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WALSH, Michael Joseph, 1942- ACETALDEHYDE- INDUCED RELEASE AND ALTERS] METABOLISM OF CATECHOLAMINES: ITS ROLE IN ETHANOL ACTIONS AND THE DISULFIRAM- ALCOHOL REACTION.

The Ohio State University, Ph.D., 1969 Pharmacology

University Microfilms, Inc., Ann Arbor, Michigan ACETALDEHYDE-INDUCED RELEASE AND ALTERED METABOLISM OF CATECHOLAMINES: ITS ROLE IN ETHANOL ACTIONS AND THE DISULFIRAM - ALCOHOL REACTION

DISSER TATION

Presented in Partial Fulfil ment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohi d State University

Michael Joseph Walsh, B. S.

The Ohio State University 1969

Approved by JS-QjuJBrkAx> Adviser ' Department of Pharmacology ACKNOWLEDGMENTS

I want to express my gratitude first and formost, to my wife, Estelle Marilyn Walsh, for her constant encouragement, thought fulness, and assistance; and to dedicate this volume to Estelle in acknowledgment of her understanding and moral support, without which this research would not have been possible or purposeful. I certainly am indebted to Professor Edward B. Truitt, Jr., for his excellent advice a id training. He has remained a good friend, advisor, and teac ler throughout my graduate school years, I hope his qualities of unc erstanding and encouragement as well as his constructive critical ability have become permanently impressed on me. I am especially gra eful to Dr. Philip B. Hollander for his willingness to be my teac ler. His friendship and training and col laboration on certain experiments has certainly benefited my back ground as well as this dis sertation. Professor Bernard H. Marks has been a constant source of enlightment. As Professor,u and head of the Department of Pharmacol ogy, he occupies a unique position and interacts in a special capacity with all graduate students. His encouragement and sincere interest in my research and all graduate student problems is greatly appre ciated. Ultimately, my gratitude to the Department of Pharmacology and the graduate committee must be expressed. I will always appreciate the readiness of this department to accept me as a student transferring from another University. In retrospect, I sincerely feel that continuing my graduate studies in the Department of Pharmacology of The Ohio State University may have been the most important decision of my life. I am sure that this change has been most beneficial to my training as a pharmacologist, and hope my work has been mutually beneficial to the department. A graduate student is subject to many helping hands, there fore, I wish to acknowledge the frequent stimulating discussions, and constructive and helpful criticisms and comradships of the other faculty members: Drs. Daniel Couri, Rose Dagirmanjian, Saradindu Dutta, Robert Gardier, Harold Goldman and John Lindower, and especially of my fellow graduate students. The permission to perform experiments and utilize the facilities of B attelle Memorial Institute was greatly appreciated, For the effort s of Dr. Joseph Boatman, head of the Division of Physiology and Pharmacology, in my behalf, I am grateful. The efforts of Mrs. Joan Rotaru and Miss Joyce Berio for their excellent technical assistance in many phases of this research is appreciatively acknowledged. The helping hand of Daria Cverna, in both preparation of this manuscript and with any problems which arose during my graduate school days, will always be remembered. Also, the frequent sec retarial help of Mrs. Carol Jones and Miss Millie Flanagan of The Ohio State University, and Mrs. Nancy Handel and Miss Sharon Wells of Battelle Memorial Institute was greatly appreciated. Finally, I would like to acknowledge and express my thanks to the National Institutes of Health ( NIH) for monetary support in the form of fellow ships during my training in Pharmacology. VITA

Michael Joseph Walsh Born: York, Pennsylvania April 18, 1942 Graduate Calvert Hall College High School, Baltimore, Maryland June, I960 B. S. in Pharmacy, University of Maryland, School of Pharmacy June, 1965 Ph. D. The Ohio State University June, 1969 NIH Predoctoral Trainee, Pharmacology Training Grant GM 01417 October, 1966 to June, 1969 PUBLICATIONS AND PRESENTATIONS Walsh, M. J. and Truitt, E. B. , Jr. , : Mechanism of the Effect of Disulfiram on the Sympathomimetic Action of Acetaldehyde, Fed. Proc, 26; 293, ( 1967) Abstract.

Walsh, M. J. and Truitt, E. B. , Jr.; Confirmation of the Catechol amine Releasing Effects of Acetaldehyde and Ethanol by a

.new Technique Utilizing 7-H 3 -Norepinephrine, Presented to the Ohio Valley Section of the Society for Experimental Biology and Medicine, Lexington, Kentucky, November 17, 1967.

Truitt, E. B. , Jr. and Walsh, M. J. : Alcohol, Acetaldehyde and the Sympathetic Nervous System, Presented at a Symposium on Alcoholism - 1968, Battelle Memorial Institute, March 13, 1968.

Walsh, M. J. and Truitt, E. B., Jr.; Release of 7-H3-Norepinephrine in Plasma and Urine by Acetaldehyde and Ethanol in Cats and Rabbits, Federation Proceedings, 27; 601 ( 1968) Abstract. Truitt, E, B., Jr. and Walsh, M. J . : The Biochemical Pharmacology

of Alcoholism, Battelle Technical Review, 17; 8 , 3-8 August, 1968.

Walsh, M. J,, Truitt, E. B., Jr., and Hollander, P. B. : Adrener gic Effects of Acetaldehyde on Mechanical and Electrical Properties of Cardiac Muscle, Pharmacologist, 10: 183 ( 1968) Abstract.

Walsh, M. J., Truitt, E.B., J r., and Hollander, P. B. : Sympath omimetic Effects of Acetaldehyde on Electrical and Mechan ical Characteristics of the Isolated Left Atria of Guinea Pigs, J. Pharmacol. Exp. Ther. 167; 173-186, 1969.

Walsh, M. J., The Biochemical Pharmacology of Alcoholism: I. Acetaldehyde and Ethanol Metabolism and Effects on Lipids and Glycogenolysis, Presented at a Special Seminar at the University of Houston, Department of Pharmacology, School of Pharmacy, Houston, Texas, October 7, 1968.

Walsh, M. J .: The Biochemical Pharmacology of Alcoholism: II. Role of Acetaldehyde in the Disulfiram-Alcohol Reaction. Presented at a Spec,ial Seminar at The University of Houston, Department of Pharmacology, School of Pharmacy, Houston, Texas, October 14, 1968,

Walsh, M. J . : The Effects of Acetaldehyde on Contractility, Intra cellular Potentials and Catecholamine Release. Special Biochemistry Seminar, Department of Biochemistry, Bay lor University, Medical School, Houston, Texas, October 21, 1968 .

Truitt, E. B ,, Jr. and Walsh, M. J. : The Role of Acetaldehyde in the Actions of Alcohol, The Biology of Alcohol, Volume I, Biochemistry, Chapter 7, to be published in 1969.

Walsh, M. J. and Truitt, E. B ., Jr. : Acetaldehyde Mediation in the Mechanism of Ethanol-Induced Changes in Catecholamine Metabolism, Federation Proceedings, 28; 543 ( 1969) Abstract,

Walsh, M. J .: Catecholamine Release and Altered Metabolism of Norepinephrine by Acetaldehyde. Presented at a Special Pharmacology Seminar, Battelle Memorial Institute, Col umbus, Ohio, May 9, 1969. v FIELDS OF STUDY

Major Field; Pharmacology Autonomic Pharmacology B. H. M arks and R. Dagirmanjian

Bioelectric Potentials P. B. Hollander

Cardiovascular Pharmacol. S. Dutta

Neuroendocrine Pharmacol. H. Goldman

Drug Metabolism D. Couri, J. Merola and R. Ober

Toxicology D. Couri

Minor Fields;

Neuromuscular Physiology E. Bozler

Reproductive Physiology C. Barraclough ( Univ. of Maryland)

Neurochemistry J. Allen and R. McCluer

Biophysics L. Mullins and R. Sjodin ( Univ. of M aryland) TABLE OF CONTENTS

Page ACKNOWLEDGMENTS ii VITA iv LIST OF TABLES x LIST OF FIGURES xiii INTRODUCTION - - 1 General Concepts 1 Scope and Purpose of the Problem 5 Acetaldehyde Measurement and Levels 7 Interactions of Ethanol and Acetaldehyde with Amine Stores 15 Adrenergic Actions of Ethanol or Acetaldehyde 21 The Disulfiram-Alcohol Reaction 27 Effects of Ethanol on Biogenic Amine Metabolism 33 Interactions of Amines, Central Acting Drugs and Ethanol Hypnosis 38

METHODS 42 Gas Chromatographic Determination of Ethanol and Acetaldehyde 42 A. General Procedures 42 B. Analysis of Biological Fluids 45 C. Excretion in Expired Air 49 D. Tissue Concentrations 50

In Vitro Release of Norepinephrine 53 A. General Procedures for Preparation of Atria 53 B. Experimental Conditions '55 C, Potential and Tension M easurem ents 58 D, Pharmacological Agents 58

vii METHODS (Continued) Page

In Vivo Release of H3-Norepinephrine 60 A. General Preparations 60 B. Animal Equilibration 62 C. Injection, Sampling and Assay Procedure 63 D. Verification of Neuronal Uptake and Release 63

Autonomic Function in Whole Animals 64 Cl4-Norepinephrine and Metabolites 67 A. Administration of C14-Norepinephrine __ and Sample Collection 67

B. Analysis Interval 6 8 C. Treatment Schedules 70 D. General Procedures 72 E. Determination of C14-Norepinephrine and Normetanephrine 73 F. Determination of Deaminated C14- NE Metabolites 75 G. Identification of C14-Norepinephrine and Metabolites. 78

Evaluation of Central Nervous System Effects 80

RESULTS 82

Ethanol and Acetaldehyde Levels 82 A. In Vitro Production in Whole Blood 82 B. Human Filtrate Levels 82 C. Blood and Tissue Concentrations in Rats 84 D. Urine and Expired Air 96

Experiments with the Isolated Guinea Pig Left Atria 105 A. Controls 105 B. Effects of Norepinephrine (N E)and Tyramine 105 C. Mechanical Effects of Acetaldehyde 108 D. Electrical Effects of Acetaldehyde 115 E. Effects of Reserpine 115 F. Propranolol Blockade 118 H3-Norepinephrine Release and Blood Pressure Measurements in Whole Animals 121 A. Plasma Tritium in Cats 121 B. Blood Levels of Ethanol and acetaldehyde 124

viii RESULTS (Continued) Page

C. Plasma Tritium in Rabbits 127 D. Blood Pressure Responses to Acetaldehyde 131 E. Interactions oMDisulfiram and Reserpine 139 F. Vasodepressor Activity of Acetaldehyde 148 C14-Norepinephrine Metabolism and CNS Actions of Ethanol and Acetaldehyde 150 A. Excretion of Total Radioactivity 150

B. Urinary Metabolites of Norepinephrine-C 14 153 C. Effect of Various Drug Treatment on the Metabolism of C14-Norepinephrine 155 D. Correlation with Blood Levels of Acetaldehyde . 161 E. Dose-Response Effects of Ethanol and Acet aldehyde on the Central Nervous System 164 F. Effect of Norepinephrine and its Metabolites on Ethanol Hypnosis 170

DISCUSSION 175 SUMMARY 209 BIBLIOGRAPHY 212

ix LIST OF TABLES

Table Page

1 Literature Survey Comparing Blood Levels of Acetaldehyde (pg/ml) in Various Species 13

2 Volatile Substances and their Retention Times as Determined by Gas Chromatography 44

3 Recovery Data for Acetaldehyde and Ethanol in Rat Blood 47

4 Recovery Data for Acetaldehyde in Plasma and Protein-free Filtrates Prepared from Human Whole Blood 48

5 Acetaldehyde and Ethanol Air Standards as Determined in a Glass Metabolism Chamber at Various Times 51

6 Comparison of Saline Standards and Tissue Standards 54

7 Experimental Conditions for Experiments with Guinea Pig Left Atria 57

8 Thin-layer Chromatography of Norepinephrine and Metabolites 79

9 In Vitro Production of Acetaldehyde in Blood of Various Species by Ethanol 83

10 Acetaldehyde and Ethanol Blood Levels in Untreated and Disulfiram Pretreated Rats 86

11 Concentration of Ethanol and Acetaldehyde in Tissues of Rats after Ethanol 88

12 Concentration of Ethanol and Acetaldehyde in Tissues from Disulfiram Pretreated Rats after Ethanol 90

x Table Page

13 In Vitro Production of Acetaldehyde following the Addition of Precipitated Rat Tissue Homogenates 91

14 - Effect of Various Procedures on the Production of Acetaldehyde in Rat Tissue Homogenates 93

15 Effect of Equilibration Time and Ethanol Concen tration on the Production of Acetaldehyde in Rat Tissue Homogenates 95

1 6 Effect of Various Agents on the Production of Acet aldehyde in Rat Tissue Homogenates 97

17 Comparison of Acetaldehyde Levels in Blood,Urine, Expired Air and within a Subcutaneous Air Pocket 98

18 Comparison of Acetaldehyde and Ethanol Levels in Blood, Urine and Expired Air after Ethanol in Un treated and Disulfiram Pretreated Rats 99

19 Control Values for the Various Parameters of the Isolated Guinea Pig Left Atrium 106

20 Effect of Norepinephrine on Various Parameters of the Isolated Guinea Pig Left Atrium 107

21 Effect of Tyramine on Various Parameters of the Isolated Guinea Pig Left Atrium 110

22 Effect of Various Concentrations of Acetaldehyde on Electrical Parameters of the Isolated Guinea Pig Left Atrium 116

23 Potentiating Effect of Various Drugs on the Pressor Response to i, v, Acetaldehyde in Rabbits 133

24 Comparison of the Sympathomimetic Activity on Blood Pressure of Tyramine and Acetaldehyde in Untreated Animals 140

25 Ratios of Blood P ressu re Responses and Scale Ratings of Nictitating Membrane Contractions after Tyramine and Acetaldehyde in Rabbits 143

xi Table Page

2 6 Ratios of Blood Pressure Responses and Scale Ratings of Nictitating Membrane Contractions after Tyramine and Acetaldehyde in Female Cats 146

27 Comparison of the Sympathomimetic Activity on Blood Pressure of Tyramine and Acetaldehyde in Disulfiram and Reserpine Pretreated Animals 151

28 Metabolic Fate of C14-Norepinephrine in Rats 154

29 Metabolic Fate of Bound CI4-Norepinephrine in Con trol Rats 156

30 Effect of Drugs on the Urinary Excretion of C14- Norepinephrine and its Metabolites 157

31 Effect of Treatments on the Relative Amounts of VMA and MHPG after Administration of C14- Norepinephrine in Rats 163

32 Acetaldehyde and Ethanol Blood Levels after Injection of Acetaldehyde in Rats 165

33 Relationship of Dose to Intoxicating and Toxic Effects of Acetaldehyde in Mice 168

34 Norepinephrine Potentiation of Ethanol Hypnosis in Mice 171

35 Effect of Metabolites of Norepinephrine on the Duration of Ethanol Induced Hypnosis in Mice 172

36 Effect of Monoamine Oxidase Inhibitors on Ethanol Hypnosis with or without Norepinephrine 173

■ * x . u LIST OF FIGURES

Figure Page

1 Metabolism of ethyl alcohol 2

2 Photograph of the physical arrangement of the instrumentation used for experiments with guinea pig left atria 56

3 Schematic diagram of parameters measured in guinea pig left atria 59

4 Concentration-response curve for acetaldehyde on developed tension in untreated and reserpine treated atria 61

5 Effect of bilateral carotid occlusion on the dis- . apperanee rate of H3-NE in plasma 65

6 Cumulative excretion of C 14 in the urine of rats following i. v. injection of C14-norepinephrine 69

7 Rate of urinary C 1 4 excretion following administration of C14-norepinephrine intravenously to rats 71

8 Schematic diagram showing the method for isolation of C14-norepinephrine and CI4-normetanephrine in urine 74

9 Schematic diagram showing the method for isolation of C14-deaminated metabolites of norepinephrine in urine 76

10 Blood levels of ethanol and acetaldehyde from four human subjects given ethanol 85

11 Average values for the accumulation and disappearance of acetaldehyde in expired air after administration of acetaldehyde 101

xiii Figure- Page

12 Average values for the accumulation and disappear ance of ethanol in expired air after ethanol adminis tration 102

13 Average values for the accumulation and disappear ance of ethanol and acetaldehyde in expired air after disulfiram and ethanol administration 104

14 Exact superimposed tracings showing the effects of norepinephrine on left atria from an untreated and reserpine pretreated guinea pig 109

15 Exact superimposed tracings showing the effects of tyramine on left atria from an untreated and reserpine pretreated guinea pig 111

16 Concentration-response curve for acetaldehyde on the contractile parameters of the isolated left atria from untreated and reserpine prctreated guinea pigs 112

17 Concentration-response curve for acetaldehyde on the developed tension in untreated and reserpine pre treated guinea pigs 114

18 Exact superimposed tracings showing the effects of acetaldehyde in various concentrations on the left atria of untreated guinea pigs 117

19 Exact superimposed tracings showing the effects of acetaldehyde in various concentrations on the left atria of reserpine pretreated guinea pigs 119

20 Comparison of the concentration-response curve of acetaldehyde in the presence and absence of propran olol 120

21 Double reciprocal plot showing apparent non-compet itive inhibition of acetaldehyde effects by propranolol 122

22 Effect of ethanol or saline infusions on the plasma tritium level after injections of H3-norepinephrine in cats 123

xiv Figure Page

23 Effect of i.v. acetaldehyde and saline injections on the plasma tritium level after injection of H3-norepinephrine in cats. 125

24 Blood levels of acetaldehyde in cats and rabbits after i, v. injection of acetaldehyde 126

25 Blood levels of acetaldehyde in rabbits after i.v. infusion of ethanol with or without disulfiram pre treatment 128

26 Blood levels of ethanol in control rabbits after i.v. infusion of ethanol 129

27 Effect of i.v. acetaldehyde and saline on the plasma tritium level after injection of H3-norepinephrine in rabbits 130

28 Effects of ethanol infusion on the plasma tritium level after the injection of H3-norepinephrine in untreated and disulfiram pretreated rabbits 132

29 Summary of the effect of saline, acetaldehyde or ethanol alone or in the presence of disulfiram on the H3-NE levels in rabbits 133

30 Effect of various treatments on the urinary levels of H3-norepinephrine in rabbits 134

31 Blood pressure tracings showing the effect of various doses of acetaldehyde on the blood pressure of an un treated rabbit 136

32 Blocking effect of phentolamine on the vasopressor action of acetaldehyde 137

33 Nictitating membrane contractions, blood pressure responses and respiration in control rabbits to i.v, injections of tyramine or acetaldehyde before and after drug infusions 142

xv Figure Page 34 Nictitating membrane contractions, blood pressure responses ancl respiration in reserpine pretreated rabbits to i. v. injection of tyramine or acetaldehyde before and after drug infusions 145

35 Nictitating membrane contractions, blood pressure responses and respiration in disulfiram pretreated rabbits to i. v. injections of tyramine or acetaldehyde before and after drug infusions 147

36 Effect of propranolol, atropine, cocaine and tripelennamine on the vasodepressor response to acet aldehyde in disulfiram pretreated rabbits 149

37 Metabolic pathway for the metabolism of norepinephrine 152

38 Effect of various drug treatments on the proportion of the amines recovered in the urine 158

39 Effect of various drug treatments on the proportion of the deaminated metabolites of Cw-norepinephrine recovered in the urine 160

40 Effect of various drug treatm ents on the proportion of

O-methylated deaminated metabolites of C 1 4 -norepinephrine recovered in the urine 162

41 Dose-response relationship for the hypnotic and toxic effects of acetaldehyde and ethanol 166

xvi INTRODUCTION

General Concepts

The disease concept of alcohol addiction (Jellinek, I 9 6 0 ) has been widely accepted in recent years. However, the complexity of cultural, psychological, biochemical, physiological and social factors contributing to its manifestation have made cause and effect relationships difficult to prove. Similarly, the myriad of pharm acologic and pathologic effects of alcohol makes it difficult to correlate biochemical findings with a particular central nervous system response. A whole spectrum of mood alterations can be elicited by alcohol from hypnosis, sedation and depression to stim ulation, euphoria and even hallucinations.

Among the biochemical factors contributing to alcohol's action is the metabolic fate of ethanol and its metabolites. It is generally agreed that the hepatic metabolism of alcohol is by far the most sig nificant mechanism for the biotransformation and elimination of ingested ethanol. Kidney, heart ( Frazekas and Rengei, 1968), lung, diaphragm (Masoro et al., 1953 ), and brain (Sutherland et al., 1958, I960; Rasken and Sokoloff, 1968), have been demonstrated to possess a limited ethanol oxidizing capacity in vitro, however, the quantitative contribution of these tissues to the overall catabolism of ethanol in the intact animal is quite small. Similarly, the renal and pulmonary excretion of ethanol accounts for a relatively small per centage of the total eliminated.

There are several hepatic oxidative systems which have been shown to be involved in the metabolism of ethanol ( Westerfeld, 1955, 1961; von Wartburg and Papenberg, 1966) . However, most of the evidence implicates the pathway illustrated in Figure I as the pre- dominant hepatic system for the oxidation and utilization of ethanol (von Wartburg, 1966).

NAD

A-DH

NADH NAD

NADH

Citric Acid Cycle

Figure I: Metabolism of Ethyl Alcohol

Recently, Lieber and DeCarli ( 1968) , and Kalant (personal communication) , have demonstrated a microsomal ethanol oxidiz ing system (MEOS)in rat liver. The specific enzyme involved has not been well characterized except that it is definitely different from the alcohol dehydrogenase system. The enzyme has a pH op timum of 7.0 - 7.4 and utilizes NADPH as a cofactor in the biotrans formation of ethanol. In this system acetaldehyde (CH 3 CHO) is also the primary metabolic product of oxidation ( Lieber and DeCarli,

1 9 6 8 ; analysis performed in our laboratory) .

A biochemical alteration in the metabolism of ethanol has been repeatedly sought in the hope of finding a characte.ristic pattern in alcoholic drinkers. However, the preponderant conclusion is that no significant acceleration in the rate of alcohol metabolism occurs in habituated drinkers (Bernhard and Goldberg, 1935; Bogen, 1936; Elbel, 1938; Carpenter, 1940; Newman, 1941; Jacobsen, 1952a; Harger and Hulpieu, 1956; Himwich, 1956; Kalant, 1962; Mardones, 1963; Mendelson, 1968). Several recent studies have supported the claim that prolonged alcohol consumption can induce a significant increase in hepatic alcohol dehydrogenase activity in animals ( Dajani et al., 1963; McClearn et al., 1964; Mirone, 1965; Hawkins et al., 1966), and in man (Mendelson et a l., 1965; Iber et a l., 1969) . Yet other investigators have failed to observe this increase (von Wart burg and Rothlisbcrger, 19 6 1; Figueroa and Klotz, 1962a, 1962b, 1964; Greenberger et al., 1965). Induction of an increase in catalase activity has also been suggested to explain alcoholic toler ance, but the quantitative importance of this pathway remains doubt ful (Kalant, 1962; Fazekas et al., 1966; Bartlett, 1952).

The microsomal ethanol oxidizing system seems to be inducable after prolonged administration of ethanol to rats ( Lieber and DeCarli, 1968) . This inducability has also been offered as an explanation of alcoholic tolerance. These same workers (Ruben and Hutterer, 1968; Ruben and Lieber, 1968) have demonstrated a proliferation of the smooth endoplasmic reticulum by chronic alcohol treatment in rats and an induction of certain microsomal drug metabolizing enzymes in both man and the rat. This could explain the frequent tolerance of alcoholics to certain sedative and hypnotic drugs and the cross toler ance to alcohol. Also, as are other drug metabolizing systems, MEOS itself is inducable by phenobarbital or benzopyrene ( Roach et a l., 1969). But, the quantitative importance of this system to the overall metabolic fate of ingested alcohol remains to be evaluated. Estimations from in vitro data indicate that this microsomal oxidizing system'would account for only a small percentage of the total hepatic oxidation of ethanol. Until more data is obtained, the alcohol de hydrogenase pathway (Figure 1) is believed to be of primary importance.

As depicted above, the initial step involves dehydrogenation of ethanol to acetaldehyde. This reaction is catalyzed by the enzyme alcohol dehydrogenase (ADH), which utilizes nicotinamide adenine dinucleotide(NAD) as the hydrogen acceptor. Several studies (Westerfeld et al., 1943; Westerfeld, 1955; Jacobsen, 1952a) have demonstrated that the metabolism of acetaldehyde in vivo proceeds at a much greater rate than its formation from ethanol, indicating that the initial oxidative step is the rate-limiting reaction.

.. ' ' ' V I ■ The second stage in ethanol metabolism involves the simul taneous dehydrogcnation of acetaldehyde by aldehyde dehydrogenase (Ald-DH) and condensation with coenzyme A. Ald-DH also utilizes NAD as a hydrogen acceptor. Similarly, the major site of CH3CHO catabolism has been relegated to the liver (Lubin and Westerfeld, 1945) mainly in the mitochondrial and some in the soluble fraction (Erwin and Dietrich, 1966) . Although other oxidative systems have

been found to oxidize CH 3 CHO, they seem to be of relativery minor quantitative importance. Thus, the amounts of this proximate metab olite of ethanol that can be detected in biological samples will logically depend on the relative activities of the generating ( ADH) and degradative (Ald-DH) enzymes. Despite the fact that ADH is the rate- limiting factor, measurable quantities of CH3CHO do escape immediate

hepatic degradation with the production of CH 3 CHO concentration in blood and tissues.

Ultimately, acetyl coenzyme A (Acetyl CoA) undergoes inter mediate metabolism through the citric acid cycle. The terminal

production of C0 2 from ethanol accounts for 71 - 90^ of the amount administered ( Himwich, 1956) . Of course, small amounts of acetyl CoA are shunted into various pathways of intermediary metabolism. For example, acetyl CoA derived from ethanol-l-C 1 4 administration may appear as free fatty acids, triglycerides, cholesterol, ketone bodies or as labeled glycolytic intermediates.

