) Patient s.e.: Congenital spherocytosis, hemo1ytic anemia, pregnancy.

50/1/64 Before sp1enectomy 7.4 25.6 1.66

20/2/64 5 wee ks att.er sp1enectomy 12.5 o.s 1.95 t:lo

TABLE XI DITHROOlD MBMBRANE SH GROUPS m PATIENTS W:rrH mON DEFICIBICY ANDIIA

NO. NAME AGE SEX SERUM,Fe UIOOT HGB RETICS. MEMBRANE SH CONTENT ~100m1 (norm. gm% % xlo-16moles/RBC rm: 160-240) 90-150)

15 A.w. 41 M 47 220 5.5 ?,tl 2.58

24 W.H. 35 F 10 348 10.3 5.8• 2.40

25 S.H. 56 F 19 310 6.0 6.8 • 2.48

26 J.I. 48 M 21 531 7.9 1.6 2.45

MEAN: 2.50

• Short1y after onset of iron therapy T UIBC = unsaturated iron binding capacity ti:1

TABLE XII

ERITim.OClTE MEMBRANE SH GROUPS IN PATlENTS WITH PERNICIOUS ANEMIA

IN RELA PSE

NO. NAME AGE BEX TREATMENr HGB. RETICS. MEMBRANE SH CONTENT gm% % x lo-16 moles/RBC

27 v.s. 54 M Before Bl2 5.5 2.5• 2.62 After Bl2 8.5 25.0 2.70

28 W.T. 78 M Before Bl2 6.1 1.0 2.75 After Bl2 7.0 58.5 2.55

MEAN: 2.65

- Shortly after onset of folie a cid therapy TABLE XIII ERYTHROCYTE MEMBRANE SH GROUPS IN MI&;ELLANEOUS HEMATOLOOICAL CONDrriONS

NO. NAME AGE SEX HEYJATOLOGICAL CONDrr ION HGB RETICS MEMBRANE SH CONI'ENT i gm% % :xlo-16 moles /RBC

29 J.M. M M Sickle cell trait 15.7 1.2 2.50

50 S.B. 58 F Hemoglobinuria, 14.1 5.6 2.65 cause undetermined

51 I.G. 59 M Anemia, possible 7.1 2.4 2.75 Pyridoxin deficiency

52 R.L. 40 F Family history of 14.8 2.2 2.50 spherocyto sis. Splenomegaly. Manie depressive

35 M.F. F Mbther of patient 15.7 1.8 !.58 s.c.(case no.l8 )

MEAN 2.48 TABLE XIV

ERlTHROClTEMEMBRANE SH GROUPSIN PATIENTSWITH SEVERE AZOTEMIA

NO NAME AGE SEX DISEASES BUN HGB. RETICS. MEMBRANESH CONTENT mg% gm% % xlo-16moles/RBC

35 R.P. 29 M Glomerulonephritis. Renal 70 11.0 o.s 2.25 failure. Uremia. Anemia ( Hemodialysis )

36 G.B. 59 M Chronic renal disease. 102 10.0 2.4• 2.44 Uremia. Hypertension. Anemia.*

37 R.D. 27 F Glomerulo-and pyelonephritis• -1.62 6.5 6.2" 2.41 Uremia. Hypertension. Anemia. 38 J.C. 33 F Mitral comissurotomw.Empyema. 150 14.5 1.0 2.50 Acute renal failure. Anemia.••

MEAN 2.40 --- - •Shortened red cell survival indicates increased red cell destruction

~4 2 weeks after transfusion for severe anemia

?.... ~- TABLE XV

ERYTHROClTEMEMBRANE SH GROUPSIN PATIENTSTREATED WITH SULFONAMIDES

NO. N.AMEAGE BEX DIAGNOSlCS TYPEAND BUN HGB. RETICS. MEMBRANESH CONTENT TŒAL DOSE mg% gm% % x 10-16 mo1es/RBC OF SULF- ONAMIDE