Eventually 90 - 98 a/0 of the C1 4 is excreted as C14Oz (Casier and Polet, 1959).

Scope and Purpose of the Problem

Acetaldehyde as a pharmacologically active agent has been scarcely examined. Our interest in this two carbon aldehyde as a possible mediator of some of the effects of ethanol arise from the following considerations:

1. Acetaldehyde, the proximate metabolite of ethanol, can be measured in biological samples after ethanol ingestion. It is definitely a circulating metabolite which has pharmacologic actions of its own (MacLeod, 1950).

2. Unlike many drug metabolism processes ( Gillette, 1963) the oxidation of ethanol produces an inter mediate metabolite which has greater lipid sol ubility than the parent compound.

3. Aldehydes as a chemical class of compounds are highly reactive. The fact that acetaldehyde is en dogenously produced from ethanol would allow it to come in contact with varied biological material. The reactivity implies that it might interact chem ically and produce effects (Collins and Cohen, 1968) .

4. Aldehydes are notoriously toxic compounds. The toxic effects of methanol on the retina are due to its metabolic product, formaldehyde (Potts and Johnson, 1952). An analogous situation may occur with alcohol, that is, acetaldehyde might play a role in the toxicities produced by ethanol. 5. The tolerance to alcohol which is seen most strikingly in alcoholics may be due to an enzyme induction mechanism. The increased oxidation of ethanol may lead to increased levels of acetaldehyde. Increased levels of

CH3 CHO in tolerant individuals may be impor tant in the etiology of the disease or in the psychologic abberations and pathologic seque lae of alcoholism,

Therefore, acetaldehyde as an intermediate metabolite of ethanol oxidation is a reactive molecule with greater volatility and lipid solubility than its precursor. Yet, despite this reasoning, the importance of acetaldehyde in the actions of ethanol have been minimized in the past. This is so, primarily because of the relativ ely low blood levels produced and the lack of sensitive and specific assay procedures. This concept is based largely on very limited amounts of blood level data obtained by methods which have been frequently criticized (Duritz and Truitt, 1964; Lundquist, 1958; Wagner, 1957).

Yet, acetaldehyde has been recognized by some investigators as having activities which might be responsible for a number of actions in the acute and chronic syndromes produced by ethanol. Acetaldehyde has a potent hypnotic activity of its own (MacLeod, 1950). Additionally, it produces marked actions on the circulation, respiration and the metabolism of many tissues, as well as inducing nausea, vomiting and sweating (Himwich, 1956). Actions such as the selective interference with pyruvate maintenance of mitochondrial oxidative-phosphorylation (Truitt et al., 1956; Rehak and Truitt, 1958; Beer and Quastel, 1958a) , .the cardiovascular activation (Eade, 1959) and the release of biogenic amines from the adrenal gland (Akabane, 1965; Perman, 1958a) and the brain stem (Duritz and Truitt, 1966) can all be elicited by acetaldehyde alone. 7 This information has led to an investigation of the effects of acetaldehyde on several systems, with particular reference to the following:

1. Levels of acetaldehyde have been re-evaluated both for blood and tissues using a gas chromatographic assay.

2. Acetaldehyde has been demonstrated to be an indirect sympathomimetic and catecholamine releasing agent both in vivo and in vitro.

3. The alterations in biogenic amine metabolism have been demonstrated to be mediated by acetaldehyde. An action of ethanol which can be attributed solely to its metabolite.

4. The interaction of acetaldehyde with norepinephrine has prompted a more plausible explanation for the disulfiram-alcohol reaction.

5. The interaction with catecholamines could explain some observed pharmacologic effects of ethanol.

Acetaldehyde Measurement and Levels

Several methods have been described for the measurement of acetaldehyde. Some of the methods have been shown to be subject to problems of specificity. Similarly, because levels have been found to be low, the sensitivity of many of the methods is insuffic ient to give quantitatively reliable results. Methods of measurement fall into five general types of procedures: Colorimetric, photometric, radiometric, enzymatic, and gas chromatographic ( GLC ) .

The colorimetric method of Stotz ( 1943) has been the most widely used procedure for analysis of acetaldehyde. The analysis in volves a cumbersome special distillation setup, at least a 1 2 ml blood sample to yield three ml of distillate, and is subject to interference by several endogenous metabolites. Lester and Greenberg ( 1950) have modified the method to analyze 0 . 2 ml of blood, which involves the distillation of whole blood. Said and Fleita (1965) have publish ed a modification of this method which also allows analysis of smaller volumes but still requires a distillation process. The Stotz method (1943) involves vacuum distillation of acetaldehyde into a bisulfite trap and reaction with p-hydroxybiphenyl in the pre sence of copper sulfate. The method is sensitive to small quantities of acetaldehyde, such that levels as low as 0.25 pg/nl of blood can be detected, Stotz noted that distillation of lactate or pyruvate from acid precipitated samples yielded a color, but did not interfere if alkaline precipitation was used. Similarly, diacetyl, paraldehyde and formaldehyde seriously interfere with the determination. The interference from lactic acid may be due to conversion to acetalde hyde under acidic conditions has been used as an assay method for lactic acid (Conway, [1947], a microdiffusion method; and Savory and Kaplan [1966], a GLC method).

Burbridge, Hine and Schick ( 1950) have published an ultra violet spectrophotometric procedure for acetaldehyde which in volves measurement of acetaldehyde - semicarbazone absorption at 224 mp, The method involves the use of Conway cells for the m icro diffusion and trapping of acetaldehyde, which poses problems with evaporation and spillage of the sample into the semicarbazide well. The sensitivity is again quite good, and allows measurement of acetaldehyde as low as 0.2 pg/ml. It involves acidic precipitation of blood, and is therefore subject to the same criticism as before. Acetone also interferes with the method, which is a serious problem since acetone levels appear to be elevated after alcohol administration (Lester, 1962). Similarly, Lundquist ( 1958) stated that under the conditions of analysis, acetoacetate may be converted to acetone and produce interference. In 1958, Casier and Polet utilized a radiochemical method for the analysis of C14-acetaldehyde which they claimed could be separated from radioactive ethanol. These investigators failed to find increases in CI4-acetaldehyde after ethanol in disulfiram treated mice. This has led them to conclude that acetaldehyde could not be responsible for the symptoms of the disulfiram-alcohol react ion. It is not clear whether a sufficient dose of ethanol was given to cause elevation of acetaldehyde, but if so, then the validity of the method for acetaldehyde determination is in serious question. Un doubtedly, acetaldehyde levels are elevated in the presence of disulfiram (See Results), and failure to find acetaldehyde under these conditions makes it doubtful that their separation procedures are adequate.

Numerous enzymatic determinations for acetaldehyde have been published ( Holzer et al., 1955; as modified by Brahn-Vogel- sanger and Wagner, 1957; Racker, 1957; Lundquist, 1958). The methods involve the oxidation of NADH and simultaneous reduction of acetaldehyde by yeast alcohol dehydrogenase or horse liver alcohol dehydrogenase. The interference by other aldehydes, contamination of the enzyme preparations with other dehydrogeneses, and chances for volatilization of the substrate are matters for concern. Further more, the values for acetaldehyde after ethanol that were reported by Lundquist ( 1958) approached the limit of detection of his method.

The usefulness of gas-liquid chromatography for the determin ation of blood alcohol levels is attested to by the numerous reports describing its sensitivity and reproducibility (Harger et al., 1950; Goldbaum et al., 1964; Bassette and Glendenning, 1968; reviewed by Savory et al., 1968). LeBlanc ( 1968) has recently published a micromethod for ethanol determination, but does not differ much from other methods. The method of Duritz and Truitt ( 1964), which 10 was developed in our laboratory, has been slightly modified and used for the determination of ethanol and acetaldehyde in this research. T he method offers several advantages. The ability to determine both ethanol and acetaldehyde simultaneously is the primary advan tage. This method utilizes alkaline precipitation to avoid some of the aforementioned problems, and vapor phase analysis to maintain column-life. The method is rapid, and allows duplicate analysis of samples every four minutes. In the analysis of acetaldehyde there is no interference from other aldehydes or alcohols normally present. Therefore, the technique is highly specific (See Methods) . The use of certain modifications in sample preparation, has allowed the accurate analysis of levels as low as 25 ng/ml. This represents a

1 0 -fold increase in sensitivity from previously available methods. Several methods have been published which also separate ethanol from acetaldehyde, but the quantitative data presented pertains only to ethanol determinations. A method similar to ours has been pro posed by Freund and O'Hollaren ( 1965) for determination of ethanol and acetaldehyde in alveolar air. For human studies, large volumes of air can be used to improve the sensitivity, which is necessary to detect alveolar concentrations. Recently, Roach and Creaven ( 1968) have described a "micro-method" for the determination of ethanol and acetaldehyde. The method offers no advantages over the one we have used. They have criticized our technique ( Duritz and Truitt, 1964), claiming that there is doubt whether acetone is separated from acet aldehyde, a point which is adequately demonstrated in the original publication. Acetone comes midway between acetaldehyde and ethanol. Even though they are using quite small samples, these are of no value for acetaldehyde determination since their stated sensitivity is only as low as 4 Therefore, our method appears to be the most sen sitive one available, which has proven to be necessary in the analysis of human blood levels (See Results). In the course of preparing whole blood standards with ethanol, it was found that the level of acetaldehyde in blood increased with time of equilibration in the water bath (Truitt, 1969) • This acet aldehyde could not have been enzymatically produced because of the alkaline precipitation of the blood and the high incubation temper ature. Similarly, inhibitors of alcohol dehydrogenase and catalase failed to prevent this production. This release of bound CH3CHO in blood has been ignored for several decades. Barker ( 1941) noted that addition of ethanol or treatment of blood with copper-lime reagent of Van Slyke and Salkowski produced large amounts of acet aldehyde (20 to 100 jig/4nl) in blood of various species. This artifactual production of acetaldehyde was confirmed by Stotz ( 1943), who used a tungstic acid treatment of a copper-lime blood filtrate and pro duced levels as high as 900 jjg/ml. Similarly, he found that treatm ent of crystalline horse hemoglobin wifli hot copper-lime yielded levels of 30-40 jjg/g.

MacLeod ( 1950) was the first to rediscover that appreciable amounts of acetaldehyde could be produced when ethanol was added to whole blood after withdrawal. He failed to find this phenomenon if plasma was used. Levels of acetaldehyde from 3.1 - 6.1 |Jg/ml were obtained when ethanol was added to deproteinized blood, Lionetti and co-workers ( 1964) further studied the acetaldehyde production in red cell ghosts and concluded that acetaldehyde was a product of deoxynucleoside metabolism. Specifically, the simultaneous production of triosphosphates and acetaldehyde from deoxyribose-5-P0 4 led him to postulate the presence of deoxyribosephosphate aldolase in erythro cytes. Whether the source of acetaldehyde after ethanol or copper- lime reagent addition to blood is the same as with pentosephosphate addition is not clear. 12 Further investigations of this phenomenon ( Truitt, 1969) have revealed that blood precipitation with ZnS0 4 -Ba( OH) 2 or other protein precipitants caused a release of acetaldehyde when ethanol was added to blood. The amount of acetaldehyde produced was pro portional to the temperature, time of equilibration, and the log of the ethanol concentration added to the blood. This release of "bound acetaldehyde" occured mainly in the erythrocyte fraction and to a much lesser degree in plasma. It coincided with the degree of hemo lysis, and therefore, the possibility of protein (hemoglobin) as the source was again suggested. It did not seem to be formed by non- enzymatic conversion of ethanol, since experiments with tri-deuter- ated ethanol did not yield tri-deuterium labeled acetaldehyde by mass spectral analysis. Formation of a protein-free filtrate of blood before heating seemed to eliminate this artifact, which constitutes a large error in reporting acetaldehyde levels in ethanol containing blood. This artifact occured both with the Burbridge technique and the GLC method. The colormetric procedure of Stotz is seemingly unaffected since the blood precipitate is separated before raising the temperature in distillation. However, this method is still subject to the shortcomings previously described.

Table 1 is a compilation of an extensive literature survey of all

CH3 CHO blood levels after ethanol in various species which have been previously reported. Human levels range from 0.3 - 30 |jg/4nl, which represents a hundred-fold range. The highest levels were obtained by non-specific chemical methods and the Burbridge procedure (3.61- 30 pg/4nl), Analysis by the Stotz procedure yielded intermediate levels ( 1.0 - 2.4 pg/tnl), while enzymatic analysis gave very low levels ( 0.3 yg/inl). Similar trends are also seen in examining other species. Levels obtained in the rat and mouse are relatively low and are in agreement with the fact that these species are not as suscept ible to this "artifactual release" (See Results) . Because of the 13

TABLE 1. LITERATURE SURVEY COMPARING BLOOD LEVELS OF ACETALDEHYDE GigM ) VARIOUS SPECIES

CK3CHO After CHjCHO A fter Drug Alcohol and Seeetea Alcohol Treatment Frotreatment Reference

Human 16.06 ■" \ Stepp, 1920 . -4-H 5- TETD 5.64 - 8.0 Hi Id and Jacobsen, 1948b 6.9 - 9.6 TBTD 15.4 - 48.4 Furtado, Chlchorro Amaral, 1951 TETD -5 .0 - 9.0 L ester, Conway, Mann, 1952 3.61 TETD 10.0 Dine, Shick, Margola, Burbridge, Simons, 1952b -- Clucoae 1.9 - 6.6 Varela, Pcnna, Alcalno, Johnson, Mardones, 1953 5.9 - 17.0 TETD 6.4 - 25.1 Raby, 1954 30.0 -- — Forster, 1956 0.3 ---- Lundquist and Wolthere, 1958 1.0 — Akabane, 1960 — TETD 8.4 Kargcr and Forney, 1965 13.75 — -- Kulpleu, Clark, Onyett, 1965 2.5 TETD 5.2 In Krantz and Carr, 1965, No reference O.I - 0.56 -- Welsh and T ru itt, unpublished data Rabbit 15.0 — -- Supnlewskl, 1927 . 1.0 - 1.7 TETD 12.0 - 26.0 Larsen, 1948 0.8 - 1.2 TETD 15.0 * 24.5 tlald, Jacobsen, Larsen, 1949 3.0 - 5.0 1ETDrt.l 20.0 • 40.0 Carratall, 1950 3.3 - 6.8 TMTD 9.5 - 28.8 Klrchhtln, 1951 49.0 - 51.7 TETD 65.0 • 73.0 Fartado, Chlchorro, Amaral, 1951 2.7 TETD 9.1 - 9.5 Skelton, KcConkcy, Grant, 1952 H *1 M 11 * VP TETD + Cyanatold 12.8 - 29.0 1.9 - 3.2 TETD 9.0 - 15.4 Akabano and Ikool, 1958 1 . 6 - 3.2 'WTD 20.0 - 25.0 it 11 n 3.4 TETD 15.4 T ru itt and D urltz, 1967 1.2 - 2.0 TETD 7.0 - 9.0 Welsh and T ru itt, 1968 Png 7.5 — — Supnlewskl, 1927 — Pyruvate 2.0 Westerfeld, Stotz, Berg, 1943 1 .0 Animal Charcoal 5.5 Asmusscn, HaId, Larsen, 1948 2 . 0 - 3.6 TETD 6.0 Nowman, 1950 0.25 TETD 1.37 Loomis, 1950 1 .8 TETD 6.3 Newman and Petzold, 1951

Cat .. Chlorpropamide 7.0 - 13.4 T ru itt, D urltz, Morgan, Prouty, 1962 Rat 0.9 - 6 . 2 TETD 3.1 - 18.8 MacLeod, 1950 1 .3 TETD 19.5 L ester and Greenberg, 1950 1.2 Cyanoratd 35.0 Warson, Ferguson, 1955 1 .5 TETD 1 . 0 - 3.0 Wa'gner, 1957 3 . 3 TETD 4.5 Ridge, 1963 4 . 5 TETD 22.4 Durltz and T ru itt, 1966 7 . 0 - 2 2 .0 NAD 5.0 - 32.0 Majchrowlcz, Bcrcaw, Cole, Gregory, 1967 1 . 2 - 2.61 TETD 14.61 - 19.6 Walsh and T ru itt, 1969 House 2 .2 B - 3.9 Cyannmld 2 3 .0 ’ Warson and Ferguson, 1955 0.97 TETD 1.84 Schleslnger, Kaklkana, and Bennett, 1966

(a) TETD ” Tetraethylthiuram Disulfide {Antabuse,®) (b) TKTD ■ Tetramethylthlursm Disulfide 14 knowledge of methodological problems in acetaldehyde measurement, part of the purpose of this dissertation was to reevaluate the levels of acetaldehyde which occur after ethanol in several species; human, rabbit and rat.

Investigation of the levels of acetaldehyde in tissues have only been performed in a few laboratories. Kiessling ( 1962a) examined the levels of acetaldehyde in the brain after injection of a 30 °/0 alcohol solution at a dose range from 1 to 3 g/Kg. The acetalde hyde was determined by the method of Stotz, and the levels were from 4.2 - 7.7 (Jg/gm of wet tissue. Interest in CH3CHO levels in brain arose because even with the use of potassium or electrically stimulated brain slices, four times the human threshold level of ethanol was needed to demonstrate any effect on nervous tissue (Ghosh and Quastel, 1954; Sutherland et al. , 1956; Fischer, 1957; Beer and Quastel, 1958b; Wallgren and Kulonen, i960). Even fatal alcohol concentrations failed to produce depressant actions on brain mitochondrial respiration (Wolpert et al., 1956). Using acetaldehyde concentrations from 4.4 pg/4nl, m any investigators have confirm ed the depression of oxidative phosphorylation produced by acetaldehyde (Kiessling, 1962a, 1963, Beer and Quastel, 1958a), which was first reported by our laboratory ( Truitt et al., 1956; Rehak and Truitt, 1958) . Acetaldehyde is much more potent than ethanol in this respect. Kiessling ( 1962b) further studied the sensitivity of various parts of rat brain to acetaldehyde. Cerebellar mitochondria were most affected, and interestingly the cerebellum had the highest con centration of acetaldehyde ( 14.8 (Jg/fem) 60 minutes after ethanol in jection. In these experiments, whole brain concentrations averaged 5*1 Mg/gni of tissue, whereas the cerebrum contained 2.7 Hg/gm and the mesencephalon 7.2 jig/gm. Duritz and Truitt ( 1966) measured whole rat brain concentrations of acetaldehyde after administration of 15 a 2 gm/Kg i. p. dose of ethanol after disulfiram pretreatment. The

levels of acetaldehyde peaked at 9 0 minutes after ethanol adminis tration and reached 9.51 ± 0,43 (Jg/nl ± S. E. By rough calculation of their data this level is approximately 28.5 pg/gm of tissue. Ridge ( 1963) found levels of about 3 }Jg/gm in rat brain after ethanol and up to 4,4 fig/^m after disulfiram and ethanol.

Interactions of Ethanol and Acetaldehyde with Amine Stores

Klingman and Goodall ( 1957) first reported that acute sublethal intoxication in intact unanesthetized dogs was accompanied by marked increases in the urinary excretion of epinephrine and norepinephrine. In contrast to intact dogs, they demonstrated that bilateral adrenal ectomy prevented the increased excretion of epinephrine. However, the urinary excretion of norepinephrine was still elevated after adrenalectomy. They further showed a significant decrease in the adrenaline content of the adrenal. Similar confirmatory reports on these amines in man have appeared { Abelin et al. , 1958; Giacobini et al., I960) . Perman ( 1958b) also demonstrated significant in creases in the urinary excretion of adrenaline after ingestion of moder ate doses of alcohol in man. In 65 subjects, there was a mean differ ence in the excretion of adrenaline in control and alcohol treated subjects of 3.2 ng/tnin., corresponding to a 51 increase above normal. Furthermore, he failed to find a significant enhancement of norepinephrine excretion with alcohol. Conversely, Goddard { 1958) studied subjects who had taken a flight in a glider plane, and measured urinary adrenaline and noradrenaline. The anxiety was demonstrated by a significant increase in the excretion of norepinephrine in the urine. Subjects were given 50 ml of brandy before the flight, failed to produce an increased norepinephrine excretion. These experi ments demonstrated a depression of autonomic nerve function as a result of some central anti-anxiety ( sedative) effect of ethanol, before 16 the amine releasing activity of this agent comes into play. These experiments imply that the sedative actions of ethanol are quite separated from the amine releasing activity of alcohol.

Further evidence of an effect of ethanol on adrenal medullary secretion was provided by Perman ( i960) using cats. Sampling left adrenal venous blood, he showed that the amounts of catecholamines increased when alcohol in doses of 0.6 g/Kg and higher were infused. There were much larger increases in the secretion of noradrenaline than with adrenaline in this species. Again, he demonstrated ( Perman, 1961a) in man that low doses of alcohol augmented the ex cretion of adrenaline during the two hours following ingestion. Fur ther studies (Perman, 1961b) compared the excretion of catechol amines after alcohol or hydration. The augmentation of norepineph rine excretion seemed to be equilvalent with the degree of diuresis produced, whereas a diuretic effect did not explain the increases in adrenaline excretion.

Interesting studies by von Wartburg and Aebi ( 1961) demon strated that treatment of rats with a single dose of alcohol (0,15 m l/

1 0 0 gm weight) produced marked increases in the excretion of epi nephrine and norepinephrine over 24 hours. However, a single challenging dose of ethanol to rats previously treated with ethanol (10 °/a solution to drink) for 50 to 200 days produced only small in creases in adrenaline and noradrenaline that were significantly lower than those observed in rats given only single doses. That is, the response to a challenging dose of ethanol appeared to be related to previous long term ingestion of ethanol, a tolerance seemed to develop, Masse et al ( 1961) also demonstrated an increase in total catecholamine excretion in rats. Catecholamines increased from 1.56 pg to 2.71 pg per 24 hours when rats drank a 1'/ alcohol solution ad lib compared to plain water. 17 Carlsson and Haggendal ( 1967) investigated plasma levels of norepinephrine after alcohol withdrawal. When alcohol is with drawn in chronic alcoholics, excitatory symptoms develop such as tremor, nervous tension, hallucinations, increased heart rate, and dilated pupils. These investigators felt that some of these abstinence symptoms might be due to increased sympathetic activity, and mea sured plasma levels as a means of getting more direct information about the activity of adrenergic structures than would urinary levels. These experiments demonstrated increases in plasma noradrenaline 13-24 hours after the last alcohol consumption in 36 male alcoholics. Recently, several broad studies in humans have been reported con cerning excretion of the various catecholamines and their metabolites in human subjects, Anton ( 1965) studied the effect of ethanol admin istration realizing that changes obtained "might well be due to a metabolite rather than alcohol itself". His results confirmed that moderate doses of ethanol result in increases in urinary catechol amines in man. In contrast to others however, ( Abelin et al., 1968; Perman, 1958b, 1961a)he observed a greater increment in the nor epinephrine fraction than epinephrine. This extensive study showed a significant increase in the urinary excretion of norepinephrine, normetanephrine, and metanephrine after ethanol. Likewise, ethanol induced an increase in dopamine excretion, and a decreased elimin ation of 5-hydroxyindoleacetic acid. Similar studies were performed by Schenker et al ( 1967) using alcoholic subjects, Ethanol produced significant increases in the excretion of tryptamine and epinephrine at 1, 2 and 4 hours after drinking compared to after water intake in these subjects. Norepinephrine and dopamine excretion, however, for these time periods were not significantly altered by alcohol in gestion. The most current report (Mendelson, Ogata and Mello, 1969), illustrated significant increases in norepinephrine, epinephrine, normetanephrine and metanephrine during alcohol ingestion, with dose- 18 response relationships to blood alcohol levels in alcoholic patients. In this study, subjects who experienced withdrawal symptoms following cessation of drinking continued to show enhanced catechol amine levels, while those without symptoms showed urinary catechol amine levels which were not significantly different from pre-alcohol baseline levels.

One provocative study by Akabane et al ( 1965), investigated the effect of acetaldehyde on the adrenal medullary secretion using perfused cat adrenals. These workers were able to show that per fusion with acetaldehyde intensified the secretion of both epinephrine and norepinephrine from the adrenal medulla. This action of acetaldehyde was not blocked by hexamethonium or atropine, and seemed therefore to be a direct effect on the medulla. Unlike the effect of acetylcholine in producing medullary cell secretion (Douglas and Rubin, 1961), acetaldehyde still produced significant increases in catecholamine output in preparations perfused with calcium-free Locke's solution. Nakanishi et al ( 1967), in Akabane's laboratory, investigated the effect of ethanol on the perfused cat adrenal. Ethanol in doses ranging from 10 - 100 mg injected into the perfusion circuit induced no increase in output of catecholamines from the adrenal medulla. These investigators felt that elevated urinary ex cretion of catecholamines after administration of ethanol reported by others could not be due to a direct action, and proposed that it was due to acetaldehyde.

Ethanol has been shown to produce effects on other amines besides catecholamines. The excretion of tryptamine is markedly enhanced by ethanol in both normal and alcoholic subjects (Schenker et al., 1967) . Westerfeld and Schulman ( 1959) reported a release of serotonin by ethanol, exhibited as a 40'/ decrease in serotonin con tent of intestine after ingestion of ethanol. Ethanol has been shown to 19 increase acetylcholine content of rat brain by Berry and Stotz { 1954, 1956) . This effect is attributed to inhibition of the release of acetylcholine from guinea pig and rat cerebral cortex slices (Kalant et al., 1967; Kalant and Grose, 1967). These authors have also shown that the acquisition of increased tolerance to eth anol in rats, as a result of chronic daily administration of a mod erately intoxicating dose, is accompanied by the development of refractoriness to the in vitro effect of ethanol on acetylcholine release by brain cortex slices.

The concentrations of y -amino butyric acid (GABA) in rat brain are also reported to be affected by ethanol. Ferrari and Arnold ( 1961) reported that a dose of 4.3 g/Kg orally decreased brain GABA 16 °/0 in one hour in Sprague-Dawlcy rats, and found a decrease of 1.6^/ in the Wistar strain. However, Hakkinen and Kulonen ( 1959, 1963) using Wistar rats, found an increase of 34 °/0 under these same conditions. Higgins ( 1962) attested to the marked strain differences, while Gordon ( 1967) failed to find any change in rat brain levels of GABA after ethanol.

Several studies have been performed to investigate the effect of ethanol on the release of histamine, mainly as an explanation for the hyperchlorhydria produced by alcohol consumption. Most invest igators have failed to find a significant difference in histamine excretion after ethanol. Saint-Blanquat and DeRoche ( 1967a) found no significant change in the levels of histamine in the gastrointestinal tract or urine of rats. Yet, after chronic treatment (21 days), these same authors ( 1967b) did find an increased excretion of histamine in the urine which they attributed to liver damage with subsequent im pairment of histaminase activity. Saindelle et al ( 1968), studied the effects of various components of cigaret smoke on the isolated guinea pig lung. They found that acetaldehyde caused a marked liberation 20 of histamine in this preparation. CH 3 CHO has been shown to liber ate histamine from rat tissues in vitro (Troquet and Lecompte, I960) and also in platelet rich plasma ( Farber et al., 1968). However, quite large concentrations were needed ( 5mM) to produce significant increases in histamine liberated in the medium, whereas formaldehyde was quite active.

Because of the close correlations which have been found be tween psychoactive drugs and their actions on amines, the effect of ethanol on brain amines was extensively examined. Two very pro vocative reports appeared (Gursey et al., 1959; and Gursey and Olson, I960) which claimed significant decreases in serotonin (40 of control) and norepinephrine ( 55 °/a of control) in rabbit brain stem after i. v. ethanol (2 g/Kg). This effect lasted several days, and the authors compared this action to that of reserpine and proposed amine depletion as a mechanism of the sedative, hypnotic and mood depressant action of ethanol. However, these claims have not been substantiated (Haggendal and Lindqvist, 1961; Pscheidt et al., 1961; Bonnycastle et al., 1962; Efron and Gessa, 1963; Duritz and Truitt, 1963, 1966). These descrepancies could possibly be explained by the fact that acetaldehyde, a major metabolite of ethanol, may be responsible for this effect, Duritz and Truitt ( 1966) demonstrated that ethanol alone produced only small decreases in brain stem nor epinephrine and serotonin. However, administration of ethanol in the presence of disulfiram or administration of acetaldehyde alone produced significant decreases in brain stem norepinephrine. The magnitude of this depletion was better related to the brain concentra tions of acetaldehyde than to blood levels,

Corrodi; Fuxe and Hokfelt in 1966 reported a failure to produce decreases in rat brain noradrenaline and dopamine after ethanol ad ministration. But, in animals also treated with an amine synthesis 21 inhibitor H 44/68 (amethyl-p-tyrosine) there was a significantly greater decrease of noradrenalin but not of dopamine in the brain compared with rats receiving only H44/68. This effect is consistent with an amine releasing action of ethanol rather than a depleting action. In this case, where synthesis can not replace any amine which has been released, the effect will be seen as a reduction in amine content. In retrospect, this is the same situation which Duritz and Truitt ( 1966) simulated by using disulfiram. This drug was able to inhibit amine synthesis at the dopamine-P-oxidase step and make it rate limiting ( Goldstein et al., 19&4), so that decreases in norepinephrine synthesis due to release were detectable.

Adrenergic Actions of Ethanol or Acetaldehyde

It is quite well known that ethanol increases lipolysis in adipose tissue (Brodie et al., 1961; Estler and Ammon, 1967; and Ammon et al. , 1966) . The effects of ethanol on lipid metabolism have been re viewed by Isselbacher ( 1966) . He stated that when alcohol was admin istered to man, the first change in blood lipids which occurs is an increase in plasma triglycerides. This is followed, as the blood alcohol levels increase, with a gradual decrease in triglycerides to normal levels and a concomitant and often marked increase in plasma free fatty acids which appear to be derived from adipose tissue. Furthermore, he finds that the lipid which accumulates in the liver under these circumstances is triglyceride. The fatty acids present in these triglycerides are derived from adipose tissue (Mallov,

1 9 6 1 ) in the fasting state, and to a lesser extent from synthesis of fatty acids within the liver.

Sympathomimetic amines produce rapid rises in plasma free fatty acid ( FFA ) levels, while p-adrenergic blocking drugs inhibit this rise. This lipolytic action of ethanol has been attributed to an 22

effect on catecholamine release (Klingman and Goodall, 1957; Abelin et al., 1958; Perman, 1961a; von Wartburg and Aebi, 1961); because it can be prevented by adrenalectomy (Mallov and Gierke, 1957), a-adrenergic blocking agents (Brodie et al., 1961) and also by p-adrenergic blocking drugS (Estler and Ammon, 1967).

Similarly, ethanol has been shown to have an effect on lipo protein lipase { LPLi) activity (Mallov and Cerra, 1967). They found that ethanol intoxication significantly increased LPL activity in the heart as does the administration of epinephrine and norepi nephrine. It' was postulated that the effect of ethanol on cardiac LPL may be mediated by the release and subsequent activity of endogenous sympathomimetic catecholamines. It was suggested that this action might.be due to acetaldehyde because of its postulated sympathomimetic activity, and the 30 minute latency in the response on LPL after ethanol. A very recent report by Myers et al ( 1969), showed an elevation of all major lipid fractions due to hyperpre-P-lipoprotein -

em ia in 1 0 0 alcoholic patients.

Truitt and Twardowicz (unpublished data) have demonstrated that relatively small doses of acetaldehyde (200 - 300 mg/Kg) pro duced prompt rises in plasma FFA in 15 minutes which were effect ively blocked by one hour pretreatment with propranolol. Similarly, other unpublished data of Truitt and co-workers demonstrated that acetaldehyde injection could also significantly increase hepatic tri glyceride levels as did ethanol.

Another activity of ethanol which has an implied sympatho mimetic mediation is the hyperglycemia produced by ethanol. In early studies ( Tennant, 1941; Klingman and Haag, 1958) it has been shown that sublethal doses of alcohol ( as low as 3.2 g/Kg) produce hyperglycemia and hypokalemia in animals. Epinephrine produces hyperglycemia and an initial but temporary rise in serum potassium 23 followed by a hypokalemia (D'Silva, 1934; Ellis, 1956). Norepi nephrine produces similar potassium changes (D'Silva, 1949) but is far less effective as a glycogenolytic agent (Schumann, 1950). Receptor mechanisms for the hypoglycemic response to adrenaline in man have been discussed (Antonis et al., 1967). Forbes and Duncan ( 1950) demonstrated that acute alcohol intoxication caused a definite reduction in liver glycogen. More recently, ethanol- induced increases in glycogenolysis in brain and liver have been demonstrated (Ammon et al,, 1965, 1966; Estler and Ammon, 1965). Akabane et al ( 1964), have shown gradual increases in the perfusing blood sugar levels, when rabbit livers were perfused with alcohol. Since catecholamines increase glycogenolysis by stimulation of the adenylcyclase system, Ammon and Estler ( 1968) examined whether the glycogenolytic action of ethanol in brain and liver was mediated by catecholamines. The intravenous injection of ethanol in mice was followed by decreases in glycogen in these organs. But, ethanol failed to produce decreases in glycogen in the presence of p-adrenergic blocking drugs.

Recent investigations on the mechanism of ethanol induced hyperglycemia (Kohei, 1967a) has illustrated that acetaldehyde can elicit this response. He has shown that intravenous injection of acetaldehyde caused a marked rise in blood glucose levels, pyruvic acid, lactic acid, and a-keto glutarate in rabbits. Also, in rats a hyperglycemia and glycogen depletion in the liver were produced ten minutes after acetaldehyde injection. It was suggested that acetalde hyde effects on catecholamines could be .responsible for this response. Further studies (Kohei, 1967b) have shown that propranolol blocked the acetaldehyde induced hyperglycemia whereas phenoxybenzamine did not. Chronic reserpine treated rabbits failed to respond to acetaldehyde with a hyperglycemic response. 24 First evidences for an amine releasing action of acetaldehyde came from investigation of the cardiovascular effects of this agent. The effects of alcohol on the heart and circulation have been frequent ly reviewed (Grollman, 1942; Loomis, 1952; Truitt, 1966; Alexander, 1967). The general conclusion that alcohol is without beneficial effect on the heart came mainly from experiments on isolated heart preparations. This was done because of the conflicting reports which have been obtained in whole animals. More recently, Regan et al ( 1966), failed to find a significant effect on coronary flow, heart rate or arterial pressure, and Conway ( 19 6 8 ) said the effect of excessive alcohol on myocardial function was depressant and ad verse.

On the other hand, the effects of acetaldehyde on the circulat ion and isolated heart are quite pronounced. As was shown early by Handovsky ( 1934, 1936), the intravenous administration of acet aldehyde in anesthetized dogs produced a sharp rise in blood pressure and cardiac acceleration. These effects persisted after removal of the stellate ganglia, ligation of the adrenals or administration of atropine, He concluded that the action of acetaldehyde was at least partially of peripheral origin. These findings were confirmed by Nelson ( 1943) who also showed that the sympathomimetic activity of acetaldehyde was potentiated by cocaine. Potentiation of the response by ephedrine and triplennamine was subsequently shown by Teague and Wingard ( 1953), and Wingard and Teague ( 1957) . The pressor response could be reversed by adrenergic blocking agents such as ergotamine tartrate (Koppanyi, 1945), dibenamine (Teague and Win gard, 1953), tolazoline and piperoxan (Romano et al,, 1954). Similarly, Christensen ( 1951) reported that the action of epinephrine upon the blood pressure was duplicated by acetaldehyde iri an amount

1 0 0 times as large, and that the pressor response could be reversed 25 by adrenolytic drugs. Nelson { 1943) and Feingold ( 1952) both adequately demonstrated that this sympathomimetic effect of acet aldehyde persisted in animals whose adrenal veins were ligated. Therefore, an action via the adrenals (Perman, 1958a) could not entirely explain the sympathomimetic action of aldehydes.

Hitchcock ( 1947) was the first to show that normal aliphatic aldehydes from acetaldehyde to hexaldehyde were mainly pressor in action, while formaldehyde and aldehydes with longer chain lengths than hexaldehydes were depressor. The most active pressor agents were acetaldehyde and propionaldehyde, the activity tending to de crease with increasing chain length or chain branching, Aldehydes in the aromatic series were mainly depressor. These findings have been repeated and confirmed (Wingard et al., 1955; Wingard and Teague, 1957) . Siebert et al ( 1952), and also Feingold ( 1952) both confirmed that this pressor response to acetaldehyde could be blocked by dibenamine and priscoline. This treatment reversed the response to a depressor action.

More recently Eade ( 1959), reported that acetaldehyde caused a fall in blood pressure in spinal cats pre-treated with reserpine. In normal animals the sympathomimetic aldehydes appeared to exert part of their action upon the cardiovascular system by the release of catecholamines from tissue stores other than the adrenal medulla. The action of acetaldehyde on blood pressure and nictitating membrane were not abolished by hexamethonium. Furthermore, he found that the releasing action of acetaldehyde appeared to differ from that of tyramine, in that cocaine potentiated acetaldehydes effects but inhibit ed the response to tyramine. Akabane et al ( 1964a), also examined the action of acetaldehyde on the blood pressure and nictitating mem brane, He confirmed that bilateral adrenalectomy reduced the actions of acetaldehyde but did not abolish them. Reversal with dibenamine, 26 potentiation by cocaine, and failure to block this response with hexamethonium or atropine were ail demonstrated. They also re ported that bretylium slightly potentiated the acetaldehyde pressor response. As was shown by Eade, these workers found a reversal to a depressor response after reserpine pretreatment which could not be restored by norepinephrine infusion. These same investi gators (Akabane et al., 1968), studied the effects of formaldehyde, and found it produced a depressor response, increased intestinal activity and a hyperglycemic effect which persisted in the chronically reserpine pretreatcd animal. One study performed by Asmussen, Hald and Larsen ( 1948b), infused acetaldehyde intravenously into human subjects at concentrations of 0.2 to O.Tmg"/, They observ ed a marked increase in heart rate and ventilation. Unfortunately, these workers did not present any data on blood pressure effects. It is quite interesting in this respect, that acetaldehyde was once used clinically with success as a cardiac stimulant and vasopressor agent in shock ( Gyorgy, 1932 ).

Investigation of the cardiac effects of ethanol and acetaldehyde have recently been reported (James and Bear, 1967). Positive inotropic and chronotropic responses were obtained in situ with coro nary sinus perfusion using concentrations from 1 0 to 1 0 0 0 (jg/nl. Similar effects were found on the isolated rabbit paired atria (Kumar and Sheth, 1962) using concentrations of 100 to 800 }jg/ml. Even more recently, James and Bear ( 1968) using perfusion of the sinus node in the open-chest dog have made a comprehensive study of the effect of aliphatic aldehydes with emphasis on structure-action re lationships. They concluded that the sympathomimetic activity of aliphatic aldehydes depends on the presence of a terminal aldehyde and a free contralateral, nearby terminal methyl group. This action was strongest in the compound containing only these two essential groups, acetaldehyde. 27 The Disulfiram-Alcohol Reaction

It was first noted in 1937 by Williams, that workers exposed to tetramethylthiuram disulfide ( TMTD) developed a hypersensitivity to ethanol.' He suggested that it might be used as a cure for alcoholism. Tetraethylthiuram disulfide ( TETD, disulfiram, Antabuse^) was used in’the rubber industry as an antioxidant, and workers exposed to it also developed a hypersensitivity to ethanol ( reviewed by Jacobsen, 1950).

The symptoms of the syndrome which has been termed the disulfiram-alcohol reaction, and is induced in humans by alcoholic bev erages after administration of TETD, were quite severe (Hald and Jacobsen, 1948a). They consisted of flushing of the face, dilation of the scleral blood vessels, "bull-eyed" sight, palpitations, dyspnea, vomiting, cephalgia and sometimes collapse. These symptoms are accompanied by a decrease in blood pressure, particularly the diastolic, and by ECG changes, namely flattening of the R-wave, de pression of the ST-segment and disappearance of a pre-existing left predomenance (Raby, 1953a; Raby and Lauritzen, 1949). Some cases of fatal TETD-alcohol reactions have been reported (Jacobsen, 1952b; Alha et al,, 1957). Several investigators initiated studies which provided the basis for use of TETD as an adjunct in the treatment of chronic alcoholism (Assmussen et al., 1948a; Martensen-Larsen, 1948). A similar sensitization is produced by eating the fungus coprinus atramentarius ( Fisher, 1945), ingestion of animal char coal (Clark and Hulpieu, 1958), and by hypoglycemic sulfonylureas (Truitt et al., 1962), citrated calcium carbimide ( Temposil^, Fer guson, 1956), and by many other substances.

Within a short time after the initial report on the effects of TETD plus ethanol, the mode of action of this drug seemed establish ed. There was no evidence that TETD in a moderate dose could 28 influence the first step in the metabolism of ethanol, that is, no one observed any accelerated conversion to acetaldehyde (Hald et al., 1948; Hald, Jacobsen and Larsen, 1949a; Loomis, 1950; Newman and Petzold, 1951). It was, however, found in human subjects (Hald and Jacobsen, 1948b) and in rabbits (Larsen, 1948; Kirchheim, 1951; Fujiwara and Kuwana, 1954) that had been pretreated with TETD and then given ethanol, that the concentration of acetaldehyde in the blood was higher than after ethanol alone. Acetaldehyde was also isolated from the expired air of human subjects (Hald and Jacobsen, 1948b) and rabbits (Hald et al., 1949) given disulfiram and ethanol. Sim ilarly, the rate of acetaldehyde oxidation in rabbits was decreased when they were pretreated with TETD (Hald and Larsen, 1949) . Then, Kjel’dgaard ( 1949) demonstrated that disulfiram in concentra tions as low as 0 . 1 pg/nl inhibited aldehyde oxidase from rabbit liver in vitro, but had no similar effect on a series of other enzymes includ ing alcohol dehydrogenase. It was demonstrated by Jacobsen and Larsen ( 1949) that the liver was the main site of formation of acet aldehyde when animals were pretreated with disulfiram by experi ments performed on perfused rabbit livers. Lubin and Westerfeld ( 1945) had already shown that the liver was the main site of acet aldehyde destruction.

The appearance and disappearance of the disulfiram-alcohol reaction coincided with the rise and fall in blood acetaldehyde ( Hine et al., 1952a; Raby, 1954a). Assmussen, Hald and Larsen ( 1948b) were able to reproduce several of the manifestations of the reaction by infusing acetaldehyde into normal human subjects not pretreated with TETD. Handovsky ( 1934, 1936) and Raby ( 1954b) had demonstrated that acetaldehyde stimulated respiration (increased rate and amplitude) which seemed to be a direct effect on the carotid body chemo-receptors, dilation of bronchial muscles, and a hypertension. It was concluded from these works that the reaction in man was due to accumulation of acetaldehyde in the blood. That TETD does inhibit the metabolism of acetaldehyde has been demonstrated many times since then ( refer to Table 1). At least twenty-five studies in at least seven species of animals in cluding man have been performed, in which the blood level of acet aldehyde from ethanol has been shown to increase 5-10 fold in the presence of disulfiram compared to when ethanol is administered alone. Therefore, a large body of evidence indicates that TETD exerts an important inhibitory effect on acetaldehyde metabolism, but there has not been a correspondingly conclusive demonstration that acetaldehyde accumulation is solely responsible for clinical manifestations of the reaction.

However, a recent report of Wagner ( 1957) has expressed doubt of acetaldehyde accumulation during the reaction, or that the reaction could be caused by acetaldehyde based on an enzymatic de termination. Casier and Polet ( 1958) using disulfiram and C 1 4 labeled ethanol found that mice formed only small amounts of radio active acetaldehyde. For this reason, Casier and Merlevede (1952) have postulated that the reaction may be due to a hypothetical con densation product between disulfiram and ethanol. These reports combined with other older works (Kirchheim, 1951; Fujiwara and Kuwana, 1954; Child et al., 1952) showing little or no acetaldehyde accumulation require a re-evaluation of this effect, especially in light of methodological problems of acetaldehyde measurement ( as stated previously) . Ridge in 1962 was able to show that cyclic fluctuations in low blood and brain acetaldehyde levels after ethanol are abolished and an elevated single peaked curve in acetaldehyde is produced by disulfiram. The excellent work of Deitrich and Heller- man ( 1963) has shown that almost all of the known disulfiram-like agents interfere with acetaldehyde metabolism by competition with NAD for attachment to liver aldehyde dehydrogenase. 30 Once TETD came into wide clinical use, it was often found that there was a marked hypotension produced after large amounts of ethanol (Raby and Lauritzen, 1949; Hine et al., 1952). This fall in blood pressure often led to serious complications and fatalities (Solms, 1951; Jacobsen, 1952b). It remains a serious question whether acetaldehyde could be responsible for this effect, especially since it had been shown to be vasopressor. Raby ( 1953b, 1954a, 1956) made an extensive clinical study of the reaction, and confirmed that it was associated with pronounced blood pressure declines. He further found the acetaldehyde levels always to be increased. As previously mentioned, Asmussen et al { 1948b) produced several of the symptoms characteristic of the reaction by infusing acetaldehyde into normal subjects; hyperventillation, tachycardia, and facial vaso dilation, but did not present blood pressure data. Raby ( reported by Perman, 1962 ) tried to reproduce these blood pressure changes by infusion of large doses of acetaldehyde, but patients experienced severe pain, and the experiments were unfinished.

Several investigators have attempted to elucidate the mech anism of this discrepant blood pressure effect. Early results indicat ed that the effect of acetaldehyde on the blood pressure of animals was altered by pretreatment with TETD ( Christensen, 1951). He dem onstrated and confirmed earlier findings that acetaldehyde injection into dogs produces a biphasic blood pressure response resembling that of epinephrine. He also found that TETD exaggerated the hypo tensive phase of the response to both adrenaline and acetaldehyde, and felt that TETD specifically altered adrenergic receptors, to change the responses to acetaldehyde and epinephrine. Feingold ( 1954) however, found that while the pressor response to acet aldehyde was diminished by TETD, it did not prolong the depressor action for as long as Christensen reported. Similarly, disulfiram failed to alter the blood pressure response to adrenaline, noradrenaline^ 31 histamine or acetylcholine in dogs. Larsen ( 1948) administered ethanol to rabbits treated with TETD, and reported a change in ventilation rate but failed to produce changes in blood pressure or heart rate. Child { 1951) and Seibert et al ( 1952), reported they were unable to demonstrate the TETD-ethanol reaction in dogs, and Zelgler and Meyer ( 1959) reported similar findings using cats.

Certain aspects of this reaction, however, have been studied in animals. Several workers observed that toxicity to ethanol (Larsen, 1948; Lecoq, 1949; Child et al., 1952) and to acetaldehyde (dejongh, 1952) were potentiated by TETD pretreatment. Joki- vartio ( 1950) reported amelioration of the alcohol-antabuse symp toms in one patient and lessening in two others by injections of soluble iron. However, no data was presented in this report. Lester and Greenberg .( 1950) thought they could test an acetalde hyde hypothesis for the reaction by using this information. They reasoned that if iron and ascorbic acid injections lowered blood acetaldehyde in animals, then this would be consistant with the idea that acetaldehyde is responsible for the reaction. They rather ill- ogically said, that if it did not, the hypothesis was untenable, which was their final conclusion. However, a year later Lester, Conway and Mann ( 1951), failed to find any improvement in the reaction in patients with iron-ascorbic acid. They concluded that this treatment was no better than placebo.

The only thorough investigation of the reaction in animals has been recently reported by Perman ( 1962). He demonstrated a clear-cut long lasting hypotension and hyperventilation by the in jection of 0.05 - 0.2 g/Kg of ethanol in rabbits pretreated with TETD. He reported that one rabbit reacted to as little as 13 mg/Kg of eth anol. This "sensitivity1’ occurred in both anesthetized and un anesthetized rabbits. The hypotension was not prevented by 32 maintaining the animals on constant ventilation, nor by pretreat ment with atropine, mepyramine, promethazine or chlorpromazine; indicating that cholinergic vasodilation, or circulating histamine were of no major importance for the hypotension. He found that a decrease in peripheral resistance was the major contributing cause of the hypotension. This hypotension was also produced by ethanol when rabbits were treated with ganglionic blocking drugs. During infusion of acetaldehyde in small amounts into normal animals, he observed small increases in ventilation, and in TETD pretreated animals there was a concomitant blood pressure fall. Yet, he still concluded that it seemed doubtful that acetaldehyde was respon sible for the hypotension per se, and suggested it may be due to a metabolite of acetaldehyde. The characteristics of the reaction in rabbits were very similar to those reported in humans (Perman, 1962) . The blood pressure fell to about 60 mmHg with a concom ittant increase in ventilation, and was, as a rule, transient with a duration of 3-4 hours. These effects appeared rapidly after the in jection of ethanol. He also confirmed that norepinephrine adminis tration may be adequate supportive therapy for the serious hypo tension which may occur during the TETD-alcohol reactions in man.

A rather brief report by Royer and LaMarch ( 1967) implicated serotonin release in the reaction. Using rabbits pretreated with TETD, they obtained hypotension and a polypnea. When they inject ed acetaldehyde, they claimed to find (no results shown) a hyper tensive effect and a respiratory stimulation, whereas serotonin pro duced hypotension without affecting respiration. Their conclusion was based on no other information. Others have implicated a release of histamine ( Farber et al., 1968) to be the cause of the hypotension, with'little evidence for histamine release by ethanol or acetaldehyde. Furthermore, the circulation of the rabbit is known to be comparative ly resistant to histamine ( Paton, 1957) , Dietrich and Hellerman(1963) 33 have proposed that the difference in symptoms reported between the disulfiram-alcohol reaction and those after acetaldehyde alone (Lester and Greenberg, 1950; Forney and Harger, 1965)might be attributed to other aldehydes such as 5-hydroxy-indole acetaldehyde ( Feldstein et al., 1964) or possible 3, 4 dihydroxymandelic aldehyde arising from the non-specific interference of disulfiram with these aldehyde oxidations. Davis (personal communication) has also suggested that the altered metabolism of amines might play a role in the disulfiram-alcohol reaction. As can be seen, some aspects of the reaction are still obscure. Investigations into the mechanism of this hypotension has failed to adequately explain this effect, There is still some question, therefore, whether acetaldehyde is solely res ponsible for the clinical manifestations of the TETD-ethanol reaction.

Effects of Ethanol on Biogenic Amine Metabolism

As long ago as 1950, Heim reported that ethyl and methyl alcohol inhibited monoamine oxidase (MAO), an enzyme which destroys both adrenaline and serotonin, as well as tyramine and other amines. No further mention was made of this until I960, when Rosenfeld measured serotonin metabolism in mouse liver homogenates, Results showed that there appeared to be an apprec iable decrease in the rate of serotonin metabolism after sublethal doses of alcohol. It was suggested that the oxidation of alcohol, competitively inhibited the oxidative metabolism of serotonin and perhaps in particular the oxidation of the aldehyde derived from it. Rosenfeld ( 1960a) also reported that ethanol in man resulted in a pronounced decrease in endogenous output of 5-hydroxy-indoiacetic acid ( 5HIAA ). Perman ( 1961a) found no significant change with ethanol. Schenker et al ( 1967) noting increased tryptamine excret ion after alcohol, attributed this to MAO inhibition. They then 34 examined the extent to which ethanol could be expected to inhibit MAO in the tissues of intact animals pretreated with alcohol (Maynard and Schenker, 1962) . It was found that ethanol did in hibit MAO in mouse liver using three substrates but not in brain. However, they concluded that the degree of inhibition found was relatively small, albeit significant, and could not account for the very marked increase in tryptamine excretion observed in their subjects. This led them to postulate release of a bound form of the amine by ethanol, thus making it available for physiological action. On the other hand, Cotzias and Greenough ( I960) suggested that ethanol stimulated MAO. Finally in 1964, Towne demonstrated that acetaldehyde inhibited both liver and brain MAO, 26 and 36 °/c respectively. He explained the previous lack of effect of ethanol in brain, on the absence of alcohol dehydrogenase in this tissue, so that little acetaldehyde was produced in vitro. Still, the degree of inhibition was not very marked and the concentration required was rather high.