39 J.B. 31 M Suprapubic prostat- Thiosulfi1 15.5 12.6 5.6 2.10 ectomy, urinary' 18 gm infection,b1eeding

40 F.o. ft3 M Carcinoma of bladder Thiosulfil 12.5 15.1 1.4 2.60 cystectomy 52 gm

41 G.D. 68 M Prostatic hypertrophy Gantrisin 32 15.0 2.0 2.50 urinary infection 20 gm

MEAN: 2.53

:....., -~ TABLE XVI

SUMMARY : ERYTHROClTE MEMBRANE SH GROUPS IN VARIOUS CONDITIONS

CONDITION NO.OF ERYTHROCYTE MEMBRANE SH CONTENT !NO.~PATIENTS DETER/ lit- x lO-l6moles/RBC MINATIONS RANGE 1 MEAN Healthy donors 52 89 2.5-2.85 2.50

ACD-stored blood 3 5 2.5-2.60 2.44

G6PD deficient subjects 4 6 1.5-2.00 1.79 Drug-induced hemolytic anemia (no G6PD deficiency} 8 15 1.27-2.48 1.81 Acquired hemo1ytic anemia (varions causes, drug- induced hemolytic anemia excluded ) 5 12 1.55-2.10 1.75 Autoimmune hemo1ytic anemia(included in above group ) 5 6 1.55-1.85 1.61 Congenital spherocytic hemo1yt ic anemia l 2 1.66-1.95 1.80

Pernicious anemia 2 4 2.55-2.75 2.65

Iron-deficiency anemia 4 4 2.40-2.58 2.50 Miscellaneous hematologie conditions 5 5 2.50-2.75 2.48 ,'

Patients with azotemia and anemia 4 4 2.25-2.50 2.40 Patients on sulfonamide therapy 5 5 2.1-2.60 2.55 • More than one exper1ment. may have been carried out on b1oods obtained from the same patient, on different occasions. Each determination was carried out in duplicata or triplicata. TABLE XVII

THE LOSS OF POTASSIUM FROM PMB INCUBATED ERTIHROOlTES ·

10 PMB CONC. POTASSIUM LOSS IN MICRO-EQUIVALENTS PER to RBC* p. moles/ml 1 RB:: NORMA- ERl'.l: l:ittW ~·l·.l!io!l'ROM RBG'S !l'ROM 2 PATIENTS GSPD DEFICIENT RBG S FROM H&ALTHY DONORS Wrl'H COOMBS1 POS.A ..H.A. 2 PATIENTS

NO.OF DETER- PATIENTS NO. MEAN PATlENTS NO. MEAN MINATIONS RANGE MEAN 15 & 17 12 & 11

0 40 0-10 0.5 4 , 10 7 0 , 0 0 1 16 0-12 6 - - - -

2 20 6-26 16 40, 48 44 27 J 55 51

5 20 20-54: 26 50, 62 56 40 , 52 46

4 20 26-40 51 52, 62 57 50 , 62 46

5 20 25-40 52 54:,62 58 50 , 62 56

6 20 52-44 57 Hemo1ysis interferred with determinations i'

7 12 38-48 40 !

8 20 42-58 46

9 10 42-60 4:6

10 10 42-60 46 :.,. ~------·-·····~---- ~--- -Cr; .. =approximate1y the same as mE/L of RBC (-::: 1ol3RBC ) :1_1_7

FIGURE 1

REACTIONS OF P-CHLOROMERCURIBENZOATE a. p-chloromercuribenzoate (PMB) COOH 0 HgCl b. Behaviour of PMB in alkaline solvent COOH coo - w· 0 + KOH-~~ 0 + KCl HgCl Hg OH c. Stoichiometric reaction with SR-groups COOH COOH 0 + RSH ) 0 + HCl HgCl Hg - SR FIGURE 2

ABSORBANCY OF P-MERCURIBENZOATE AND ITS MERCAPTIDE WITH

GLUTATHIONE IN 0.1 M. PHOSPHATE pH 7.4 AT 257 m~.

e = 0.0.257 PMB o = 0.0. 257 PMB + GSH x = à 0 . D. 2 57 ( P M B + G S H ) - ( P M B )

2.0

1. 6 . 0 . 1. 2 0

'+- .8 0 - .4 0~--~---r--~--~---.--~r---.--. 0 2 3 4 5 6 7 8

conc. of PMB x 10_4 M

O.D.257 of PMB in 0.1 M. phosphate buffer, pH 7.4. O.D.257 of PMB-GSH mercaptide. The concentration of GSH is constant. AO.D.257 of mercaptide formation (O.D.PMB-GSH- O.D.pMB)· :t1.9

FIGURE 3

PMB BINDING BY ERYTHROCYTES

cJ ..à incub. ti me 30 mins ~ 12 (/) 'Q) = PMB upto k e meon 0 10 Il Il E = ronge

~ 8 -1 0 6 x

CD 0 :lE a.. 0 2 3 4 5 6 7 8 9 10

PM B ,Al moles 1 ml. r.b.c.