In 1964, Feldstein et al re-examined this effect by adminis tering serotonin-C 1 4 orally to six subjects. They were able to demonstrate that ethanol blocked the metabolism of serotonin to 5HIAA. The alteration in metabolism showed a dose-response relationship, and the duration of the block was less than 24 hours. On the basis of these experiments, it was not possible to detect which enzyme (MAO or aldehyde dehydrogenase) was altered by alcohol. Feldstein concluded that ethanol intoxication might be due in part to altered levels of brain biogenic aldehydes, biogenic alcohols or biogenic amines.

Further studies by this same group ( Feldstein et al., 1967)

again demonstrated marked decreases in C 1 4 -5HIAA after various alcohol doses. It seemed reasonable for him to assume that 5-HIAA after various doses was decreased because of an acceleration of the 35 alternate pathway for the formation of 5-hydroxytryptophol ( 5HTOH) and its conjugates, although he did not m easure these. He felt that the most plausible explanation for this effect was due to alteration in the NAD/NADH as a result of ethanol oxidation. This alteration in pyridine nucleotide levels in rat liver is well established as a consequence of acetaldehyde and acetate formation (Buettner et al., 1961; Cherrick and Leevy, 1965; Horn and Manthei, 1965; M ir- one, 1965) . He postulated that such a shift would force the 5-hydroxyindoleacetaldehyde derived from serotonin to be preferentially re duced to 5-HTOH by an NADH-linked alcohol dehydrogenase. Simultaneous studies by Davis et al ( 1967a) demonstrated that in man there was a definite increase in 5-HTOH formation after alcohol con sumption. These investigators were able to show that low doses of ethanol produced marked shifts in the metabolism of C 1 4 -serotonin.

They found that normally 2.3 °/f of the excreted C 1 4 appeared as 5-HTOH and 82.3 /£ as 5-HIAA. After ethanol, the percentage of carbon-14 excreted as 5-HIAA had fallen to 42 while the amount metabolized by the reductive pathway to 5-HTOH had risen to 42 °/0 . Feldstein and Williamson ( 1968) administered ethanol to rats and homogenized the liver and incubated it with serotonin-C 1 4 ( 5 -H T ) . In livers from ethanol treated rats the conversion of 5HT to 5-HIAA was decreased from 85 to 48 °/0 with concomitant increases in the for mation of 5-HTOH from 11 to 43 e/g . Administration of disulfiram produced similar effects in vitro. These same investigators adminis tered 5-hydroxytryptophane in order to raise brain levels of serotonin. Administration pf ethanol to these rats and examination of the m et abolites of serotonin in the brain, showed no significant change from control. More recently, Tyce, Flock and Owen ( 1968) investigated this shift in serotonin in brain. C 1 4 -5-HT was injected into the cau date nucleus of rats, and the MAO metabolites 5-HIAA, 5-HTOH and 36 5-HTOH glucuronide were formed. A significant shift in the met abolism of 5-HT after ethanol could not be demonstrated.

Lahti and Majchrowicz ( 1967) investigated the effect of acet aldehyde on this system in vitro. They demonstrated that acetalde hyde at an initial concentration of 1 mM inhibited the oxidation of 5-hydroxyindoleacetaldehyde to 5-HIAA. They could not demon strate any effect on MAO by acetaldehyde. Despite the fact that these workers used excessive concentrations of NAD, a shift to the reductive pathway occurred. They postulated that the shift in ser otonin metabolism is due to substrate inhibition of aldehyde dehy drogenase by acetaldehyde, the metabolite of ethanol.

A similar effect was shown on tryptamine metabolism with formation of tryptophol in rats treated with disulfiram, another aldehyde dehydrogenase inhibitor (Smith and Wortis, 1960a) . These same workers (Smith and Wortis, 1960b) also demonstrated that disulfiram could alter the metabolism of C14-normetanephrine in the rat, Disulfiram caused an increase in 3-methoxy-4-hydroxyphenyl glycol sulfate (MHPG) and a reduction in 3-methoxy-4-hydroxy- m andelic acid ( VMA ).

Davis (personal communication) has demonstrated a marked alteration in 5-HT and norepinephrine (NE) metabolism in man, dur ing disulfiram therapy. There was a decreased conversion of C14-

5-HT to C 1 4 -5-HIAA and of C14-NE to CI4-VMA with an increase in the excretion of C 1 4 -5-HTOH and C 1 4 -MHPG. Disulfiram also dimin ished the excretion of endogenous VMA and augmented the excretion of the corresponding glycol. However, the excretion of endogenous 5-HIAA was unaffected, and suggested an alternative pathway for the production of this acid, 37 It was concluded that disulfiram modifies the metabolism of 5HT and NE by inhibiting aldehyde dehydrogenase and thus divert ing the intermediate aldehyde derivative of the amines from the major oxidative pathway to a collateral reductive route.

Smith and Gitlow ( 1967) have also demonstrated that small amounts of disulfiram decrease VMA formation while markedly en hancing glycol synthesis after infusion of 7-H 3 -norepinephrine. Endogenous VMA was also substantially reduced from 1-3 pg/ing of creatnine down to 0 . 1 pg/tng of creatnine when disulfiram was administered.

Several studies have also been made on the effect of ethanol on the metabolism of norepinephrine (Davis et al., 1967b, 1967c; Smith and Gitlow, 1967; Bertani et al., 1969) . Smith and Gitlow ( 1967) administered dl-7-H3-NE to subjects one hour after the in gestion of ethanol. Ethanol ingestion was found to produce a re v ersal in the VMA/felycol ratio, as did disulfiram . These investi gators noted also that when large amounts of ethanol were given, the excretion rate of unchanged norepinephrine during the first hour collection was nearly twice that of the controls, They post ulated that catecholamines released by ethanol "spared" the de gradation of the isotopic species resulting in this increased excretion.

Experiments performed by Davis et al ( 1967b) using C14- labeled norepinephrine obtained similar results. Administration of

6 0 cc of alcohol caused a marked shift in the two major metabolites of this amine. They failed to find an alteration in the excretion of total C14, CI4-NE or C14-NM as a result of ethanol administration. However, urine samples were collected over eight hour periods, and any effect may have been masked by this time period. Similar 38 experiments were performed ( Davis et al., 1967c) in which the end ogenous metabolites were monitored. There was a decrease in excreted VMA from 60.8 to 35^ of the total O-methylated catechol amine metabolites and a corresponding increase in MHPG from 32.1 . to 55.5 °/0 . It was thought to be desirable to see if ethanol could produce a similar effect on brain norepinephrine metabolism. These same investigators (Davis, personal communication) pre treated rats with ethanol, and then injected CI4-NE into the lateral ventricles of the brain. Irregardless of the time of brain assay, no shift in the metabolism of NE could be detected. However, re cent experiments by Bertani et al ( 1969) have demonstrated indirect ly that ethanol does seem to produce a similar shift in NE metabolism within the central nervous system of man.

Interactions of Amines, Central Acting Drugs and Ethanol Hypnosis

Numerous studies have shown that the degree of depression produced by ethyl alcohol closely parallels the concentration of alcohol in the brain, and that this concentration quickly follows that of the blood. Several decades ago, Friedman ( 1942) repeatedly described an apparent increased permeability of brain capillaries produced by i. v. injection of epinephrine. He called this phenomenon the auxoneurotropic action of adrenaline and studied it in relation to alcohol, various toxins, dyes and drugs. In the case of alcohol, he found that a dose which produced no symptoms by itslef, caused marked depression if injected shortly after adrenaline. Hulpieu and Cole { 1946) confirmed this finding but questioned the mechanism. They reasoned that if the potentiating action of adrenaline was to in crease the permeability of the blood-brain barrier to alcohol, this was in opposition to the belief that the brain capillaries were nor mally very rapidly permeable to alcohol. 39 Lee (1962) re-evaluated the effect of alcohol on the blood- brain barrier permeability. Using radioautographic techniques with iodinated bovine albumin, he studied alterations in cerebral vascular permeability. The brain vasculature was impermeable to albumin molecules or trypan blue particles, but injection of 2 cc of 7.5 °/0 alcohol concentrations increased the permeability of these compounds. Using higher alcohol concentrations, the radio activity in brain was intense at one hour and reached a peak after two hours. The radioactivity and blue dye were very pronounced in the thalamus, hypothalamus and amygdaloid nuclei but less in the cortex. No histopathological change in the cerebral capillaries or brain tissue could account for the overall increase in permea bility. This effect was seen with relatively low blood levels of alcohol ( 0 . 2 °/0 ).

Studies were performed by Rosenfeld ( 1960b) to see if there was an interaction with attendant neuropharmacological or toxicol- ogical effects between alcohol and various naturally occurring amines. In view of the rather selective effect of alcohol on the brain and lack of a satisfactory explanation of this effect, he reason ed that there was a good possibility that the narcotic action of alcohol could derive from a specific effect on the specialized biochemicals in neurons (viz; amines) rather than to actions on metabolism com mon to all cells (Larrabee et al., 1950). Rosenfeld found that as much as 3.64mM/Kg of serotonin, dopamine, and tyramine or 1.21 mM/Kg of tryptamine could be injected i, p. in the mouse without pro voking hypnosis or death. And, 0.9 mM/Kg of each amine could be administered before 4.5 g/Kg of ethanol without causing death. But, if these amines were administered 30 minutes after the alcohol, mortalities ranged from 50-83 °/9 as well as a decisive prolongation in the sleeping time of survivors. These primary aromatic mono 40 amines markedly potentiated the hypnotic effects of alcohol, re induced sleep in mice which had recovered from an alcohol-induced hypnotic state and serotonin caused sub-hypnotic doses of ethanol to become hypnotic. In contrast, a primary aliphatic amine, GABA, which is not metabolized by MAO, caused no deaths nor did it affect the ethanol hypnosis. Similarly, a secondary amine, amphetamine slightly reduced the ethanol sleeping time. This author postulated that the aldehydes of these amines may have hypnotic actions and that acetaldehyde from ethanol could inhibit their metabolism. He had previously shown a decrease in 5-HIAA due to ethanol (Rosen feld, 1960a). Therefore, alcohol would increase the aldehyde inter mediates of these amines formed by the action of MAO. He proposed a causal relationship between the effect of alcohol on the metabolism of one or more of these endogenous aromatic monoamines in the CNS and the neuropharmacological effects of alcohol.

Kalant et al ( 1967) postulated that the central depressant effect of alcohol could be related to an anti-cholinergic effect in the area of the reticular activating system. They postulated that the effect of alcohol in preventing release of acetylcholine, might well cause a diminution in an effective concentration of this transmitter at central cholinergic synapses and may contribute to its central depressant action. A recent examination of this hypothesis by Burn ham and Erickson ( 1969) has been made. Injection of physostigmine (0,01 |jg/Kg)five minutes before ethanol was given, shortened the sleeping time from 97 to 73 minutes. Pretreatment with atropine did not alter this effect of cholinesterase inhibition on ethanol hyp nosis. This suggested that increased acetylcholine can overcome the central depressant action of ethanol. However, this data is not very convincing. 41 Ethanol causes marked potentiation in the depressant action of many sedative and hypnotic drugs. This is well documented in the case of tranquilizers and the barbiturates ( Danechmand et al., 19f>7; Mirsky and Giarman, 1955). However, potentiation of the depressant action of ethanol by anti-depressant compounds is not as well documented (Milner, 1967). These drugs do not alter the ethanol blood levels (Casier et al., 1966). Again, Milner ( 1968a) and Landauer et al ( 1969) have shown that amitryptyline potentiated the toxic effects of alcohol in mice, and that this tricyclic anti depressant added to the deleterious effect of alcohol in man. Holli- well et al ( 1964) , demonstrated that amitriptyline, imipramine and desipramine (in descending order of potency) increased the mean sleeping time of mice under ethanol. Theobald et al. ( 1964), found that imipramine and desipramine caused only weak and variable en hancement of the toxicity to ethanol. An examination of the effect of various neuroleptics and anti-depressants on the response of mice to ethanol has recently been reported by Milner ( 1968b) . This same author found that imipramine caused no significant changes in ethanol effects, while methylphenidate and desipramine protected mice against ethanol induced coma. However, amitriptyline, trim- ipramine, nortriptyline and diazepam induced statistically signifi cant potentiation of the depressant and toxic effects of ethanol in mice. He suggested that potentiation of alcohol by some psycho tropic drugs may add to the hazard of drug overdosage and may be dangerous (Milner, 1968b; Landauer et al., 1969). These reports have led us to investigate the interactions which occur between catecholamines and ethanol in producing hypnosis. METHODS

Gas Chromatographic Determination of Ethanol and Acetaldehyde

A. General Procedures Gas-Liquid chromatography was used for the simultaneous determination of ethanol and acetaldehyde in biological specimens. The method was originally published by Duritz and Truitt ( 1964), and has been modified both to avoid the problem of spontaneous acetaldehyde production (Truitt, 1 9 6 9 ), and to analyze tissues. An F-M Model 400 programmed temperature gas chromatograph was used for these analyses with a Model 1609 hydrogen flame ionization detector and a lmV Honeywell Brown Electronik record er. The column measuring 0.25 inch in diameter and four feet in length is U-shaped and packed with 5°/0 Carbowax 1500 on Haloport 60-F. New columns are always preconditioned for at least 18 hours at a temperature of 130°C.

The instrument was operated isothermally under the follow ing conditions: 1. column oven temperature 70°C 2. flame monitor temperature 120°C 3. injection port temperature 105°C 4. detector temperature 100°C

Helium was used as the carrier gas at a flow rate of 35 ml per minute, the flow rate of hydrogen was 23 ml per minute, while that for compressed air was 330 ml per minute, all at 50 p. s. i. Standards were prepared from stock solutions of ethanol (Absolute Alcohol in ampules, Abbott, List No. 3772) and CH3CHO

42 43 (redistilled in nitrogen sealed ampules, Matheson, Coleman and Bell) prepared as 2.0 percent solutions in isotonic saline. Daily dilutions were prepared in isotonic saline to obtain concentrations of acetaldehyde, 2 y/nl, and ethanol, 2 0 0 0 y/ml, which were used as standards and were interspersed among experimental samples to correct for any changes in detector sensitivity. The peaks at these concentrations were obtained on range 1 and attenuation of 2 for acetaldehyde and range 10 attenuation 32 for ethanol. All sam ples for analysis were deproteinized with 5 e/0 ZnS 0 4 and 0.3 N BafOHjz. The samples for analysis were prepared in 5 ml glass vials fitted with self-sealing rubber stoppers. Samples were equi librated in an Aminco-Dubnoff constant-temperature water bath. After 15 minutes at 55°C the head space gas of the vials was sampl ed by a pre-warmed plastic tuberculin syringe. A 500 (jl air sample was equilibrated in the syringe by moving the plunger up and down at least 8 - 1 0 times and was then rapidly transferred to the injection port of the gas chromatograph. The method has general applicability for the determination of several volatile substances. Table 2 lists various substances tested and their retention times in seconds. Using the column specifications which are reported here, it can be seen that ether would interfere with the acetaldehyde peak; whereas isopropyl alcohol and chloroform would interfere with the ethanol peak. However, since these substances are not normally in biologic specimens the method is ideal for the analysis of the other volatiles listed, especially acetaldehyde, acetone and ethanol. For these three compounds, there is excellent separation with no tailing. All samples are read twice, so that a different sample may be analyzed every four minutes. The rapidity of elution of acetaldehyde and eth anol from the column has allowed peak height analysis because of the sharp peaks obtained, and area measuremejits are not necessary. TABLE 2

VOLATILE SUBSTANCES AND THEIR RETENTION TIMES AS DETERMINED BY GAS-CHROMATOGRAPHY

Relative Retention Time Retention Time Compounds (seconds) Ethanol = 1 .0

Ether 14.2 0.37

Acetaldehyde 14.75 0.39

Formaldehyde 17.35 • 0,46

Acetone 22.00 0.57

Methanol 32.00 0.89

Isopropanol 37.20 0.98

Chloroform 38.00 1.00

Ethanol 38.00 1.00

Propanol 65.40 1.72 45 B. Analysis of Biological Fluids Because of the in vitro spontaneous production of acetaldehyde in precipitated blood when ethanol is present ( Truitt, 1969), modi fied methods for the measurement of this metabolite have had to be developed. It was found that obtaining plasma or a protein-free filtrate of whole blood would avoid this problem and prevent the artifactual increases in acetaldehyde. . Glass vials with a'volume of 5.0 ml have been used to analyze biological fluids for ethanol and acetaldehyde. The contents of a vial for analysis would be as follows:

0 . 2 ml sample ( aqueous standard, plasma, urine or tis sue homogenate, etc, )

0 . 1 m l saline

0,05 ml ZnSC >4

0.05 ml Ba(OH ) 2 0.4 gm NaCl 0.4 ml Total Volume of Fluids

For blood analysis, rats were decapitated and blood was collected in a heparinized tube, which gave identical results to heart puncture blood. Samples were divided to prepare both a protein-free filtrate and plasma. Two ml of the blood is added to 1 ml of ice-cold saline and 0.05 ml of ZnSC >4 contained in a rubber-stoppered plastic centri fuge tube. After the blood has been added, 0.5 ml of Ba( OH ) 2 is added through the cap with a syringe. The balance of the blood sample is placed in another chilled tube to obtain the plasma. Both the precipitated and unprecipitated bloods are centrifuged in a cold room at 4°C and 1500 x g for ten minutes. In the case of plasma, duplicate 0 . 2 ml plasma samples are transferred to the capped vials

already containing the saline, ZnS 0 4 and NaCl, and then 0.05 ml of

B a(O H ) 2 is immediately added. Saline standards or urine samples and other biological fluids which are practically protein-free are 46 handled in a similar manner, that is, 0 . 2 ml of the fluid is added directly to the vial and precipitated. For the protein-free filtrate determinations, duplicate 0.4 ml samples of the clear filtrate above the packed, precipitated red cells are transferred to capped 5 ml vials already containing 0.4 gm of dry NaCl by means of a syringe. Vials are then placed in the water bath and equilibrated for analysis of the head space gas. Inclusion of NaCl in the vials more than doubles the sensitivity of the methods and gives excellent duplication for sub-microgram quantities of acetaldehyde.

When using a plasma fraction or a protein-free filtrate of blood for the determination of acetaldehyde or ethanol it is neces sary to make corrections for recovery. Recovery data has been obtained by adding known amounts of acetaldehyde and ethanol to blood and taking it through the processes described above. This has been done separately for rat and human blood because there was a statistically significant difference between the % recovery for these two species. Table 3 shows seven experiments performed over a 30-day period. The values are peak heights in arbitrary units for saline standards and filtrates made from blood standards, both of which were made at concentrations of 2 y/nl for acetaldehyde and

2 0 0 0 y/inl for ethanol.

The data in Table 3 demonstrates the constancy of the machine sensitivity over prolonged periods of time and the manner in which a correction factor for recovery was obtained. In this manner, experi ments with rats were performed using aqueous standards to calculate the concentrations of acetaldehyde and ethanol, and these values were multiplied by 1.71 and 0,97; respectively, to relate them back to whole blood concentrations. Similar data was obtained for the human experiments. The recovery data for acetaldehyde in the plasma and filtrate along with the correction factors are shown in Table 4. 47

TABLE 3

RECOVERY DATA FOR ACETALDEHYDE AND ETHANOL IN RAT BLOOD

Acetaldehyde Ethanol Aqueous0- Aqueous Standard Filtrate” Standard Filtrate

61.75° 37.75 48.2 51.88

62.9 28.6 49.8 44.3

5 ^ 7 32.8 48.7 40.8

57.2 43.5 52.9 52.8

54.7 30.0 46.5 59.1

59.9 30.6 51.8 52.25

p6,6 41.6 45.13 54.6

X 59.68 34.98 49.0 50.8

Kean $ Recovery 58.5 * 103.1 $

Correction Factor 1.73. 0.97

ft Aqueous standards were prepared by adding 0,2 ml of a stock solution of acetaldehyde (2 //ml) or ethanol (2 mg/ml).

^ Filtrates represent protein-free filtrates prepared from vhole rat blood containing acetaldehyde (2 //ml) or ethanol (2 mg/ml).

c Values represent peak heights In arbitrary units. TABLE h

RECOVERY DATA FOR ACETALDEHYDE IN PLASMA AND PROTEIN- FREE FILTRATES PREPARED FROM HUMAN WHOLE BLOOtf1

(Acetaldehyde, $ Recovery) Sub.lect Plasma Filtrate

E.T. 67.5 78.6

E.L. 62.9 7^.0

J.B. 5 M 66.0

M.W. 70*8 100.5

X-percent 63.9 79.77 Recovery

Correction 1.56 1.25 Factor

1 Tf/ml of acetaldehyde was added to whole blood and the plasma and protein-free filtrate obtained as described (see text). 49 The recovery in the filtrate of human blood is better than rat blood. In all experiments reported in this paper protein-free filtrates were used and corrected to blood levels unless otherwise stated. This was done because in vitro production sometimes occurs in poorly separated plasma, filtrate values were much more consistent, and the recovery of acetaldehyde in the filtrate was in general much better.

C. Excretion in Expired Air Because of the problems with blood level determinations of acetaldehyde, a method was devised to monitor acetaldehyde in expired air. A nine liter glass metabolic chamber was constructed which could be sealed air-tight. This glass metabolism cage con tains a wire mesh floor suspended four inches above the glass bottom. The animal is placed on the wire flooring, so that any urine or feces can drop through. At the roof of the chamber is a sleeve with an opening to the outside. Rubber tubing is placed over the sleeve and a 1 ml tuberculin syringe with a half-inch, 25 gauge needle attached, was inserted into the other end of the tubing. Be tween the syringe and the sleeve is a pinch clamp which forms a tight seal, and can be easily removed for sampling. Samples of 0,5 ml of air can be removed periodically to monitor the excretion of acetaldehyde and ethanol into the expired air. Again, the plunger should be equilibrated at least ten times before removing the sample for analysis by gas chromatography. The chamber should be kept in a room where the temperature is more or less constant. All analyses were done at room temperature,

Male Wistar rats weighing 175-225 g were used, and were in jected intravenously with acetaldehyde, 30 mg/Kg or given ethanol, 4 gm/Kg, orally and immediately placed into the metabolic chamber. Levels of these substances in the expired air were measured at 50

various intervals. In order to measure the amount of acetaldehyde or ethanol excreted by this route, standards were run for compari son. A glass petri dish was placed on the shelf of the chamber, into which was placed small amounts of each substance. Standards which were found to be most convenient consisted of 25 pi of absol

ute ethanol which is equivalent to 2 0 , 0 0 0 pg, and 50 pi of a 2 0 , 0 0 0 pg/nl stock of acetaldehyde which is equal to 1000 pg. These volumes were placed in the petri dish with micropipettes and the chamber immediately sealed. The chamber then was shaken slight- .ly to hasten evaporation of the standard solutions, which was us ually effected in five minutes. It was found that 15 minutes of equi libration were required before the peak heights stabilized and remained at a constant level as can be seen in Table 5. Standards for calculations were usually read at 15, 20, 25 and 30 minutes to insure equilibration, and the mean value used.

D. Tissue Concentrations The method of Duritz and Truitt ( 1964) has been modified so that tissue concentrations of ethanol and acetaldehyde could be de- i termined. Male "VVistar rats weighing 175-225 g were used. All animals were fasted overnight before testing. They received ethanol orally at a dose of 4 gm/Kg, and some were given disulfiram orally, 200 mg/Kg, at 20 hours and at four hours prior to the alcohol dose. Disulfiram was prepared as a suspension (2 gm/100 m l), using 10 m l of 5 % methylcellulose as the suspending agent. Animals were sacrificed at one or two hours after the administration of ethanol.

All procedures were performed in a cold room at 4 8 C. Animals were sacrificed by decapitation, and nine tissues removed. These included: brain, heart, lung, kidneys, spleen, liver, adipose tissue,' small intestine and skeletal muscle. All tissues were 51

TABLE 5

ACETALDEHYDE (CH^CHO) AND ETHANOL (EfcOH) AIR STANDARDS AS DETERMINED IN A GIASS METABOLISM CHAMBER AT VARIOUS TIMES5

Time In Minutes CH3CHO Attenuation EtOH Attenuation

5 55.5b 6 68.6 6k

10 MS.O 16 59.5 160

15 5*uO 16 69.5 160

20 53.0 16 70.0 160

£5 53.5 16 67.5 160

30 52.0 16 65.0 160

k5 5^.0 16 69.5 160

60 5^.0 16 69.0 160

a 1 mg of acetaldehyde and 20 mg of ethanol were placed on a shelf in a glass metabolism chamber and allowed to evaporate, see text for details of standard preparation,

^ Values represent peak heights in arbitrary units. 52 removed and frozen in less than ten minutes. Each tissue was triturated in a porcelain mortar with liquid nitrogen. It was im portant that the particles of tissue be of medium size, so that the entire tissue was frozen, and alternatively so that when it was later weighed it did not thaw rapidly. After freezing with liquid nitrogen, each tissue was packed in a plastic air-tight container and stored in a freezer at -20°C. Tissues were always assayed immediately, that is, on the same day on which they were taken. The entire tis sue was used except in the case of liver, small intestine and skele tal muscle where from 1 - 2 grams of tissue was assayed.

Each tissue was analyzed separately and in triplicate. The tissue was weighed, and a 20°^ v t/v homogenate was prepared. The tissue was placed in a graduated cylinder and brought to the calculat ed volume with cold isotonic saline. Then, the mixture was trans ferred to a glass tube, and homogenized with a teflon pestle at a speed of 2000 r, p. m. After homogenization, 0.2 ml of the homogen ate was pipetted into 5 ml glass vials containing 0.05 ml of ZnS0 4 and 0.05 ml of Ba(OH ) 2 was immediately added and the vial was cap ped. The remainder of the homogenate was discarded. Then samples were handled as described for blood, they were equilibrated for 15 minutes at 55°C and the gas phase was analyzed by gas chromato graphy for ethanol and acetaldehyde.

Tissue standards were run for each tissue several times during the course of these experiments. Loss of acetaldehyde due to met abolism could be avoided if the blank tissue homogenates were pre pared and precipitated in the vial, and then acetaldehyde was added. Tissue blanks were also run at this time. The acetaldehyde standards for each tissue were prepared by adding 5 >. of a 20y/fcnl aqueous acetaldehyde solution to the vial. The final concentration in the vial for each tissue then represented 2.5 y/% of tissue. Ethanol standards 53 were also run separately on these tissues, in this case 5 \ of a 20,000 y f a i l stock was used to give a final concentration of 2,500 y / g of tissue. Typical standard values are illustrated in Table 6 . It was found that actual tissue standards only had to be run every couple of weeks, A saline standard was used with each experiment and compared back to one which had been prepared on the day the standard tissues were analyzed. If the saline standard deviated by

m o re than 1 0 °/0, then new tissue standards were prepared to cor rect for changes in sensitivity. The relative constancy of the in

strument in this regard is also shown in Table 6 , the saline stand ards did not vary for about three weeks, but by day 30, a new set of tissue standards had to be prepared.

In Vitro Release of Norepinephrine

A. General Procedures for Preparation of Atria Left atria from 14 male guinea pigs weighing 22 5-340 g were used in this study. The whole heart was removed from decapitated animals by making a midline incission through the sternum. The heart was gently lifted with blunt-end tweezers without stretching, and severed of all connecting vasculature. The whole heart was placed in a beaker containing Krebs-Henseleit (K-H) bicarbonate

and equilibrated with 95 % 0 2 and 5 % C02. The purpose of the beaker was to wash the heart of any blood trapped in the chambers. After

6 0 seconds the heart was transferred to a second beaker and allowed to rinse for another 60 seconds. The heart was then lifted by hand and both atria severed with a single cut. The paired atria were placed in a petri dish containing K-H media and the left atrium was cut away from the right one. Suture silk was used to Becure the Beptal end of the left atrium to a stimulating punctate electrode as sembly. Atria with the endocardial surface upward were then trans ferred to a tissue bath and attached to a Statham force transducer by 54

TABLE 6

COMPARISON OF SALINE STANDARDS AND TISSUE STANDARDS FOR ACETALDEHYDE AND ETHANOL

Weight Sample in Grams Acetaldehyde Ethanol

Brain 1.69 31.5b 49.38 Kidney 1.81 23.8 41.75 Heart 0.86 28.75 44.0 Lung 0.52 46.5 51.5 Liver 7.35 17.1 4o.o Spleen 0.53 48.25 41.5 Fat Pad 1.04 24.5 48.5 Small Intestine 1 .1 43.5 44.0 Skeletal Muscle 1.31 30.25 45.0

Saline Standards

Day 0 26.5 45.38 Day 10 26.75 43.2 Day 20 26.0 49.1 Day 30 16.3 63.0 \ / RUN NEW TISSUE STANDARDS

a The acetaldehyde concentration was 2.5 V/gm of tissue and the ethanol concentration was 2.5 og/gm of tissue.

^ Values represent peak heights in arbitrary units. 55 means of stainless steel hooks (Hollander and Webb, 1955). The physical arrangement of the mounting, stimulating and bath assemb ly are illustrated in Figure 2. The entire system as pictured was housed in a walk-in copper-wire mesh Faraday cage.

B. Experimental Conditions Left atria ( average wet weight = 42 mg) were maintained in the constant temperature bath ( 30“C) filled with 20 ml of K-H

medium at a pH of 7.41 and aerated with a gas mixture of 95 °/ 0 2 and 5 */9 C02. Tissues were equilibrated for 60 minutes, and the tissue medium changed at 30 minute intervals during this period. ^Punctate type of stimulation was used because of the minimal effect it produces on the nervous elements in the myocardium (Amory and West, 1962; Blinks, 1966), This was very important in this study, in which we were trying to demonstrate a chemo-releasing effect of acetaldehyde on the adrenergic transmitter stores within nerve terminals within the tissue. The atria were electrically stimulated with threshold voltages ( 0.2 - 0,74 V; 2 msec duration square waves) at a frequency of 100/nin. This stimulation frequency is on the up ward slope of the force-frequency curve for guinea pig left atria,

which has a peak tension at about 2 0 0 per minute and deteriorates at 300 per minute (Hollander, unpublished results).

The initial tension was standardized by stretching the tissue to 1.5 times its resting length, which resulted in an average diastolic tension of 645 mg. This degree of stretching is about midway on the increasing slope of the length-tension curve for guinea pig left atria. The rest length was determined with the aid of a microscope ( see Figure 2) and an optical micrometer, and is defined as the maximum tissue length at which no measurable contractile response was eli cited at threshold stimulation, A summary of the conditions under which these experiments were performed is shown in Table 7. 56

Figure 2: Photograph of the physical arrangement of the instrumenta tion used for experiments with guinea pig left atria*

1. Bioelectric CA 5 Calibrator. 2* Bioelectric ISA Voltage Isolation Unit. 3* Instrument Labs 180 FET Impedance Matching Amplifiers. 4. Calomel Cell (Corning I6 U98-2 ) and Microelectrode holder, located on the Micromanipulator unit. 5. Constant temperature chamber. 6 . 25 ml. glass tissue chamber containing a ground return calomel cell and stimulating assembly. 7. 565 Tektronix Oscilloscope. 8. Microscope with optical micrometer. TABLE 7

EXPERIMENTAL CONDITIONS FOR EXPERIMENTS WITH GUINEA PIG LEFT ATRIA

ANIMALS; Male Guinea Pigs

WEIGHTS: 225 - gm.

TISSUE: Isolated Left Atrium

WEIGHT: 42 mg* Average Wet Weight

MEDIUM: 20 ml* Krebs-Henseleit Bicarbonate

pH: 7.^1

TEMPERATURE: 30 aC

AERATION: 9 5 # 02 - 5# COg

STIMULUS: Tareshold Pulses = 0.2 - 0*75 V. Duration « 2 msec.

Rate = 100 per min.

EQUILIBRATION: 60 minutes

INITIAL LENGTH: 1.5 x Rest Length

DIASTOLIC TENSION: Average e 645 mg.

MICROELECTRODES: Tip Diameter £ 600A

Resistance £ 25 megohms

EXPERIMENT: 1 Hour Duration Following Equilibration 58 C. Potential and Tension Measurements Microelectrodes were filled by boiling under reduced pressure in methanol, replacing the methanol with distilled water for several hours and then soaking in 2.5M KC1 overnight. Electrode tip dia- * m eters averaged less than 600 A and when filled with 2.5M KC1 had an electrical resistance in excess of 25 megohms. The micro electrode is connected to a calomel electrode through a bent tube filledwith 2.5M KC1 agar. The microelectrode is pushed through a rubber membrane occluding the end of the agar bridge in order to allow for free movement which is often needed for strongly contract ing atria. Once fastened into the holder, the electrodes could be positioned for penetrations with a micromanipulator ( see Figure 2).

In these experiments, transmcmbrane potentials and isometric contractions were simultaneously recorded with the use of glass microelectrodes and a Statham G-10-B force transducer. Signals from the microelectrode and transducer system were amplified and differentiated with Philbrick operational amplifier units prior to re cording, Results were recorded at selected time intervals through the use of a Honeywell 1508 multi-channel oscillograph. The type of data recorded, the measurements made, and the acronyms for the various parameters are illustrated schematically in Figure 3.

D. Pharmacological Agents In these experiments, cumulative concentration-response curves were produced using acetaldehyde concentrations ranging from 0.1 to 30mM. Acetaldehyde solutions were prepared in K-H media and injected into the bath in a volume of 0.2 ml. The test solutions were checked by gas chromatography and acetaldehyde was found to be very stable in K-H media. Similarly, we checked the tissue bath media at the time of peak-r espouses because of the high volatility of CHjCHO, and found the concentrations to agree PARAMETER ACRONYMS UNITS

0 . Isoelectric Potential Zero mV 1. Resting Potential RP mV 2. Action Potential AP mV 3. Action Potential Duration AP-D m sec 4. Action Potintiol Areo AP-A reo m V -sec 5. Depolarization Rote d V /d t V /sec 6. Conduction Time CT m sec 7. Latent Period LP msec 8. Developed Tension DT mg 9. Developed Tension Peak DT-P m sec 10. Developed Tension Duration DT-D m sec 11. Mokimum Rale of Tension d F ( g /s e c Increose 12. Maximum Rate of Tension d F j g /se c Decreose

Figure 3: Schematic diagram of the electrical and contractile parameters which were measured in Isolated left atria of guinea pigs 60 closely with the theoretical concentrations even when aerated with Oz -C02. Injections of acetaldehyde were made every four minutes so that the duration of each experiment was 24 minutes. At least 5-10 membrane potential recordings were made in each tissue prior to exposure to acetaldehyde and then at intervals during the cumulative acetaldehyde additions.

Prior to exposure to acetaldehyde, tissues were tested for responses to norepinephrine { 10” 7 M) and/or tyramine {7.2 x 10’ 5M ). Some animals were pretreated with a dose of 3 mg/Kg reserpine (Serpasil, CIBA) i. p., 24 hours prior to the experiment. This dose was selected because doses of 2 mg/Kg were not sufficient to block positive inotropic responses to tyramine. Figure 4 illustrates experiments in which reserpine pretreatment with 2 mg/Kg only depressed the maximum inotropic effect to acetaldehyde by 50 whereas 3 mg/Kg essentially eliminated the response. Propranolol (inderal, Ayerst) was added to the bath in concentrations of 1.5 x 10”7M and 1.5 x 10"6M to block beta receptors. Tissues were ex posed to propranolol for 30 minutes prior to addition of any other drug.

In Vivo Release of H3-Norepinephrine

A. General Preparations To further substantiate the catecholamine-releasing effect of acetaldehyde, we have developed an in vivo technique to demon strate this phenomenon. The method involves the monitoring of plasma and urine levels of tritiated norepinephrine after intravenous injection. These experiments utilized a total of 20 animals, 11 cats and nine New Zealand rabbits, of either sex and weighing 2-3,5 Kg. Animals were anesthetized with pentobarbital { 30 mg/Kg) . Rabbits were anesthetized by ear vein injection, whereas cats were injected intra-thoracically. After the animals had reached a surgical depth of 61

CONTROL

800-

RESERPINE Zmg/Ku f J 600-

ic/> I 500- RESERP1NE 3mg/Kg

O 300-

200 -

100-

2min 3min 3.0 10.0 CH3CH0 (mM)

Figure Concentration-response curve for acetaldehyde on the developed tension of the Isolated atria from an untreated, and two reserpine pretreated guinea pigs. 62 anesthesia both femoral veins were cannulated. Preparation of the animal required one-half to three-quarters of an hour, and experi ments lasted as long as two and one-half hours. It was observed that both species remained anesthetized during the entire experi ment and supplemental administration of the anesthetic was not n ecessary .

B. Animal Equilibration Animals were injected intravenously with dl-norepinephrine which was labeled with tritium in the 7 position (H 3 - N E ) , and w ere given a dose of 100 \*c/Kg. The isotope was obtained from New England Nuclear Corporation (Boston, Massachusetts) and had a specific activity of 10.1 curies/nillimole. The animals were equi librated for an hour after injection of H 3 -NE, during which time blood samples were taken every ten minutes to follow the plasma de cay pattern. It was found that after 60 minutes, a slow rate of de cline of radioactivity was achieved in the plasma (See Results), That is, the processes of uptake, release, and excretion seemed to have achieved equilibrium in the animal, because the level of iso tope remained relatively constant over the experimental period, which in several experiments lasted as long as an additional one and one half hours. The achievement of steady-state levels was im portant in these experiments, because the purpose was to monitor changes in the plasma isotope level, If the effects of drugs had been observed during the time when the isotope level was rapidly decay ing in the plasma, results would not have been as definitive. Accord ingly, alterations in the plasma tritium level were measured above these background levels. Even though the background counts were high, in all probability they consisted mainly of the unphysiological d form of the isotope. The isomer involved in uptake processes in the nerve ending, and that which is released on nerve stimulation has been shown to be the 1 isomer (Withy, et si., 1961; Hertting and Axel rod, 1 9 6 1 ). 63 C. Injection, Sampling and Assay Procedures Upon withdrawal of the 60 minute sample which was the end of the equilibration period, an intravenous injection of acetaldehyde or ethanol was made and plasma tritium was again monitored at various time intervals. Acetaldehyde was administered as a 10°/ solution at a dose of 30 mg/Kg. Ethanol was infused over a ten- minute period as a 10’/ solution at a dose of 2 gm/Kg, and at a rate of approximately 6.5 ml per minute. Several animals were pretreated with an oral regimen of disulfiram (200 mg/Kg) at 20 hours and again at four hours prior to anesthesia.

All injections including the radiolabeled norepinephrine were made into one femoral vein and blood samples of 2 ml were drawn from the contralateral limb at intervals with replacement of blood volume by isotonic saline. In several of the animals larger blood samples were taken for analysis of acetaldehyde and/kr ethanol levels. Two ml of the sample was immediately injected into a plastic centrifuge tube and precipitated as previously described in order to obtain a plasma-free filtrate for gas chromatographic analysis. The remainder of the sample was likewise centrifuged at 1500 x g for approximately ten minutes, and 0.2 ml of the clear plasma was transferred to a counting vial containing a toluene: triton ( 10 m l:5 ml) scintillation mixture using POPOP and PPO as the phosphors. Duplicate samples were counted for ten minutes in a Packard 400 Tricarb liquid scintillation counter. The values were corrected for efficiency of counting and most of the data is expressed as percentage of the initial (ten minute sample) level of dpm's in the plasma.

D. Verification of Neuronal Uptake and Release One important aspect pertaining to the validity of the method was to demonstrate that the administered HJ-NE was taken up into 64 adrenergic nerve terminals and could be released, as are the endogenous stores of this transmitter substance. For the pur poses of these experiments, one would like to show a generalized

release of H 3 -NE, as would be the case upon injection of a chem ical agent that released norepinephrine from its storage sites in adrenergic nerve terminals. The other criterion for this type of generalized activation was that the degree of response (in this case the rise in plasma tritium) should be of a comparable magnitude as that which would be produced by a chemo-releasing activation.

This type of experimental evidence was obtained by bilateral carotid occlusion, Both carotid arteries in a rabbit were exposed and clamped for one minute intervals as illustrated in Figure 5 by the arrows. This treatment causes a generalized reflex sympathe tic discharge in the animal. This overall activation of the sympathe tic nervous system, was detected as an immediate rise in the H3-NE levels in the plasma. The magnitude of the response to two con secutive clampings at 30 minute intervals was a rise in plasma trit ium of 20 and 10°/g respectively. Since the activation of sympathe tic nerves produced a release of radioactivity into the plasma, this was taken as evidence that the administered H3-NE was taken up into adrenergic nerve terminals and could be released upon stimulation. These results were compared with the response produced by acet- ■ 4 • aldehyde and ethanol administration.

Autonomic Function in Whole Animals

For these experiments, 32 animals were studied. Ten cats and 22 New Zealand rabbits of either sex weighing between 2.5 and 4 Kg were anesthetized with pentobarbital, 30 mg/Kg. Blood pressure was measured from a femoral artery using a pressure transducer. Con tractions of the nictitating membrane were measured by an isometric Figure 5: Effect of bilateral carotid Effect of occlusionbilateral on disappearance the 5: Figure H3 - NE (%0F CONTROL dpm/ml) tONE DIVISION = 10% CHANGE) 20 rate of tritium oftritium rate in plasmathe of a injection after rahbit of 7-H3-norepinephrine of IAEA AOI OCCLUSION CAROTID BILATERAL TIME (MINUTES) TIME ( h ’- e n ) at zeroat time. 089 0 65

66 strain gauge transducer against 4 gm of tension, and respiration was monitored by means of an oscillograph. All three parameters were recorded simultaneously on a Model 5D Grass Polygraph. The heart rate was obtained by changing the chart speed and count ing the blood pressure pulse. All drugs were injected or infused through a femoral vein cannula.

Reserpine pretreatment was given at 48 hours and 24 hours prior to the experiment in doses of 3 mg/Kg i. m. for cats and 5 mg/Kg i. v. for rabbits. Disulfiram premedication was given in 10 % acacia suspension at a dose of 200 mg/Kg, through a stomach tube at 20 hours and 4 hours before the experiment. In the case of re serpine pretreatment, the anesthetic dose was reduced by one-half and for disulfiram by one-third of normal because of the synergistic action of these drugs.

The vasopressor and nictitating membrane responses to standardized i. v. doses of tyramine (800 pg/Kg) and acetaldehyde (30 mg/Kg) were compared to the average effect of four or more doses of norepinephrine (2 |Jg/Kg). Maximum blood pressure rises were calculated as a ratio to the average control norepinephrine res ponse ( i. e., maximum increase in mean blood pressure ( mm Hg) of test drug divided by average maximum increase of norepinephrine control response). Contractions of the nictitating membrane were rated semi-quantitatively on a scale from 0 to 4+ in comparison to the average control contraction for norepinephrine (4+ = about 100 % of control, 2+ = 50%, etc. ). The responses to tyramine and acet aldehyde were checked within 30 minutes prior to and within 30 minutes to one hour after the completion of ’brepleting" infusions of norepinephrine bitartrate (0.5 mg/Kg as base), dopamine hydro chloride (10 mg/Kg) and methyldopate hydrochloride (Aldomet^ = 50 m g/K g). All three 'depleting" infusions were applied to most 67 animals with a test of tyramine and acetaldehyde following each, while the order of infusions was varied to control carry over effects. The interaction of a number of pharmacological agents with acetaldehyde and tyramine was examined with regard to their cardiovascular effects. The doses of drugs administered can be found with the individual data under results.

Cl4-Norepinephrine and Metabolites

A. Administration of C 1 4 -Norepinephrine, and Sample Collection Thirty-six male Wistar rats, weighing 250-310 grams, were fasted overnight and housed in stainless steel metabolism cages. The cages were so constructed as to allow separation of urine and feces. All cages were sprayed with silicone to completely coat the metal surfaces, and prevent metal contamination of the urine.

dl-Norepinephrine { carbinol-C14) bitartrate with a specific activity of 31.0 mC/inillimole was purchased from Nuclear-Chicago division of Amersham-Searle Corporation. The isotope was obtain ed in crystaline form and dissolved with sterile isotonic saline at the time of use. Vials of 50 pc were diluted to 2.5 ml yielding a con centration of 1 }ic/50 pi. Aliquots were stored at -20°C.

Animals were pooled according to weight for a given drug treatment. Each animal was injected via the tail vein with 50 pi of the C14-norepinephrine stock solution. Theoretically, each animal should have received 1 |jC, but actual counting of this volume of radioactivity revealed that on the average 1 , 0 9 pC was administered. The animal received the injection at time zero and was immediately placed in a metabolism cage for the collection of urine samples.

Urine was collected at two hour intervals for 18 hours. A sus tained diuresis was induced by giving the animals 1 0 m l of a 2 °/ glucose solution made in isotonic saline at the beginning of each collection period. Five ml was also given to each animal 30 min utes prior to the injection of radioactivity to initiate the diuresis. By loading the animal, approximately 10 ml of urine was recovered during each two hour period. The urines were collected in poly ethylene bottles, which contained 25 mg of ascorbic acid, 50 mg of EDTA, and 0.5 ml of a pool of norepinephrine and its five major metabolites, each at a concentration of 100 pg/nl. For each col lection period, the metabolism cage was thoroughly rinsed into the plastic bottle and the urine sample was diluted to 50 ml. This dilution resulted in a final concentration of carrier compounds of 1 ng/ml. From this sample, two 0.5 ml aliquots were withdrawn for total urine counts, while the remainder of the sample was frozen until analyzed.

B. Analysis Interval By glucose-saline loading of the animals, approximately 75 °/0 of the total administered radioactivity was excreted in 30 hours ( see

Figure 6 ). A lm ost 60 °/0 of the isotope had been excreted in eight hours, while it took almost an additional 22 hours for 15 9/0 m ore of the C1 4 to be excreted. This exponential type of excretion pattern is typical in the case of injection of both optical isomers of nor epinephrine (Kopin and Gordon, 1963) .

It has been demonstrated that a considerable portion of admin istered Hs-norepinephrine is bound to tissues (Whitby et al., 1961) and appears in the nerve ending (Wolfe et al., 1962 ). The fate of the bound portion of the injected H3-norepinephrine has further been shown to best approximate that of the endogenously formed neuro transmitter (Kopin and Gordon, 1963). For this reason, control experiments were run in order to decide which time period should be analyzed for the metabolites so it could best reflect that of endogenous Figure Figure CUMULATIVE % OF ADMINISTERED RADIOACTIVITY EXCRETED lOO-i 50- 25- 75- 6 : Cumulative excretion of in the urine of of urine in the .ratsfollowing of excretion Cumulative the intravenous injection of 1 pc of d,l-C^ -norepinephrine. d,l-C^ of of 1 pc injection intravenous the 4 8 12 HOURS 16 20 69 0 3 70 norepinephrine. For this reason, it was desirable to analyze radio activity excreted in the urine which had been bound and therefore, most likely represented the natural 1 form which had been taken up into nerve ending and released. A kinetic analysis of the excretion pattern of radioactivity which is depicted in Figure 7 revealed at least two apparently distinct phases of isotope excretion. Following an initial rapid excretion of radioactivity, the rate of excretion be comes slower, and after 1 0 - 1 2 hours the rate decreases exponen tially, in a single phase with a half-life of about seven hours. The period of initial rapid excretion represents that norepinephrine which was circulated and metabolized and also includes the bulk of the d isomer which is rapidly excreted (Kopin and Gordon, 1962), The slower phase of excretion represents C 14 -norepinephrine which was continually released from the tissues and had mixed with endogenous stores of this catecholamine (Whitby et al., 1961; Kopin and Gordon, 1963). From the curve, it can be seen that curve B can be construc ted by subtracting curve A ( slow exponential process) from the entire curve. Curve B represents the pure curve for the rapid process, and shows that the d form of the isotope is essentially eliminated at about 12 hours. For this reason, the samples excreted from 1 2 - 1 6 hours after injection of the isotope were used to analyze for the metabolites of norepinephrine. Therefore, the two 50 ml samples (12-14 hours and 14-16 hours)were combined, and the entire 100 ml sample was assayed. These samples were combined in order to insure sufficient counts for a valid analysis, on the aver age the combined sample contained about 1 0 0 , 0 0 0 cpm 's.

C. Treatment Schedules After selection of the interval for analysis, various drug treatments were given to observe effects on the metabolism of C14- norepinephrine. One group of rats received ethanol, 4 gm/Kg, 71

J 0.5-

HOURS

Figure 7: Rate of urinary cp-bon-14 excretion following administration of 1 pe of d,l-C^-norepinephrine Intravenously. The total radioactivity excreted during the collection interval is expressed as percent of the administered dose excreted per hour* The mean values obtained in six experiments are •plotted at the midpoint of the collection intervals I.______•). Curve A - represents a tangent to the slow exponential process extrapolated bach to time zero* Curve B - represents the difference between curve A and the actual rate of excretion. 72 orally at the beginning of the analysis interval ( 12 hours). Another group was given three consecutive intraperitoneal injections of acet aldehyde, 300 mg/Kg, These animals were dosed at 12,. 12 1/2, and 13 hours. Other animals were pretreated with an aldehyde dehydro genase inhibitor, disulfiram or calcium carbimide. Some animals were given disulfiram 200 mg/Kg by mouth, 20 hours and at four hours prior to the analysis period, which represented eight hours before and eight hours after the administration of C 1 4 -norepinephrine. A portion of the animals which received disulfiram were also given 4 g/Kg of ethanol at the 12 hour time period. Similarly, citrated calcium carbimide ( Temposil^ ) was administered i. p. at a dose of 50 mg/Kg at six hours and again at two hours prior to the analysis period. Control animals were either treated with i. p. injections of saline, 3 ml/Kg, or given oral saline, 16 ml/Kg. All controls were finally pooled (See Results) because the two treatments did not significantly alter the results.

D. General Procedures The method used for the separation of norepinephrine and its five major metabolites is that of Davis et al ( 1967b) . It consists of a combination of various techniques, which by judicious alteration of columns, for the first time allows a precise and simple method for determination of all the metabolites of radiolabeled norepinephrine. Essentially, the methodology encompasses the alumina adsorption method of Weil-Malherbe and Bone ( 1952) for the estimation of free catechols, combined with an assay for C14-normetanephrine reported by Maas, and Landis ( 1968) . Radioactive catechol deaminated m et abolites were assayed for alumina eluates by modifications of the method of Kopin, Axelrod and Gordon ( 1961). 73 A weakly basic anion exchange resin, Amberlite CG-4B, 100 to 200 mesh, which is available only as the hydroxide form (Mallin- ckrodt No. 3343) was used. This is converted to the acetate form before use and at a pH 4.5. This form allows for easy exchange of the acid metabolites. Similarly, a cation exchange resin,- Dowex- 50 in the hydrogen form (x2, 100-200 mesh) was converted to the ammonium form with 3 N ammonium hydroxide treatment overnight. The resin was then washed with deionized water until the effluent was pH 7.0, and then resuspended in water and refrigerated. "Woelm" Neutral Chromatographic grade of aluminum oxide was used for cat echol adsorption. The chromatographic columns which were used consisted of a 6 by 120 mm tube with a 16 by 150 mm reservoir. All materials were assayed by counting by liquid scintillation spectrometry in 20 ml of a mixture of toluene; triton (2;1) . The phosphors were dissolved in toluene before mixing with triton, 1 2 g of 2, 5-diphenyloxazole (PPO) and 150 mg of 1, 4-bis-2-( 5-phenyl- oxazolyl) benzene ( POPOP) .