The shaded area representa the range, the solid line the mean of PMB uptake by the erythrocyte membranes at various concentrations of PMB. The cells were incubated with the reagent at 37°0. for 30 minutes. :l20

FIGURE 4

EFFECT OF VARIOUS CONCENTRATIONS OF PMB ON THE ERYTHROCYTE

GSH CONT:.;:;E:.:..:NT-=------,

a. Erythrocyte GSH levels

---- 70

(..) 60 ..0 normal ~ 50 erythrocytes N incub. time 30 mins 0 40

...... 0'1 30 E G6PD defie. ·::r: 20 (/) erythrocytes (!) 10

0 0 2 3 4 5 6 7 8

PM B ,AJ moles 1 ml. r.b.c.

Black circles ( • ) represent the mean values of GSH deter­ minations on the PMB-incubated erythrocytes of 12 healthy subjects. Open circ les ( o ) represent the mean GSH content of PMB-incubated G6PD-deficient erythrocytes from 4 patients.

The intracellular GSH level is expressed in mg. 1 s of GSH per 1012 erythrocytes, the approximate equivalent of lOO ml. packed red cells (see text).

- . :~ :-. .. :t21.

FIGURE 5

EFFECT OF VARIOUS CONCENTRATIONS ON PMB ON THE ERYTHROCYTE GSH CONTENT

b. Per cent decrease of GSH level after 30 minutes' incubation with PMB

incub. time 30 mins

<1> 60 > <1> 50 ::r::: CJ) 40 (!)

'+- 0 30 <1> (/) 20 0 Q.) t.... (.) 10 <1> '"0 0 ;;-...0 0 2 3 4 5 6 7 8 9 10

PMB ;1J moles/ml.r.b.c.

The per cent decrease is calculated on the basis of GSH level in erythrocytes incUbated in phosphate buffer for 30 minutes at 37°0. without addition of PMB. :1.22

FIGURE 6

TIME COURSE OJ:!1 PMB UPTAKE BY ERYTHROCYTES

·------

. norma 1 erythrocytes (.) .d ~ 3 'en Q.) -0 • E • U) 2 1 0

)(

Q.) ~ 0 +- CL :::::J

Cil 0 ~ 0 10 20 30 40 50 60 Cl.. ti me rn minutes

Experiments conducted at 37°C. Concentration of PMB in the suspension: 3 ~· moles/ml. RBC. The triplicate circles represent three different patients. ..f03·..J_,

FIGURE 7

TIME COURSE OF PMB UPTAKE BJ:: ERYTHROCYTES IN ACQUIRED HEMOLYTIC ANEMIA

--~--~ ~

acquired he mo lyt ic anemia . 0 .d ~ 3 (/) 'Q,) -0 E U) 2 1 0

)(

Q,) ..::.:; 0 +-a. = normal ;:::, - ... = patient al 0 ::E 0 10 20 30 40 50 60 a. . ti me 1n minutes

Solid line represents the PMB uptake by normal erythrocytes. Dotted line represents the course of PMB uptake by erythro­ cytes in a patient with Coombs positive auto-immune nemolytic anemia. Experiments conducted at 37°C. Concentration of PMB is 3 ~. moles/ml. RBC. FIGURE 8

TIME COURSE OF PMB UPTAKE BY ERYTHROCYTES FROM PATIENT WITH CONGENITAL SPHER;...:O:...:C-=.YT..::....:.;OS::..:I::::..;;S;;...______