E. Determination of Cl4-Norepinephrine and Normetanephrine

F ig u re 8 outlines the procedure for the determination of total urine norepinephrine (NE) and normetanephrine (NM) in acid hydro- lyz ed samples. Four 5 ml aliquots of the total 100 ml urine samples were placed in beakers, 5 ml of deionized water added, and adjusted to pH 1 with 6 N HC1. The acid hydrolysis was carried out in a steam bath for 20 minutes. The samples were cooled to room tem perature, two of the aliquots adjusted to pH 5.5 with NaOH, and each sample of hydrolyzed urine passed through a 6 by 6 0 mm column of Dowex-50-NH4 , The columns were rinsed with two consecutive 5 ml water washes. C14-NE and C14-NM which had exchanged on the resin were eluted with 20 ml of 3 N NH 4 OH into counting vials. 74

ACID HYDROLYSIS

I urime ! ADJ: pHl, 6 NKCI STEAM BATH; 20MIN.

1 ALUMINA~| REMOVES DIHYDROXY COMPOUNDS pHj5.5 NE . ELUTE WITH + „ ^ I L ^ l i iDOWEX^o1 |DOWEX.-SOj-' H 3N NH4OH '------J NM

. Figure 8: Schematic diagram illustrating the method for Isolation of Cl^-norepinephrine (NE) and C^-normetanephrine (NM) in urine. The two other aliquots were quantitatively transferred to 0.7 grams alumina columns which removed NE and other catechols. The effluent was then passed through a Dowex-50 column which picked up NM, the only free amine remaining. For this procedure, the two aliquots were adjusted to pH 8.4 and then passed through the alumina. The effluent urine and a 5 ml sodium acetate buffer (pH 8.4) wash was collected, adjusted to pH 5.5 and quantitatively transferred to Dowex-50 columns. Columns were washed and eluted as described above. Thus, for each sample duplicate analyses of NM, and the combination of NE plus NM were determined. The val ues for C14-NE were obtained by the differences for the data obtained from these two Dowex-50 eluates.

F. Determination of Deaminated Cw-Metabolites For the determination of all deaminated metabolites, 65 ml of urine underwent enzymatic hydrolysis. After adjusting the urines to pH 5.5, 1.0 ml of Glusulase^ (Endo Laboratories) was added to each containing 1 0 0 , 0 0 0 units of beta-glucuronidase and 50, 000 units of sulfatase per milliliter of preparation. A graphic outline of the general isolation procedures is illustrated in Figure 9. Glusulase was added and the samples were incubated at 37°C for 32 hours. Four 15 ml aliquots of the hydrolyzed urine were placed on Dowex-50 resins and the effluents collected. Dowex-50 quan titatively removed the free amines, NEand NM.

Two of the aliquots were adjusted to pH 4,5 and passed through duplicate amberlite-CG-4B resin columns with the dimensions of

6 by 100 mm. The effluents from the CG-4B columns were collected with a 3 ml water wash, and the columns were then washed twice with 5 ml of water and this rinse was discarded. This weakly basic

anion exchange resin picked up C 1 4 -3-mcthoxy-4-hydroxymandelic 76

GLUSULASE HYDROLYSIS

I"" URIfJE I ADJ: pH 5.5 ADD:lm t GUUSULASE

[d 5 w EX-501 REMOVES FREE AMINES: NE + NM pH 8.4/ Y H4'5 DHMA ELUTE WITH ELUTE WITH. VMA | a l u m in a | f CG-4B 1- DHPG 0.5N HCI 3N NH40H DHMA pH|4.5 pH|B.4

ELUTE WITH r ^ n <-e l u t e w ,th 1 CG-4b1 I a lu m in a I > |DHPG | 3N NH4OH 0.5N HCI pH 17.0 PHJ7.0

ETHYL ACETATE ETHYL ACETATE |m h p g |< EFFLUENT EFFLUENT » 1mhpg| EXTRACTION EXTRACTION

Figure 9! Schematic diagram illustrating the method for isolation of C1 -deaminated metabolites of norepinephrine in urine (see text for abbreviations). 77 acid ( VMA) and C 1 4 -3, 4-dihydroxymandelic acid (DHMA) . These compounds were subsequently eluted with 20 ml of 3 N NH 4 OH into counting vials.

The effluent from the CG-4B columns was adjusted to pH 8,4 and placed on aluminum oxide columns. The effluents from these were collected with a 5 ml acetate buffer wash in a 25 ml vol umetric flask. These columns were washed with water and dis carded, The alumina column picks up dihydroxy compounds, the only one which is remaining in these samples is C 1 4 -3, 4-dihydroxyphenyl glycol (DHPG). These columns were subsequently eluted into counting vials with 20 ml of 0,5 N HCI. The effluent from the alumina column was diluted to 25 ml with water, the pH adjusted to 7.0 and two 10 ml fractions extracted with 40 ml of ethyl acetate for one-half hour. Four grams of NaCl was added to the aliquot for extraction. This extraction removed CI 4 -3-methoxy-4-hydroxyphenyl glycol (MHPG), 35 ml of which was evaporated to dryness in a counting vial. The efficiency of this extraction was 80 0/o

The remaining two 15 ml hydrolyzed urine samples which had been passed through the Dowex-50 columns were adjusted to pH 8.4 and passed through duplicate alumina columns. The effluent urine with a 5 ml acetate wash was collected. The columns were washed twice with 5 ml of water which was discarded. The alumina picked up the catechol compounds, DHMA and DHPG, which were eluted into counting vials with 20 ml of 0.5N HCI.

The effluent was adjusted to pH 4.5 and passed through a column of CG-4B resin. The effluent and a 3 ml water wash was collected in a 25 ml volumetric flask and adjusted to volume. The VMA exchanged on the CG-4B and was eluted with 20 ml of 3 N N^OH* Two 10 ml fractions of the effluent were again extracted for MHPG as described above. 78 The use of these exchange resins, alumina, and solvent ex traction in the sequence described allows for the isolation of the individual C14-NE metabolites: VMA, MHPG and DHPG. Besides duplicate samples for each determination, a double check is made on the amount of DHMA. This metabolite is calculated in two ways. It can be obtained by subtracting the alumina eluates containing DHPG from those which represent DHPG plus DHMA. Comparable values for DHMA were also calculated by subtracting VMA values from CG-4B eluates containing both VMA and DHMA. Similarly, there was a double check on the MHPG which had been isolated by two different techniques. All eluates from the columns were evapor ated almost to dryness ( about 0.5 ml) in a vacuum oven before addition of the scintillation mixture. All data were corrected for the percent recovery of standard compounds taken through the procedures, The recovery of compounds from the ion exchange resins and alumina was approximately 90 /£. All results were likewise corrected for counting efficiency.

G. Identification of C14-norepinephrine and Metabolites The material isolated on the ion exchange resins, alumina columns, and with ethyl acetate extraction were identified by thin- layer chromatography. The material used for the plates was 10 g of cellulose ( 300) with 75 ml of water. The materials from the various fractions were compared with nonradioactive standards which were obtained from Cal-Biochem. The column eluates were all treated with 50 ml of acetone which was evaporated to near dryness and plated with capillary pipettes. Zones of radioactivity were de tected by a Packard Chromatogram Scanner or by spraying the plates with p-nitroanilin. A comparison of the Rf values for the various fractions of C14-metabolites with that of standard non-labeled com pounds is shown in Table 8 . TABLE 8

THIN LAYER CHROMATOGRAPHY OP NOREPINEPHRINE AND METABOLITES0,

Ff. C3^ % Chemical Compound Standard

NE 0 .1*2 0 .1*2 + NM 0.52 0.51

DHMA 0.59 0.57

DHPG 0.65 0 .6 *

DHPG 0.63 0 .6 1*

VMA (acid) 0 .7 I* 0.73 + DHMA (acid) 0.57 0.58

VMA (NH^ salt) 0 .1*8 0 ,1*8

MHPG 0.79 0.80

a The solvent system used consisted of butanol: 5N acetic acid (3:1) while the plate material consisted of 10 gms of cellulose (300) in 75 ml of water (see text for abbreviations). 8 °

The best solvent system found for separation of all metab olites was a butanol; 5N acetic acid (3:1) which was described by Giese et al ( 1967) . For sharp separation of the substances it is important to saturate the cellulose plate for 30 minutes above the solvent. The column eluates or extracts containing VMA, MHPG or NM showed single peaks corresponding in Rf to authentic stand ards, which had been treated in a similar manner. Elution of VMA and MHMA from the anion exchange resin with ammonium hydroxide resulted in the formation of the ammonium salt having R /s less than the free acids. Acidic properties could be restored by treating the salts with HCI, in this way Rf's identical to the free acid stand ards were obtained. All eluates which contained two metabolites correspondingly demonstrated a double peak. These eluates have also been identified by paper chromatography using three other solvent systems and those findings concur with the results shown here (Davis et al., 1967b).

Evaluation of Central Nervous System Effects Adult male albino Swiss mice, weighing 20-26 g were used for these studies. In all, 250 mice were utilized, all of which were maintained three days prior to use and permitted free access to food and water. Ethanol was administered orally as a 20°/ w/v solution, whereas acetaldehyde was given intraperitoneally as a 2 w/v solution. The doses of these agents were varied in order to . construct dose response relationships and compare potencies in producing hypnosis and in causing toxicity.

In studies on the potentiation of alcohol induced narcosis, the duration of hypnosis or "sleeping-time" was recorded as the in terval between loss and return of the righting reflex. Animals were placed on their backs and were not handled until they had spontaneous ly righted themselves three consecutive times within 6 0 seconds. 81 Central nervous system effect of these agents were assessed mainly by observation, and recorded as producing ataxia and motor incoordination, sedation and depression, sleep and hypnosis, or death. In this way the effects of various agents on the central actions of acetaldehyde and ethanol were evaluated. RESULTS

Ethanol and Acetaldehyde Levels

A. In Vitro Production in Whole Blood Truitt ( 1969) has rediscovered that acetaldehyde is pro duced when ethanol is added to precipitated whole blood (See In troduction) . We have examined this ’’release” of acetaldehyde by incubating precipitated blood of various species with ethanol ( 2 , 0 m g/nl). The results are shown in Table 9.

Blood samples were drawn, precipitated with ZnS 0 4 -Ba( OH) 2 and incubated at 55°C for 15 minutes before GLC analysis. There seems to be a greater production of acetaldehyde in man, monkey and cow, than in the dog, rabbit, rat or mouse. However, with longer incubation times, large amounts of acetaldehyde are pro duced in all species. Because of the possibility of artifactual pro duction of acetaldehyde in blood when ethanol is administered in vivo and then assayed, protein-free filtrates have been analyzed in all species studied in these experiments,

B. Human Filtrate Levels This artifactual production of acetaldehyde in whole blood prompted a re-evaluation of the levels of acetaldehyde in humans after ethanol. Four human subjects received BO proof Vodka mixed 2;1 with grapefruit soda yielding a 17 a/0 v/v ethanol concentration. Each subject received 0,75 gm/Kg of ethanol which was consumed over a 20 minute period. Duplicate protein-free filtrate samples

82 TABLE 9

EFFECT OF ETHANOL (2 ,0 0 0 ^ /m l) ON THE IN VITRO PRODUCTION OF ACETALDEHYDE IN WHOLE BLOOD SAMPLES OF VARIOUS SPECIES

Acetaldehyde (^/ml) Blank-no Ethanol n Ethanol Added

Mouse 10 (pooled) 0 0

Rat 7 (pooled) 0.17 0 .1

Rabbit 5 0 0.12

Dog 5 0 .1 0 .1

Cow 6 0.13 4.51

Monkey 8 0.11 4.86

Human 12 0.36 3.42 84 w ere analyzed at 0, 30, 60, 90, 120, 180 and 240 m inutes by GLC as previously described.

The data obtained in four human subjects are illustrated in Figure 10. Ethanol blood levels appeared to peak from 30 to 60 minutes. The peak ethanol levels in these subjects ranged from 880 to 1,325 In general, the blood alcohol curve demonstra ted a linear decline over the course of the next three hours. Fitting the best curve to the mean values for ethanol in these subjects, and examination of the slope yields an approximate rate of decline of the ethanol level in blood of 130 pg/nl/hr and demonstrates a plasma half-life of about 3.5 hours.

The acetaldehyde levels in these subjects range from 0 to 0.55 \sg/mh There is no correlation in the sex or age of the subject as to the levels of acetaldehyde produced. In two of the subjects, CH3CHO levels seem to rise at the four hour period, while two fall at this time. The marked differences in levels in these subjects, poses the question of what these levels represent. One would expect a relatively constant circulating level as long as ethanol oxidation occurs, but this is not the case. Figure 10 demonstrates a marked variability in the acetaldehyde blood level even in a single individual as well as among various subjects.

C. Blood and Tissue Concentrations in Rats

Rats were given ethanol by stomach-tube at a much higher dose {4 gm/Kg)than were human subjects. This dose produced symptoms of intoxication in all rats. Blood and tissues were anal yzed at one and two hours after the administration of ethanol. The blood levels of ethanol and acetaldehyde produced in rats are tab ulated in Table 10, The ethanol levels at these two time periods were essentially the same, whereas, acetaldehyde levels increased 85

1400 * 1200

2 6 0 0 “

iu 0 . 2

6 sfo & 9*0 120 MINUTES

Figure 10: Blood levels of ethanol and acetaldehyde (V/ml) obtained from protein-free filtrates from four human subjects given ethanol per os, 0,75 gm/Kg, The type of line drawn for the ethanol and acetaldehyde curve Is the same for a given subject. For example, (•— — — — •) represents the ethanol and acetaldehyde levels obtained from one subject. TABLE 10

ACETALDEIDTDE AND ETHANOL BLOOD LEVELS FROM PROTEIN-FREE FILTRATES AFTER ETHANOL (4 gm/Kg p.o.) Ill UI'ITREATED AND DISULFIRAM PRETREATED RATS

if/ml ± S. E. n Ethanol Acetaldehyde

CONTROL

1 hour 12 1,968 ± 281 1.17 ± 0.36

2 hours 12 1,925 ± 169 2,65 ± 0.32

DISULFIRAM

1 hour 10 2,918 ± 196 14.63 ± 1.19

2 hours 10 3,431 ± 196 19.6k ± 2.84 87 at two hours as compared to the one hour levels. The acetaldehyde levels were quite consistant in these animals, and individual var iability was much less than with the human data. The mean one hour levels were 1.17 ± 0.36 pg/nl, while the two hour levels were m ore than doubled, 2.65 ± 0,32

Administration of disulfiram to rats caused a significant in crease in both the ethanol and acetaldehyde blood levels. The ethanol levels at one hour ( 2 , 9 1 8 ± 1 9 6 }jg/4nl) and at two hours (3,431 ± 196 jjg/nl) represent a 50 and 80 °/0 increase respectively in the ethanol levels of disulfiram pretreated rats as compared to untreated rats (p < 0,001), Unlike the controls, where the ethanol levels remained constant at one and two hours, the level increased at two hours in the disulfiram pretreated animals,

Similarly, disulfiram, which acts by inhibiting aldehyde de hydrogenase, produced elevated acetaldehyde levels. Pretreatment with disulfiram increased the one hour blood levels 12.5 times (1.17 to 14.63 |jg/4nl), and the two hour blood levels 7.4 times (2.65 to 19,64 pg/knl). These increases in acetaldehyde blood levels produced by disulfiram were significant at the p < 0 , 0 0 1 level.

On the premise that tissue levels may be more pertinent than blood levels to the pharmacologic actions of acetaldehyde, nine different tissues in rats were examined. Table 11 gives the mean values of twelve animals of ethanol and acetaldehyde concentrations (in (Jg per gram of tissue) . The ethanol concentrations were similar in any given tissue at one and two hours. Tissue levels of ethanol were slightly less than blood levels; that is, ethanol did not concen trate or accumulate in any of the tissues examined. The tissue to blood ratio for ethanol was less than 1.0 in every case. In two of the tissues, the epididymal fat pad, and the gastrocnemius muscle, eth anol concentrations were considerably less than the blood concentrat ion. TABLE 1 1

CONCENTRATION OF ETHANOL AND ACEIALDEHTDE IN TISSUES OF RATS AFTER ETHANOL (4 gm/Kg p.o.)

fw,ni Tissue One hour (n^6) TVo hours (n=6) Tissue______Ethanol______Acetaldehyde______Ethanol______Acetaldehyde

Brain 1612 1.86 1315 3.32

Liver 1366 13.18 1212 19.16

Kidney 1660 4.63 1559 5.34

Small Intestine l4i6 4.51 1643 7.24

Lung 1648 0.401 1273 1.075

Heart 1525 0.466 1072 1.65

Spleen 1584 0.43 1338 0.61 .

Fat Bad 523 0.337 474 0.442

Skeletal Muscle 599 0 660 0

a> 00 89 Concentrations of acetaldehyde in the tissues demonstrated a distribution quite different from ethanol. Acetaldehyde appears to concentrate or be produced in several tissues. Four tissues demonstrated a tissuerblood ratio greater than 1 . 0 at one and two hours respectively after ethanol administration: (Liver 11.2 and 7,2), kidney (4.0 and 2.0), small intestine (3.9 and 2.7 ) and brain (1.6 and 1.25). The other tissues showed much lower concentrations of acetaldehyde than are found in the blood, with no detectable amount found in skeletal muscle.

The distribution of ethanol and acetaldehyde in the tissues of rats pretreated with disulfiram is illustrated in Table 12. As with untreated animals, the concentration of ethanol in these tissues more or less reflected that in the plasma except for adiposetissue and skeletal muscle. As a result of pretreatment with disulfiram, the levels of ethanol in the tissues were very much increased. Also, like the blood concentration, the tissue levels at two hours are slightly higher than the one hour level for every tissue examined.

The acetaldehyde levels were high again in the same four tissues (Table 12) . The values were at least double that found in unpretreated animal tissues at both one hour and two hours after alcohol administration. Again, the two hour concentrations tended to be higher than the one hour. The tissue levels did not increase as much as the blood levels, when disulfiram was given. In this case all tissues tended to be less than the blood concentration, except for the liver at one hour, and the liver and small intestine at two hours.

Because of the artifactual production of acetaldehyde which occurs in blood, it was thought that blank tissues from control animals as well as tissues with ethanol added to them should be checked. The results of these types of tissue controls are shown in Table 13. Most of the tissues have negligible to no acetaldehyde detectable by the TABLE 1 2

CONCENTRATION OP ETHANOL AND ACETALDEHYDE IN TISSUES PROM DISULFIRAM PRETREATED RATS AFTER ETHANOL (4 gm/Kg p.o.)

^/gra Tissue One hour (n=6) Two hours (n=6) Tissue Ethanol______Acetaldehyde______Ethanol Acetaldehyde

Brain 2662 3-32 2937 4.12

Liver 2322 26.63 3797 33-08

Kidney 2357 9-87 3020 14.76

Small Intestine 2412 9-24 4039 23.16

Lung 2502 1.05 3520 2.18

Heart 2231 1.85 4133 3-80

Spleen 1944 0.826 2493 1.71

Jfct Rad 535 1.24 1281 1.61

Skeletal Muscle 941 0.07 2021 1.37 TABLE 1 3

IN VITRO PRODUCTION OP ACETALDEHIDE FOLLOWING THE ADDITION OP ETHANOL TO PRECIPITATED RAT TISSUE HOMOGENATESa

Acetaldehyde Tissue Ethanol Blank Added Tissue (*=k)

Brain 0 0.12

Kidney 0 6.65

Lung 0 0.764

Heart 0 0.695

Liver 0.273 10.20

Spleen 0 0.208

Fat 0 0.210

Small Intestine 0.197 0.278

Skeletal Muscle 0 0.208

a All tissues vere equilibrated at 55 #C for 15 minutes before analysis by GLC.

^ Ethanol was added to precipitated homogenates at a concentration of 2*5 mg/gm of tissue. GLC method. However, when ethanol is added to certain tissues, acetaldehyde is produced. Ethanol in a concentration of 2,500 \ig/g was added as a mean concentration found in all tissues after ethanol (both untreated and disulfiram pretreated) . The addition of this amount of ethanol caused substantial amounts of acetaldehyde to be produced in liver and kidney and less in other tissues. This occurs despite the fact that the ethanol is added after the homogenate has been precipitated with ZnS0 4 -Ba( OH) 2. The concentration of acetaldehyde produced by the in vitro addition of ethanol was very similar to (liver, spleen) or greater than (kidney, heart, lung, skeletal muscle) that produced in vivo. Only in the case of brain, small intestine, and the fat pad was the production of acetaldehyde less than that found at one hour after ethanol administration to rats. This in vitro production was not due to an interaction of the precipi tant with the tissue, since the blank tissues failed to show acet aldehyde production. Also, ethanol was needed in the homogenate in order to have acetaldehyde produced. Since the in vitro product ion of acetaldehyde was greatest in the liver, several experiments were performed using this tissue only. Of course, this assumes that the source of acetaldehyde in all tissues is the same, and that the liver can be used as a prototype. A compilation of these results is shown in Table 14. To rule out a non-specific effect of the type of precipitants used, precipitation with ZnS0 4 and B a(O H ) 2 was used along with acid precipitation using trichloroacetic acid ( TCA) . It can be seen that regardless of the precipitant used, the results were comparable. If standards were prepared by adding 2.5 (Jg/g of acet aldehyde to the liver homogenate, the peak heights obtained were similar (within 10% difference). Precipitation with base (Zn-Ba) gave a peak height of 16.9, while precipitation with acid (TCA) yield ed a peak of 18,5, Thus, the actual level of acetaldehyde was not TABLE l 4

EFFECT OF VARIOUS PROCEDURES ON THE PRODUCTION OF ACETALDEHYDE IN RAT TISSUE HOMO SENATES6,

Vial Contents**'c Zn-Ba TCA

Rrecipltants Only 0 0

Ethanol + Precipitants 0 0

Liver + Precipitants 0.24 0.1

Liver + Ethanol + Precipitants 9.1 8.13

Supernatant of Liver + Ethanol + 5.1 5.* ftrecipitants - 1500g x 10 roin.

Liver from Ethanol Treated Rat 12.13 9.85 (4 gm/Kg for one hour)

Brain + Ethanol + Precipitants 1.97 2.64

Brain from Ethanol Treated Rat 2.29 2.32 (4 gm/Kg for one hour)

a Two protein precipitants are compared, 5$ ZnSO^ plus 0.3N Ba(OH)p-(Zn-Ba), and 10# trichloroacetic (TCA).

In all cases, ethanol was added at a concentration of 2.5 nig/gm of tissue.

c Addition of ethanol to an unprecipitated liver homogenate resulted in the production of 29.3 ps of acetaldehyde/gm of tissue. 94 affected by these two precipitants. GLC analysis of the precipitants alone or precipitants plus ethanol did not produce acetaldehyde. Control liver tissue levels were low with both precipitation pro cedures. However, when ethanol was added to the precipitated liver homogenates, a marked production of acetaldehyde was seen. These levels were comparable to those found in liver homogenates from rats given ethanol (4 g/Kg) one hour prior. The in vitro and in vivo levels ( also in brain) were similar, regardless of the type of precipitation used. Centrifugation of the precipitated homogenate containing ethanol and analysis of the supernatant seemed to decrease the amount produced, but the data is insufficient and would need further verification.

Table 15 demonstrates that the amount of acetaldehyde pro duced spontaneously is directly related to the concentration of ethanol added. In general, the acetaldehyde produced increases with the length of time the samples are maintained in the water bath at 55°C. The lower concentration of ethanol was used, to see if we could show definitely that the acetaldehyde that was produced was or was not generated from ethanol. The sensitivity of this GLC method for ethanol was not sufficient to obtain this information. The peaks ob tained when ethanol is added at a concentration of 1 0 0 y/g were ex tremely small and not sharp. Yet, if larger concentrations of ethanol were used, a change due to the formation of acetaldehyde from ethanol could not be detected. Ten to twenty \ig/g of acetaldehyde

produced would be less than a 1 0 °/0 change in the ethanol concentration, and would be within the normal range of error of the machine.

Attempts were made to block the production of acetaldehyde by homogenizing the tissue in saline containing either ascorbic acid

( 1 m g/nl) or one of the chelating agents Ethylenediamine tetra- acetic(EDTA, 50 mg/100cc), or dimercoprol ( BAL, 50 mg/100 cc), TABLE 1 5

EFFECT OF TIME OF EQUILIBRATION AND CONCENTRATION OF ADDED ETHANOL ON THE IN VITRO PRODUCTION OF ACETALDEHYDE IN RAT TISSUE HOMOGENATES?

Added Ethanol = 100 Vtfm. Added Ethanol = 2500 'ffm. Tissue 15 min. 45 min. 15 min. 45 min.

Brain 0 . l 4 0 . 4 8 0.12 0.66

Liver 2.96 4 . 1 7 10.20 1 4 . 3 0

Kidney- 0.76 1 . 1 1 6.65 7 . 2 3

Small Intestine 0 0 0 . 2 8 0 . 3 5

Lung 0 0 0.761* 0 . 9 7 0

Heart 0 . l 4 0.28 O .69 1.11

Spleen 0 0. 0.18 0.21

Fat Bad 0 0 0.21 0 . 4 8

Skeletal Muscle 0 0 . l 4 0.21 0.28

a This experiment represents the tissues of a single rat, with each time period carried out in triplicate. 96 None of these agents were found to block the production of acet aldehyde as is shown in Table 16. In most cases the concentration produced was greater than the control tissue homogenized only in saline.

D. Urine and Expired Air In order to avoid the problems of artifactual production of acetaldehyde in blood, urinary and expired air concentrations were obtained. Table 17 compares results obtained by measuring acet aldehyde at various time periods after the injection of acetaldehyde (30m g/Kg). One method ( Lester and Greenberg, 1950), is to in ject 25 ml of air subcutaneously in the back of the rat and to remove 0,5 ml samples from this air sac at various time intervals. Acet aldehyde concentrations can be measured in this manner. After intravenous injection, acetaldehyde is rapidly metabolized and is undetectable in blood after ten minutes. These values are more or less in agreement with the blood levels obtained in rats, but the air pocket concentrations are several fold higher. Measurement of ex pired air and analysis by GLC ( Freund, 1967) also yeilds comparable results showing the rapid rate of metabolism of acetaldehyde. Urinary levels seem also to reflect blood concentrations quite close ly. Values obtained in the urine at 10, 30 and 60 minutes were 4.3, 2.54 and 1.84 yg/ml of urine respectively. The slow decline implies that acetaldehyde is slightly reabsorbed, but the urine in general tends to trap the excreted acetaldehyde, and is detectable there even when blood levels have fallen to essentially zero,

A comparison of chamber,blood and urine levels after admin istration of ethanol was also made (Table 18). The concentration of ethanol in blood and urine is approximately the same. Acetaldehyde levels in urine were about one-third that of the blood concentrations. TABLE 1 6

EFFECT OF ASCORBIC ACID AMD CHELATING AGENTS ON THE IN VITRO PRODUCTION OF ACEEALDEHTDE IN RAT TISSUE HOMOGENATES^

Acetaldehyde (Tf/pjn Tissues) Tissue Control Ascorbic Acid EDTA BAL

Brain 0 . 4 8 5 0 . 6 5 8 0 . 6 1 8 0 . 5 2

Liver 1 0 . 3 5 2 1 . 4 2 5 . 2 2 2 . 2

Kidney 1 1 . 0 8 8 . 2 5 1 3 . 4 1 2 . 1

Small Intestine 0.250 O .89 0 . 6 7 0 . 8 3

Lung 1.94 1 . 1 8 2 .5 2 3*34

Heart 1.18 I.805 1 . 9 3 0 . 4 6

Spleen 0 . 2 1 0 . 4 4 O .85 1 . 1 0

F a t P a d 0 . 2 9 1 . 4 2 0 . 3 5 1.50'

Skeletal Muscle 0 . 1 3 0 . 2 7 0 . 5 5 0 . 3 7

a This experiment represents the tissues of a single rat with each value the mean of triplicate samples.

^ Ethanol was added to precipitated homogenates at a concentration of 2.5 mg/gm of tissue. TABLE XT

COMPARISON OF ACETALDEHYDE LEVELS IN BLOOD, URINE, EXPIRED AIR AND WITHIN A SUBCUTANEOUS AIR POCKET AT VARIOUS TIMES AFTER i.v. ACETALDEHYDE, 30 mg/Kg

Acetaldehyde '^'/mL Time Air Pocket Expired Air*1 BloodC Urine

2.5 min. 1 & . 0 0.0033 (30)* 1 0 . 6 —

5 min. 15-0 0 . 0 1 6 (126 ) 2 . 3 7 —

1 0 min. 0.0 0.0 — ^-3 (9-5)

30 min. 0.0 — 0.0 2 .5^ (k.H)

60 min. -- — -- 1.8h (3.68)

a As measured in 9 , 0 0 0 ml glass metabolism chamber.

^ Values in parentheses are total micrograms excreted in expired air or urine for that time period.

c Blood acetaldehyde was measured in protein-free filtrates. TABLE 1 8

COMPARISON OF ACETALDEHYDE AND ETHANOL LEVELS IN BLOOD, URINE AND EXPIRED AIR AFTER ETHANOL (4 gm/Kg p . O . ) IN UNTREATED AND DISULFIRAM PRETREATED RATSa

Ethanol Ethanol + Disulfiram

Blood* Urine Air Blood Urine Air

Volume (nil) 6.2 7.2 9,oooc 6.2 7.5 9,000 Acetaldehyde ('T/ml) 2.20 0.71 —0.0001 17.55 7.5 0.01 Total (Y) of Acetaldehyde 12.84 5.13 —1.0 108.8 70.4 93.5 Ethanol ('T/ml) 1,733 1,756 0.32 3,447 3,193 0.6 Total (f) of Ethanol 10,744 12,643 2,831 21,371 23,943 5,190

a All values are taken at one hour after ethanol administration.

^ Blood represents protein-free filtrate measurements, c Represents total volume of glass metabolism chamber.

>o 100 This was true both in rats receiving ethanol only, and in those re ceiving ethanol with disulfiram. These data also illustrate the fact that in the presence of disulfiram the levels of ethanol in blood, urine and breath are about twice that normally attained after alcohol administration alone. Acetaldehyde levels were also increased 8-10 fold in blood and urine by disulfiram pretreatment. Levels in the expired air were markedly enhanced by disulfiram pretreatment.

The reliability of measuring pulmonary excretion in small experimental animals was more closely examined. Animals were housed in a glass metabolism cage and measurements made as pre viously described. Figure 11 illustrates mean acetaldehyde values obtained from six rats injected with 30 mg/Kg of acetaldehyde i. v. when the chamber air was sampled at various time intervals. The total amount of acetaldehyde increased linearly for five minutes and then began to decline, After 60 minutes the level in the chamber had reached zero (undetectable < 1.0 ptg) . Acetaldehyde which had been excreted was presumed to be reabsorbed by the lung and metabol ized. The peak air level coincides well with the blood concentration curve, because five to ten minutes after injection of this dose the blood level has reached almost zero. There is quite good repro- duceability and reliability in the values obtained as is shown by the relatively small standard errors.

Figure 12 shows data similarly obtained using six rats which were treated with 4 g/Kg of ethanol. The chamber air was sampled for two and one-half hours. The amount of ethanol excreted seemed to reach a maximum between 120 and 150 minutes. An approximate measurement of the rate of ethanol excretion by the lung can be cal culated from the slope of the curve. About 2,500 pg of ethanol were excreted per hour during this linear phase of excretion. The reach ing of a plateau may indicate that pulmonary reabsorption, urinary Figure 11: Average values for the accumulation and Average valuesaccumulationfor the and disappearance of 11:Figure I g> ± S.E. 100 125- 150-1 50- 75- - 1 3 5 4 3 2 1 0 acetaldehyde with in acetaldehydea airtime 9*000 with tight ml glass administration of 30 ofacetaldehydeadministration mg/Kg.i.v. chamber singlemetabolism containing rats(n=6) after MINUTES 10 TREATMENT: 0 2 ACETALDEHYDE 30mg/Kg I.V. 30 40 50

60 101 .Figure 12: Average values for the accumulation and values accumulation the for and disappearance Average of .Figure12:

EXCRETION OF ETHANOL (mg) t &E. 0 0 060 30 20 10 while the chamber air was sampled chambersampled intervals. while at variousthe air was ethanol with time in in aethanoltime tightwith 9,000 air ml glassmetaholism orally and immediately orally in airand the placed chamber,tight (n=6) ethanol Rats atreceived a dose of ^ gm/Kg. chamber. TREATMENT: ETHANOL p.o. A

Gm/Kg MINUTES 90 0 2 1 150

102

103 excretion and hepatic metabolism are working at approximately this rate to keep up with alveolar excretion. Using this method, no acetaldehyde was detectable in the chamber air because the sen sitivity of the method was insufficient to detect quantitites < 1 |Jg in the chamber (9.000 ml) . The relative sensitivity of the method, however, is quite good considering that concentrations of acetalde hyde as low as 0 , 1 nanogram/4nl of air could be detected.

The same type of experiment was performed using six rats previously treated with disulfiram and then given ethanol. These data are depicted graphically in Figure 13. First, it can be seen that the rate of alveolar ethanol excretion has doubled to about 5,000 fJig/hr. This is further evidence that disulfiram is altering the rate of metabolism of alcohol. The maximum amount that is excreted by the lung is essentially the same ( about 2 °/0 of the ad ministered dose) for untreated and disulfiram pretreated rats. However, the maximum amount excreted is reached much more quickly in disulfiram pretreated rats (60 - 90 minutes) . The amount of acetaldehyde excreted reaches a maximum about the same time as ethanol. The fact that the slope and shape of the acetal dehyde curve is so close to the ethanol curve, is indirect evidence that all of the acetaldehyde is coming from the oxidation of ethanol, and the artifactual source of this metabolite seems to have been eliminated by this method of measurement.

Another pertinent point is that the maximum cumulative amount of acetaldehyde excreted (93.5 ± 9.8 pg) after the dose of ethanol in the presence of disulfiram is practically the same as that excreted when acetaldehyde ( 30 mg/Kg)was injected alone ( 125 £ 3. 1 |ig). Therefore, the levels of acetaldehyde after i, p. injection of 30 mg/Kg of acetaldehyde are close to the range of acetaldehyde values produced after an ethanol dose in rats pretreated with disul firam. . Figure 13: Average values for the accumulation and of disappearance the.and accumulation for values Average 13:Figure

CUMULATIVE PULMONARY EXCRETION OF ETHANOL — ( mg) ± S .E . TREATMENT! 0 0 3 0 2 10 DISULFIRAM + ETHANOL ETHANOL + DISULFIRAM .. Kg /K m G 4 P.O. ETHANOL hours and of the ethanol Uand prior administration hoursto at20 stomach tube of 200 mg/Kg* disulfiram received by Rats chamber. tight glass metabolism 9,000 ml air ethanol and asmeasured in acetaldehyde(n=6)a ethanol rats and hy (^gm/Kg.) at zero(^gm/Kg.) time.at EAL HYDE D Y EH LD CETA A 0 6 0 9 120 150

5 7 - u -100 -50 2

104

105 Experiments with the Isolated Guinea Pig Left Atria

A. Controls Baseline electrical and mechanical values obtained on left atria from eight untreated guinea pigs are shown in Table 19. The mean value for the magnitude of the action potential was 81.0 mV, These atria demonstrated a resting potential of 64.2 mV and there fore an overshoot of about 17 mV. The duration of the action potential was 124 msec, with an average area of about 2.1 mV-sec. The average length of the atria was 3.5 - 4.5 mm, with the electrode usually placed near the end of the atria that was attached to the strain gauge at about 3.0 mm from the stimulating electrode. Since the mean conduction time was 15.8 msec, the rate of conductance of the electrical impulse can be calculated, and is approximately 0 . 2 meters per second. The mechanogram for these atria showed the following characteristics: the mean developed systolic tension was 527 mg, while the average diastolic tension was approximately 645 mg, yielding a total tension of about 1172 mg. The time to peak tension or duration of the active state was 69.3 msec, while the total duration of the mechanical event lasted 228 msec. This was. almost twice as long as the electrogram. The mean latent period, time from stimulation to initiation of contraction, was 32 msec.

B. Effects of Norepinephrine (NE) and Tyramine

Responses to NE ( 10" 7 M) on atria from normal and reserpine pretreated guinea pigs are shown in Table 20. Norepinephrine administration altered only certain tissue characteristics. The area of the action potential (AP-area) increased in both untreated and reserpine pretreated atria, whereas the duration (AP-D) was not consistantly prolonged. This seeming discrepancy was due to the fact that changes in the A P-area occurred mostly in the upper one- half of the electrogram. This type of change is illustrated in the TABLE 19

CONTROL VALUES FOR THE VARIOUS PARAMETERS OF THE ISOLATED GUINEA PIG LEFT ATRIUM8,

Electrical11 c Mechanical Parameter Mean ± S.E. Parameter Mean ± S.E.

RP 64.2 ± 3.7 mV DT 527 ± 70 mg

AP 81.0 ± 4.5 mV dFi l4.6 ± 1 . 6 g/sec

dV/dt 51.4 ± 4.0 v/sec 1 0 . 1 ± 1 . 0 g/sec " g AP-D 1 2 4 . 0 ± 6 . 9 msec DT-P 6 9 . 3 i 2 . 6 msec

AP-Area 2.1 ± 0.4 mV-sec 33T-D 2 2 8 ± 25 msec

CT 1 5 . 8 ± 1 . 6 msec LP 32 ± 4.8 msec

a Atria were stimulated at a rate of 1 0 0 /min.

^ See Figure 3 for explanation of abbreviations.

c Values represent means for nine atria. TABLE 20

Effect of NE on various parameters of the Isolated guinea pig left atrium

Normal { n ■ 5) Rescri>ineb (n = A) P a r a m e te r * m e a s u r e d C on tro l NE 'f. Change Control NE 2£_

M e c h a n ic a l P T - m g 5 8 6 .0 1010. 0 4 7 3. 0* 6 6 6 .0 1 0 1 1 .0 4 5 2 .0 * < IF i-g /se c 15. 3 21.8 443. 0* 1 6 .0 .2 2 , 1 4 3 8 . 0* d * V g / s c c 8 . 9 1 2 .7 443. 0* 9 .7 1 3 .2 4 3 6 . 0* DT-F-mscc 80.0 80.0 0 6 8 ,0 6 8 .0 JO D T - D - m s c c 240, 0 240.0 0 240.0 240.0 0 LP-mscc 35.0 3 5 .0 0 3 2 .0 3 2 .0 0

*Scc figure 3 for explanation of abbreviations. ^Rcscrpinc [3 mg/kg] 1. p ., 24 hours prior to experiment, Indicates a significant change from control by Student t test, [p{0,05], 108 record tracings of Figure 14. Norepinephrine also produced a significant increase in the depolarization rate (dv/dt) , developed tension ( D T) and the maximum rate of rise of the contraction ( dFj ). The effect of NE on the mechanical and electrical param eters of atria from reserpine pretreated animals were qualitatively sim ilar to those of untreated animals. Peak changes in the electrical and mechanical activity were found to occur between 30-60 sec after injection of NE into the tissue bath.

Effects quantitatively similar to those of NE were produced by tyramine (7.2 x 10" 5 M) on the isolated atria of guinea pigs (Table 21) . Tyramine induced positive inotropic effects in normal atria. This indirect acting sympathomimetic amine produced mark ed increases in developed tension, and in the dFj and dF2. Like wise, there was an increase in the depolarization rate, duration and area of the action potential. Atria from reserpine pretreated guinea pigs showed significant alterations in responses due to tyramine. The contractions normally induced by tyramine were re duced to one-tenth or less of control responses, and the changes in dV/dt, AP-D and AP-area were significantly attenuated (Table 21). There was also a longer latency in the response to tyramine when compared to norepinephrine. Peak responses were recorded at 1,5 - 2.0 minutes after exposure of atria to tyramine. Tracings from the oscillograph records of the effects of tyramine on the isolated atria are illustrated in Figure 15.

C. Mechanical Effects of Acetaldehyde (CH 3 CHO) Figure 16 summarizes the effects of CH3GHO on contractile responses of the isolated left atria of guinea pigs in control and reserpine pretreated animals. This two carbon aldehyde induced positive inotropic responses in normal atria when low doses were used. The control contraction was unchanged by 0,1 mM (4.4pg/£nl) 109

CONTROL NE

5 0 mV

T ~ v 5 0 0 mg

UNTREATED

100 msec I ------1

5 0 mV

5 0 0 mg

RESERPINE PRETREATED

Figure 14: Exact superimposed tracings from oscillographic records showing typical effects of norepinephrine ( 1 0 " 7 m ) on the action potential and contractile responses of Isolated left atria from untreated and reserplne pretreated guinea pigs. Two representative experiments are shown. 110

TABLE 21 Effect of tyraminc [7. E x-I0"*M] on various parameters of the isolated guinea pig left atrium

Normal ( n ■* A) Eeserpine** (n *3) P a r a m e te r ® V. m e a s u r e d C on trol T vtamine C h an ge Control Tyraminc* Change E le c t r ic a l R P - m V ( 0 . 0 ( 1 . 7 + 2 . 8 (7.0 (9.5 +3.7 A P - m V 7 5 .7 7 6 .2 + 0 .7 6 0 .0 8 0 .6 + 0 .7 5 dV/dt-V/scc 1 1 1 .4 1 3 5 .6 + 2 1 .7 * 7 8 . 5 7 9 . 3 +1. 0 AP-D msec 99.8 132.7 +33. 0* 146.0 143.7 - 1 . 6 A r c a - m V ^ e c 2 . 2 0 3. 19 + 4 5 .0 * 2 . 7 5 3 .0 0 + 9 .0 C T - m s e c 14. 0 1 4 .0 0 1 8 .6 1 6 .6 0

• M e c h a n ic a l D T - m g ( 2 0 . 0 1095. 0 +77.0* ( 4 6 . 0 ( 9 7 . 0 + 7 .9 d F j - g / s e c 1 6 .8 5 2 9 .7 + 7 6 .3 * 15. 3 1 6 .6 + 8 .5 d F j - g / s c c 1 1 .5 2 0 . ( + 79. 1* 9 . 4 3 9 . 7 + 2 .9 • D T - P - m s c c 7 4 . 0 7 4 .0 0 (2.2 64.0 +2.2 D T - D - m s c c 2 4 0 .0 2 5 0 .0 + 4 . 2 207. 0 212.0 +2.4 L P - m s e c 3 8 .0 38.0 0 40.0 40.0 0

*See figure 3 for explanation of abbreviations, b Rescrpine (3 m g/kg]i. p ., 24 hours prior to experiment, c. No significant change from control in any of the characteristics measured. In d ic a te s a significant change from control by Student t test, [p^O, 05], I l l

■CONTROL TYRAMINE

5 0 . mV

5 0 0 mg Sx UNTREATED

100 msec h

5 0 mV

5 0 0

RESERPINE PRETREATED

Figure 15s Exact superimposed tracings from oscillographic records shoving typical effects of tyramine (7.2 x 10”5m ) on action potential and contractile responses of isolated left atria of normal and reserpine pretreated guinea pigs. Tvo representative experiments are shown. 112

140-1 dF, ■ • Unlf«aled ± SE — Ritirpln* Pr»tnat*d i SE 100 •O

40-

+ 20-

4 0 -

•0 - kl

S 60- g eo-

* 40

+ 20-

0 0.1 0.3 1.0 3 0 10.0 3 0 0 0 0.1 0 .3 1.0 0.3 10.0 3 0 .0 Z Smln 2 Smin CHjCHO

Figure 16: Concentration-response curve for CHoCHO on the contractile parameters (DT, dFp, and LF) of the Isolated left atria from untreated ana reserpine pretreated guinea pigs. There are two points shown for lOmM because of the temporal blphaslc effects of this concentration. Untreated tissue experiments = 9; reserpine pretreated tissue experiments « 5. 113

CH3 CHO, whereas 0,3mM ( 13.2 iJg/nl) significantly increased the contractile responses. Incremental increases in the DT, dFj and dF 2 were obtained with concentrations up to lOmM. There was a mean maximal increase in the developed tension of 131 °/0 above the control. The maximum positive inotropic effects occurred at about two minutes after addition of CH 3 CHO. The lOmM concen tration induced a time-related biphasic effect on the atria. Init ially, there was an increase in the DT which reached a maximum at two minutes. However, with increased time of exposure this positive effect faded so that it was reduced or reversed at about three minutes. This depressant action of CH3CHO reduced the con traction to approximately that of the control. The mean absolute values for the effects of acetaldehyde on the developed tension ( DT) in both control and treated atria are shown in Figure 17.

A further increase in the concentration of CH3CHO to 30 mM resulted in a further depression of the DT response, which was re versible with washing if exposure time was limited to ten minutes. t Exposure to this concentration of CH3CHO for longer than ten min utes resulted in irreversible depression of the mechanical activity of atria. The mean depressant effect of 30mM CH3CHO in nine control atria was to reduce the response to 40 °/0 of the initial DT. In some cases, the contractile responses were almost completely eliminated ( Figure 18). In other experiments ( four out of nine), an arrhythmia was elicited by the 30mM dose. This consisted of spontaneous beating which occured between each pair of electrically induced beats. This arrhythmogenic action of CH3CHO could be re versed by washing. CH3CHO in higher concentrations also prolonged the latent period by as much as 87 ( Figure 1 6 ). No significant alterations in the duration of the mechanical event or the time to peak tension occurred in these experiments. 114

I20CH ■■■♦ - Untreoted — Reserpine Pretreoted 1000H

800H

- 600-j

400-1

2 0 CH

0.3 1.0 3 .0 10.0 3 0 .0 3 min

Figure 17: Concentration-response curve for CH^CHO on the developed tension (rag). The numbers are mean values from nine untreated atria and five reserpine pretreated tissues.

i 115 D. Electrical Effects of Acetaldehyde Changes in the electrical activity of atria were also observed with CH3 CHO (Table 22). The resting potential and action poten

tial magnitudes were not significantly altered by CH 3 CHO, whereas, *

the AP-area was significantly increased by CH 3 CHO in concentrations ranging from 0.1 to 30mM (Figure 18), Low concentrations of

CH3 CHO induced an increase in the AP-area which was the result of a decrease in the slope of the repolarization phase of the potential and the production of what appears to be a plateau in the action poten tial, These changes closely resemble those produced by norepineph

rine and tyramine. However, higher concentrations of CH 3 CHO ( lOmM at three minutes and 30mM ) produced more consistant changes in the AP-area by prolonging the time of repolarization, i. e., the action potential duration (Figure 18) . Lower concentrations of

CH3 CHO produced a concomitant increase in the depolarization rate (dv/dt) with a maximal effect at 3 mM (+54^). The higher con

centrations of CH 3 CHO did not cause the dv/dt to increase. The rate of rise of the action potential returned to control values or was decreased when concentrations of 10 and 30 mM were administered.

CH3 CHO at higher concentrations caused a marked prolongation in the conduction time and resulted in a decreased conduction velocity in atria. These changes along with the values of the other electrical

param eters which CH3 CHO significantly altered are listed in Table 22.

E. Effects of Reserpine Atria from reserpine pretreated guinea pigs (3mg/Kg/24 hr) did not show any significant positive inotropic effects after adminis

tration of CH 3 CHO. Similarly, CH3 CHO produced little or no change

in the dFj or dF 2 in these tissues ( Figure 16). The negative inotropic

effects of CH 3 CHO however, were not eliminated by reserpine pre

treatment. Concentrations of 10 and 30mM CH 3 CHO still produced TABLE 22

Effect of Various Concentrations of CH5CHO on Electrical Parameters of the Isolated Guinea Pig Left Atrium®* ^

dV/dt (v/sec) AP-D (msec) AP-Area (mV-sec) CT ( m se c ) CH,CHO N orm al Reserpine N orm al Reserpine N orm al Reserpine N orm al Reserpine (m M ) ( n = 5 ] c Tn « 3 ] [n = 3] [n = 3 ] [n - 3] [n = 3] [n = 3] [ n = 3 ] C ontrol 51.4 ±4. 0U 64. 6 ± 7. 0 124. 0 ± 6. 9 146 ± 14. 0 2. 10 ± 0 .4 0 2. 80 ± 0. 09 15.8 ±1.6 20. 0 ± 2. 3 0. 1 70. 9 ± 4. 75* 66.4 ± 5. 3 138.5 ± 18 173 ± 6.6* 2.73 ± 0.41 2. 85 ± 0. 15 15. 3 ± 2. 1 20. 0 ± 2. 3 0.3 76. 1 ± 3. 2 * 70. 7 ± 7. 8 150.0 ± 16.1* 140 ± 8. 3 3. 66 ± 0. 02* 2.91 ± 0.21 14. 0 ± 1.4 18. 0 ± 3. 0 1.0 75. 8 ± 9.4 * 62.4 ± 7. 1 153. 2 ± 7. 3* 100 ± 10. 5* 3.95 ± 0. 14* 2. 5 ±0. 24 13. 0 ± 2. 3 19. 0 ± 5. 0 3.0 81.2 ± 1.4 * 61.2 ±8.8 143.2 ± 11.8 126 ± 6.0 4. 16 ± 0.8 * 2. 84 ± 0. 67 13. 3 ± 3. 1 18.0 ± 3. 0 10.(2 min) 52. 9 ± 5. 6 6o. o ± iao 125.6 ± 10.9 132 ± 18. 1 3. 85 ± 0. 24* 3.41 ± 0.29* 16. 8 ± 2. 0 17. 3 ± 1.3 10(3m in} 4 3 . 2 & 6, 0 40. 7 ± 2.1* 1 4 2 .0 ± 15 180 ± 2 .6 * 3. 86 ± 0. 26* 4. 25 ± 0. 02* 23. 0 ± 1. 5* 24. 0 ± 2. 0 30 4 3 .4 ± 6 .1 43. 6 ± 2 . 3* 1 9 2 .0 ± 12.1* 200 ± 1 0 .0 * 3.73 ± 0.48* 4. 3 ±0. 07* « 29. 0 ± 4. 0* 25. 0 ± 3. 0

Abbreviations: depolarisation rate (d v /d t) , duration of action potential (AP-D) , action potential area ( AP-area) , conduction time (CT) , b The resting potential ( 64,2 A 3.68 mV) and action potential magnitude (81 ± 4.5mV) did not show any significant change. c [ ], number of animals at each concentration, d Values ± S. E, ^ ♦ ^ Indicates significant difference from control values by Student t test (p^ 0.05} . O 117

UNTREATED ATRIUM CONTROL CHjCHO 100 msec 1 1

IOmM(3min)

Figure 18: Exact superimposed tracings from oscillographic records showing typical effects of various concentrations of CHgCHO on the action potential and contractile responses of isolated left atria from untreated guinea pigs. The data illustrated here was obtained from a single experiment. 118 depressant effects on isolated atria. Reserpine pretreatment also prevented the prolongation of the latent period which was usually seen at higher concentrations of CH 3 CHO.