. conge n ito 1 spherocytosi s (.) ..d ~

' en 3 Q) -0 E U) 2 1 0

)(

Q) ..:111:: :: normal ...... 0 - a. •••• = patient ::J = 0~----~----T-----~----~----r---~ 0 10 20 30 40 50 60 . ti me 1n minutes

Solid line represents the PMB uptake by normal erythrocytes. Dotted line wi th solid circ les ( • ) represents the time course of PMB uptake by erythrocytes in a patient with congenital spherocytosis. The duplicate black circles represent the values before and after splenectomy. Dotted line with empty circles (o) depicts the time course of PMB uptake by the red cells of the patient 1 s mother. Experi­ menta conducted at 37°C. Concentration of PMB: 3 ~· moles/ ml. RBC. 1.. 25

FIGURE 9

ABSORBANCY OF RED CELL HEMOLYSATE DILUTIONS AT VARIOUS WAVE LENGTHS

~ 1.2 1 5. E

0 1.0

v % . 8 1 o . Ci) m -0 3 . .6 IQ 0 ~ 0 0 . 4 5 .

"+- 0 .2 2.5

(/) 0 -c ::::s 0 2 3 units of 0.0. at 25 7 rn JU.

Red cell hernolysate is diluted in phosphate buffer. The absorbancy of a given dilution at 257 rn~. is plotted against sarne at 410 rn~. The ordinate at the right-hand side shows the approximate hernoglobin concentrations of the sarnples as determined by the rnodified benzidine rnethod of Crosby (42). :t26

FIGURE 10

LOSS OF POTASSIUM FROM RED CELLS INCUBATED WITH VARIOUS CONCENTRATIONS OF PMB FOR 30 MINUTES AT 37°C.

. incub. time 30 mins () .ci .... 60 0 50 0 40 '(;') Q) 30 -0 E 20 ~ (/) (/) 10 .._. = K+ loss mean 0 Il range + 0 ~ 2 3 4 5 6 7 8 9 10· PM B ,..u moles 1 ml. r.b.c.

The shaded area representa the range of K+ 1oss obtained by 10 or more determinations; the so1id line is the mean value of the determinations. FIGURE 11

LOSS OF POTASSIUM FROM PMB-INCUBATED RED CELLS: COMPARISON OF PMB EFFECT ON NORMAL AND DEFECTIVE ERYTHROCYTES

<..! hemolysis ..c. 60 i '- hemolysis 0 50 0 ...... 40

V) -Q) 30 0 E 20 ~ = K+loss by normal r.b.c. s V) - Il 10 Il V) 0...0 = Il Coombs + r. b.c. s 0 Il Il Il G6PD defie. r.b.c.si + 0 ...... = ~ ' 0 2 3 4 5 6 7 8 9 10'

PMB ,u moles/ml. r.b.c.

Solid line represente the mean ~ loss ~rom normal erythro­ cytes. Dotted line with open circ les ( o ) represente the mean ~ loss ~rom the erythrocytes of two patients with Coombs positive auto-immune hemolytic anemia. Dotted line with black circles (•) depicts the mean ~ loss ~rom the PMB-incubated red cells o~ two G6PD-de~icient subjects. :1.28

FIGURE 12

THE MEAN AND RANGE OF ERYTHROCYTE MEMBRANE SULFHYDRYL CONTENT

IN VARIOUS CONDITIONS

__ - normal ronge -=mean values

CD ï l~lll-- -.-• • 0 • • • •. mmm · ___...... __ ::,:..:..;.,;,;..:_---- . . -• • 2 ••• • • •• • • • • :.p . • • --• • :r. • 1 u) • •

..c rï rï rï ,--, E r--:'"1...... Q) . <( E

Determinations are represented by black circles ( • ) • Solid lines represent the mean values in each group of patients. The limita of normal range are given by interrupted lines. Abbreviations: Norm. normal erythrocytes G6PD def. G6PD deficient red cells d.i.H.A. red cella from drug-induced hemolytic anemia A.H.A. red cells from other acquired hemolytic anemias H.S. red cells from hereditary spherocytosis P.A. red cells from pernicious anemia Fe-defie. A.- red cells from iron deficieney anemia Mise. red cells from miscellaneous hematological conditions Azot. red cells from azotemie patients BIBLIOGRAPHY

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