Tracing of the records from a reserpine pretreated atrium experiment are illustrated in Figure 19. It can be seen that re serpine pretreatment blocked the increase in AP-area produced by low concentrations of CH 3 CHO(seen also in Table 22).. Increases in the AP-area produced by high concentrations of CH 3 CHO, however, were unaffected by reserpine pretreatment. The increase in AP- area along with the depression in the mechanical activity could be reversed if the tissue medium was changed and the tissue was wash ed several times ( Figure 19) . Increases in the dv/dt produced by low concentrations of CH 3 CHO were also blocked by reserpine pretreatment, but the decrease in dv/dt due to higher concentrations was not blocked. After reserpine pretreatment the slowing of the conduction by higher concentrations of CH 3 CHO was not statistically significant ( Table 22 ) .

F. Propranolol Blockade Pretreatment of isolated left atria for 30 minutes with prop ranolol caused a measurable attenuation in contractile responses in duced by CH 3 CHO ( Figure 20) . The graph is plotted as percent of the maximal increase in developed tension. Propranolol ( 1. 5 x 10" 7 M) caused a shift in the concentration-response curve of CH 3 CHO to the right with a decrease in the slope and a reduction in the maximum to

50 /£. Higher doses of propranolol ( 1.5 x 10“ 6 M) caused a further reduction in the maximum contraction to 1 1 e/0 of the control response.

Kinetically, CH 3 CHO produces its contractile responses with a pD* = 3.0 (A riens, 1964) which can be calculated from the curve. Propranolol inhibits this response to the extent that the ratio of: 119

RESERPINE PRETREATED ATRIUM CONTROL CH3CHO 100 msec I------1

50m V 0 .3 mM 0.1 mM 1

— T SOO^mg

1.0 mM 3 .0 mM

vN |\ 10 mM (2 min) 10 mM (3m ln)

v 3 0 mM WASH

.K,

Figure 19: Exact superimposed tracings from oscillographic records showing typical effects of various concentrations of CH3CHO on the action potential and contractile responses of Isolated left atria from reserpine pretreated guinea pigs* The data illustrated here was obtained from a single experiment. Figure 20: Comparison of the ooncentration-response curveofCH^CHO Comparison ooncentration-response of the 20:Figure % % OF MAXIMUM 00" K ■ . M IO * 1.0mM ■ KD "| 0 l0 - 0 6 - 0 8 20 - Dj 3 * j pD pD'g * * pD'g were obtained in one atrium, and were typical of the of typical atrium,obtained in one and were were point vas based on the mean Increase obtained in in nine obtained Increase on mean the point vas based ments. dueto depression The in atria.results three produced addition of 10 and 30mM CH3CH0 (Figures 16 and 16 and (Figures CH3CH0 30mM addition of 10 and and and control experiments. The results shown for propranolol for Theshown results control experiments. calculated Hie intension.of change the developed maximum in the presence and absence of propranolol (l.Jj in and oftheabsence propranolol presence x is not shown here, was eliminated in propranolol experi in ispropranolol eliminated shown not washere, 0.1 6.8 1.5

x “ 0 1 0.3 “3 M

CH M).

3 The ordinate represents the percentage represents Theordinate H (mM) CHO 3,0 luae Point olculated C RPAOO 15 10 x 1.5 PROPRANOLOL RPAOO 15 IO x 1.5 PROPRANOLOL 10.0 CONTROL 30.0 17), “6 7 ' 10-7, which

120

121

E ( CH3 CHO ______max ______

E ( CH3 CHO + Propranolol) and thus has a pD/ value equal to 6 . 8 ( -log[l,5 x 10" 7 m]) . A Lineweaver-Burk plot ( Figure 21) of the same data also demon strates that propranolol behaves like a non-competitive inhibitor of CH 3 CHO on the DT responses of isolated atria of guinea pig. It is important to note that propranolol also blocked the negative ino tropic effects of CH 3 CHO. The depressant action of 10 and 30rhM

CH3 CHO was not exhibited in the presence of this beta adrenergic blocking drug ( Figure 20).

H3-Norepinephrine Release and Blood Pressure Measurements in Whole Animals

A. Plasma Tritium in Cats Figure 22 illustrates the time course of the effect of ethanol

( 2 g/Kg) on plasma levels of 7-H3-norepinephrine (H 3 -NE) . One hundred (Jc/Kg of H3-NE was injected into the animal by vein at zero time and allowed to equilibrate for 60 minutes. Ethanol or saline were continuously infused over a ten minute period. The curves are actually superimposable, and they all start at approximately the same level of radioactivity. In all experiments the initial ten min ute sample ranged from 21,2 x 10 4 to 25.8 x 10 4 disintegrations per minute per milliliter of plasma. In plotting the data, the curves are shifted to facilitate interpretation.

Plasma tritium (H3) measurements after the infusion of eth

anol were made every ten minutes. A rise in the H 3 plasma level was produced by ethanol, which occurs after a latency of about 30 minutes from the start of the infusion ( Figure 22). The plasma de cay pattern was markedly altered by ethanol infusion. This is hot due to a volume injection effect since an equal volume of saline failed 122

.05 t

% RESPONSE

.04--

.03--

- ~ • -1000 .02-- Kd » tO "3 M

1000 300 -100 +100 300 1000 I tCHjCHO]

, Figure 21: Double reciprocal plot showing apparent non-competitive inhibition by propranolol of the positive inotropic effects of CHoCHO on the isolated left atria of guinea pigs. O 2 “ o ETOH (2 Gm /Kg) o * 2 £ 1

£, Z “• *=3 , » o X ^

CONTROL 4 a (SALINE) i m i i w i « — w j u m a *V* ir — ^jk. 2 0 40 eo ioo 120 140

h 3- n e TIME(MINUTES) (100/iC/Kg)

Figure 22: Effect of ethanol (EtOH) (2 gm/Kg.) or saline infusions (indicated by the arrow) on the plasma tritium level after injections of E^-NE in cats. Curves are plotted as $ of initial ten minute level with each division on the ordinate representing a 10$ change. 124 to alter the curve. The degree of change produced by ethanol was an 8 ° ( rise of H3-NE in the plasma which was prolonged for the duration of the experiment. The levels of ethanol ranged from 0.24 to 0.3 V in these animals. ' 0

A sim ilar plot is shown in Figure 23 of the effect of acetaldehyde injection on plasma tritium in cats. Measurements were made at 1, 2.5, 5, 10, 20 and 30 minutes after the injection of acetaldehyde (30 mg/Kg)or an equivalent volume of saline. In these four experiments a marked rise in plasma H 3 of 10-22 % was noted one minute after intravenous injection of acetaldehyde. The peak response is much more immediate ( one minute sample) than that produced by ethanol. This effect is directly attributable to acetaldehyde, which falls off rapidly as do the blood levels of acet aldehyde after injection. This is in contrast to the latent and pro longed response produced by ethanol infusion, when acetaldehyde is continuously produced.

B. Blood levels of Ethanol and Acetaldehyde Blood level data obtained in cats and rabbits after intravenous injection of 30 mg/Kg of acetaldehyde is shown in Figure 24. The peak concentrations of 80 to 1 0 0 |Jg/inl seem high compared to normal levels produced in these species after ethanol alone. However, some of the values reported to occur in humans after disulfiram and al cohol are in this range ( refer to Table 1). As can be seen, the dis appearance from blood is rapid with an initial plasma half-life of 2,5 and 4 minutes in rabbits and cats respectively. This rapid half- life is consistant with rapid tissue uptake due to lipid solubility, high chemical reactivity with cell substituents, rapid alveolar excretion due to high volatility and rapid metabolism. iueS* Effectof S3*Figure

H3-NE(% OF CONTROL dpm /m l) (ONE DIVISION = 1 0 % CHANGE) j w a m g r plotted plotted as$ ofinitial the ten minute level witheach f7H-oeiehie(^K)i as Curvesof7-H^-norepinephrine are (H^-KE) in cats. division division on the ordlnant representinga 10$ change. injectionson the plasma tritium level after injection 0 2 30 l.v, acetaldehyde(A), 30 mg/Kg. and saline TIME (MINUTES) TIME 40 A / g) g/K M 0 3 ( ® • 090 60 ---- (SALINE) CONTROL

125 126

1 0 0 *,

7 5 *a

TIME (MINUTES)

Figure 24: Blood levels of acetaldehyde (^/rol) achieved in cats (n=6) and rabbits (n=3) after intravenous injection of 30 rog/Kg. of acetaldehyde. Levels of acetaldehyde in blood (protein-free filtrate mea surements) were also measured ( Figure 25), after the infusion of 2 g/Kg of ethanol in six rabbits. Three of the rabbits had been pretreated with disulfiram. A five to fifteen fold increase in acet aldehyde levels was affirmed due to the action of disulfiram in the presence of ethanol. The upper curves indicate acetaldehyde pro duction from ethanol after disulfiram pretreatment while the lower curves show acetaldehyde after ethanol only. Peak levels of acet aldehyde ranging from one to two Hg/ml were seen in rabbits after ethanol alone. Pretreatment with disulfiram increased the amount of measurable acetaldehyde to initial levels of 7 - 9 fJg/nl.

The levels of acetaldehyde in the untreated animals occurred in the presence of ethanol levels ranging from 2,110 Hg/nl to 3,662 lig/5nl. The levels of ethanol occuring with time in these three un treated rabbits are plotted in Figure 26. The levels of ethanol occuring in disulfiram pretreated rabbits over this same time period ranged from 2,760 to 4,780 (Jg/tnl. These levels after 2 g/Kg of ethanol were significantly higher than those in untreated animals, as was true of rats. Disulfiram again seems to increase the levels of ethanol as well as acetaldehyde.

C. Plasma Tritium in Rabbits One saline control and two acetaldehyde injected rabbit experi ments are shown in Figure 27. These results were analogous to those obtained in cats. One important correlation which should be pointed out is the great similarity between the sharp rise in plasma tritium produced by acetaldehyde and that produced by reflex sym pathetic activation ( refer to Figure 5) . The magnitude of the res ponse produced, the immediate onset of the effect and the duration of the rise in H3 were exactly equivalent to that which was produced by bilateral carotid occlusion in the rabbit. In rabbits, the immediate ' Figure 25: Blood levels of acetaldehyde with ‘time with which was acetaldehyde of levels Blood ' Figure 25:

ACETALDEHYDE BLOOD LEVEL (fig /m l.) 0i .0 9 aoi 5.0' 6,0 2 , 0 ' rabbits. Ethanol was infused infused as 10$ solution a over Ethanol w/v was rabbits. obtained from intravenous infusion infusion of ethanol (EtOH)intravenous obtained from a ten minute period indicated indicated the by ten period a minute brackets. (2 gm/Kg.), with or without disulfiram ordisulfiram in(2 without pretreatment with gm/Kg.), 040 30 I ( NUTES) S E T U IN (M E TIM 0 60 50 70 60 PRETREATMENT ' PRETREATMENT DISULFIRAM O O NO

128

129

3 0 0 0

E 2 0 0 0 » cn

1000

30 4 0 50 90 EtOH TIME (MINUTES)

. Figure 26: Blood levels of ethanol (V/ral) after the intravenous Infusion of ethanol (2 gm/Kg.) in control rabbits (n=3). 130

o z U-O ow J * S > I

Z K •0 . 0 CONTROL a (SALINE INJECTION)

2 0 3 0 4 0 5 0 / 7 0 6 0 9 0 TIME(MINUTES)

(100/iC/Kq) A(30 mg/Kg)

Figure 27i Effect of i.v. acetaldehyde (A)# 30 mg/Kg,, and saline on the plasma tritium level after injection of 7 - h 3- norepinephrine (H3-NE) in rabbits. Curves are plotted as percent of the initial ten minute level with each division on the ordinate representing a 10$ change. 131 effect of acetaldehyde was to alter the decay pattern in such a way so as to produce a 15 - 25 °/0 rise in the plasma level of radio activity.

Figure 28 represents data observed after ethanol infusions. Ethanol (bottom two curves) had a sim ilar effect in this species as it had in cats. The response was quantitatively similar in both mag nitude ( 5-10 % change) and duration ( 50-65 minutes) to that produced in cats. However, after disulfiram pretreatment (top two curves), ethanol produced a pronounced effect which was quantitatively similar to the response obtained with an acute injection of acetalde hyde. Disulfiram pretreatment potentiated the action of ethanol on plasma H3 at least 3 - 4 fold. The fact that acetaldehyde is contin uously produced from ethanol ( see Figure 25) accounts for the longer duration of this response. One would expect a prolonged elevation of H 3 -norepinephrine, if the levels of the causative agent were also elevated. A summary of all of these treatments in rabbits is illus trated in Figure 29.

Further demonstration of norepinephrine release was afforded by measuring urine tritium by canulation of both ureters. In these experiments ( Figure 30), urinary levels were monitored every ten minutes after injection of H 3 -NE. The top section shows a control animal. Saline injection had no significant effect on the concentra tion of radioactivity in the urine. Eventually, rather constant amounts were excreted into the urine. The center graph shows a three-fold increase in the concentration of H3-NE in the urine after bilateral carotid occlusion. The lower bar graph demonstrates a similar effect after the injection of acetaldehyde.

D,. Blood Pressure Responses to Acetaldehyde The effects of acetaldehyde were examined on blood pressure, nictitating membrane contractions and respiration in both rabbits and Figure H3-NE (% OF CONTROL dpm/m! (ONE DIVISION = 10% CHANGE) 2 8 : : 8 2 Effect of ethanol (EtOH) infusions (indicated (indicated infusions the arrow)(EtOH)ethanolby of Effect treated treated rabbits. disulfiram and in untreated (H3-HE)pre norepinephrine injectionthe 7-H3-of after level tritium plasma on the 0 2 0 4 0 6 ME ( NUT ) S TE U IN (M E IM T TH(GM/ ) G /K M (2G ETOH mi

8 0 100 100 0 8 b

iiii

i in i in 120 RTET ENT PRETREATM ENT PRETREATM DISULFIRAM NO 140

m X -NE IN PLASMA (%0F INITIAL LEVEL=IOO%) too 100 .Figure 70 NO 0 8 0 9 0 8 0 6 0 9 0 5 - 0 7 29 : Summary of the effect of saline, acetaldehyde (CHgCHO) acetaldehyde saline, of effect the of Summary : of the initial level. initial of the aspercent in rahhits levels H^-NE the on disulfiram of in the presence or alone (EtOH) or ethanol r.v.CHsCHO Mg/Kg 30 i.v. SALINE TIME (MINUTES) 0 10 4 160 140 120 100 0 8 0 6 0 4 0 2 0 I.v.ETOH FE DISULFIRAM AFTER i.v. Gm/Kg ETOH 2

133 SALINE (CONTROL)

BILATERAL CAROTID OCCLUSION

ACETALDEHYDE 1 i 1 I 0 10 20 30 40 50 60 70 80 90 100 TIME (MINUTES)

Figure 30: Effect of various treatments on the urinary outnut 7-H3-norepinephrine (h B-u b ) in rabMts ^ w ! ? occlusion is indicated by the arrows. »«• t L o of c S o M d 135 -cats. The responses produced by acetaldehyde once again compared to those produced by tyramine. Figure 31 illustrates the effect of various doses of acetaldehyde on the blood pressure in a female rabbit. Intravenous injection of acetaldehyde produced dose-related increases in the systolic and diastolic blood pressure. Injection of 10, 20 and 30 mg/Kg of acetaldehyde produced increases in the mean blood pressure in three experiments on rabbits of 23, 42 and 63 mmHg respectively. These pressor responses represent increases of 30, 50 and 72 °/a above the control mean pressure. Similarly, the duration of the pressor response demonstrated a dose-related incre ase after the administration of these doses of 54, 72 and 87 seconds respectively. In general, acetaldehyde produces a tachycardia in control animals. However, at the peak of the pressor response there is a reflex bradycardia. Frequently, there arc skipped beats during the blood pressure effect but a return to normal after the acetaldehyde disappears ( refer to Figure 24).

The pressor response can be reversed by administration of alpha-adrenergic blocking agents. Figure 32 illustrates the effect of phentolamine ( Regitine^, 10 mg/Kg, i. v. ) on the pressor response produced by acetaldehyde. Fifteen minutes after the injection of a dose of phentolamine, which was sufficient to block completely the pressor response to norepinephrine (NE, 2 pg/Kg), the rise in blood pressure normally produced by acetaldehyde was reversed to a de pressor effect. This seems similar to the reversal seen with epi nephrine after the injection of alpha blocking drugs. The blockade of the pressor response by this adrenergic antagonist is further evi dence for the sympathomimetic action of acetaldehyde. Several drugs have been found to produce a marked potentiation of the pressor res ponse produced by acetaldehyde (Table 23), Cocaine has been shown 136

| / ' ... Ilium*'!'';!,.* * Jt'tn i * 1J ■ -...... ”) |...... v mnr ...... P ,....J I...... j

t 10 rrc/Ke t

iMiuw

* 30BU/KC ‘

Eigure 31t Blood pressure tracings shoving the effect of various intravenous injections (10, 20, 30 mg/Kg.) of acetaldehyde (A) on the blood pressure of an untreated rabbit. 137

time (sec) •203

iu before g l * M3‘.1 3S ! <3 a j

•503*3

u after g& ! ss “ ICO \ V .j (3 \_____

•Figure 32: Blocking effect of phentolamine on the vasopressor action of acetaldehyde administered at a dose of 30 mg/Kg. i.v. The response to acetaldehyde is shown before and after alpha adrenergic receptor blockade with phentolamine (10. mg/Kg.). TABLE 2 3

POTENTIATING EFFECT OF VARIOUS DRUGS OR THE PRESSOR RESPONSES TO INTRAVENOUS ACETALDEHZBE (30 mg/Kg) IN RABBITS

Average Rise in Mean Blood Pressure in mmHg Mean Potentiation Number of Before After Difference Index Drug® Dose Experiments Drug Drug (after-before) (after/before)

Cocaine HC1 2 mg/Kg 3 66 108 +42 1.64

Guanethidine SOi, 15 mg/Kg 3 68 105 +37 1.54 Ismelin®

Chlorpheniramine 2 mg/Kg 3 46 78 +32 1.86 Chlortrimeton

Tripelennamine. 5 mg/Kg 3 57 122 +75 2.14 pyrib enzamine®

a Acetaldehyde was administered prior to and 30 minutes after the infection of the agents listed. 139 to potentiate sympathomimetic amine responses by blocking neuronal uptake of released or administered NE. Injection of cocaine potentiated the pressor response produced by acetaldehyde causing a 42 '/ increase in the mean pressure. This is in contrast to the depression of the response produced by tyramine when cocaine is given ( Burn and Hand, 1958), Likewise, injection of guanethidine causes the pressor effect of acetaldehyde to increase 37 % compared to that evoked by acetaldehyde before this drug. Two antihistaminic agents were also investigated, and were found to greatly enhance the pressor effect of acetaldehyde. Chlorphen iramine at a dose of 2 mg/Kg caused the mean pressure rise to in crease from 46 to 78 mmHg, an increase of 32 °/0. Tripelennamine also potentiated the vasoconstrictor action of this aldehyde by pro ducing a 75 % increase in the acetaldehyde induced pressor action. This effect of these antihistamines can readily be explained by their ability to block NE neuronal uptake, and thus have a cocaine like action (Isaac and Goth, 1967) . The effects of other drug treatments on the blood pressure responses to tyramine and acet aldehyde are summarized in Table 24. Several substances were ex amined only once to confirm the literature reports. Injection of propranolol ( 5 mg/Kg), atropine, or hexamethonium failed to alter the blood pressure response to acetaldehyde. Generally, the in creased pressure produced by acetaldehyde may be slightly augment ed by blockade of muscarinic receptor sites with atropine or by ganglionic blocking drugs.

E. Interactions of Disulfiram and Reserpine A sa basic protocol, the action of acetaldehyde on blood pressure and the nictitating membrane was studied by comparing it with the classical catecholamine releasing agent, tyramine. Theo retical aspects of the adrenergic system were studied in whole 140

TABLE 27

COMPARISON OF THE SYMPATHOMIMETIC ACTIVITY ON BLOOD PRESSURE OF TYRAMINE AND ACETALDEHYDE IN DISULFIRAM AND RESERPINE PRETREATED ANIMALS

TREATMENT TYRAMINE ACETALDEHYDE

DISULFIRAM:

CONTROL RESPONSE PRESSOR DEPRESSOR

CX BLOCKADE NO RESPONSE

BLOCKADE 0

GUANETHIDINE REVERSAL

*SP1NAL ANIMAL 1 0 0

ATROPINE 0 0

RESERPINE;

CONTROL RESPONSE PRESSOR DEPRESSOR

Oi BLOCKADE NO RESPONSE

BLOCKADE 0 0

SYMBOLSincrease or decrease In control response; 0 = no change in control response, * = Musacchio 1965. 141 animals by inducing release, depletion, or repletion of trans mitter substances by using precursors and false transmitters or norepinephrine itself. The effects of acetaldehyde were examined using these manipulations. Figure 33 shows the pressor responses and strong nictitating membrane contraction obtained both with tyramine (800 pg/Kg) and acetaldehyde (30 mg/Kg) in untreated control rabbits. These doses give approximately equivalent pres sure responses, however, acetaldehyde was more potent in causing contraction of the nictitating membrane. It should be noted that acetaldehyde produces a slight secondary depressor phase in con trol rabbits. The effects are quantitatively summarized in Table 25. The values are ratios of the blood pressure change produced by tyramine and acetaldehyde compared to injections of 2 y /K g of norepinephrine (NE) in each animal. This was done in order to normalize the values, on the assumption that the differences in res ponses among animals would reflect differences in receptor activity. Therefore, the natural adrenergic transmitter, norepinephrine, was utilized as a standard for comparison with agents releasing NE from its storage sites. Thus, the mean rise in blood pressure produced by tyramine in eight animals was about 11% o l that produced by NE. Infusions of NE failed to appreciably alter these responses as would be predicted. Dopamine ( DA ) infusion decreases the pressor and nictitating membrane response to both tyramine and acetaldehyde. This is to be expected, since even after one hour equilibration time before administering challenging doses of tyramine or acetaldehyde, it is possible that DA could be taken up and released by these agents. Release of DA at the nerve terminals could account for the slight reduction in the blood pressure and nictitating membrane contractile responses, since DA is not as potent a pressor agent as NE. Infus ion of methyldopate (Aldomet^, MeDOPA) considerably attenuated these actions of tyramine and acetaldehyde. The pressor action of 142

NICTITATING -7 — r - n— ,, H0.IBRANE d zrjr!±L~dj::. ... i.

f t ILDOO pressure ~:~y^zz:r.:. tfeb-Jlizl:;;;, ^ h v n s a as«ts;:r.:-.-.u5y

RESPIRATION i £ 3 I j. 1 I j*l | I ’ (I '1 I “ ■MFPM.CDunuttNOREPINEPHRINE , ■ MpW|HE-' t ALO^CT J KEPLETING INFUSION ' INFUSION . T INFUSION ' INFUSIONS 1.5 Ri/Kt • DOSES 10 iti(/K{ ] Mnf/Ki " l • . . i ' i :: sr- uf-» » i

LS5J fi. V*“i'rp-~^SI -"T. ll_>J------I ...... L:j:.T-i— u-v-~::v^ - ->f r\ - • •'-1 - : ■''~ -—^ ; ■ -\ • ■'".•'::.:: :;iu- P7“^

, . , . ,i 1 f | . . . I.. | .; . . f ' ■ • • • ! •''♦ * : 7 ; J ‘; : [• -: /: -:: a ::: /"::" ^:: v * *

..;. ■ ill' :_LLL,.. .'.: I i Ti' N }, n I-.T - ■!: U11111;;: 111; il A A A TYRAMINE (T| BOO If/Kg. CONTROL RABBITS j ACETALDEHYDE ‘(Ap"30 mg/Kg.

• Figure 33: Nictitating membrane contractions, “blood pressure responses, and respiration recorded in control rabbits given i.v. injections of tyramine (t) 800 V/Kg, (upper tracings) or acetaldehyde (A) 30 mg/Kg, (lover tracings) before and after drug infusions, lime line ( i ( ) Indicates 25 seconds* TABLE 2 5

RATIOS OF BLOOD PRESSURE (BP) RESPONSES AMD SCALE RATINGS OF NICTITATING MEMBRANE (MM) CONTRACTIONS AFTER TEST DOSES OF TYRAMINE 800 V/Kg AND ACETALDEHYDE 30 mg/Kg TO AVERAGE OF CONTROL RESPONSES TO NOREPINEPHRINE 2 V/Kg BEFORE AND AFTER VARIOUS "REHLETING" INFUSIONS IN RABBITS

Pre-Infusion Responses "Depleting"3, Post-Infusion Responses TVramine Acetaldehyde Infusion Tyramine Acetaldehyde Pretreatroent BP NM BPNMBPNMBP NM

V NE 0.70 (5) 2+ 0.79 (5) 3+ Control o .7 i (8 r 2+ 0.76 (7) 3+ DA 0.44 (3) 2+ 0.66 (3) 2+ MeDOPA 0.27 (3) 1+ 0.53 (2) 2+

NE 0.72 (3) 1+ - 0.28 (3) 3+ Reserplne 0.50 (4) 0+ -0.17 (3) 1+ DA 0.37 (2) 1+ -0.J*0 (2) 2+ MeDOPA 0.24 (2) 0+ -0.29 (2)

NE 0.52 (4) 2+ -0.18 (3) 2+ Disulfiram 0.17 (5) 1+ -0.31 (4) 2+ • DA 0.30 (4) 1+ -0.34 (2) 1+ MeDOPA 0.21 (3) 1+ -0.30 (2) 1+

(NE) Norepinephrine Bitartrate, (DA) Dopamine HC1, (MeDOPA) alpha Methyldopa (Aldomet^)

Numbers in parentheses represent the number of observations* 144 tyramine was reduced to 30 % of the control response, and acet aldehyde was decreased to a lessor extent. This is in agreement with the finding that MeDOPA is converted in vivo to alpha-methyl - norepinephrine and is stored in adrenergic nerves (Musacchio et al., 1966a). This false transmitter substance is only about 50 °/0 as potent as NE (Muscholl and Maitre, 1963).

Animals pretreated with reserpine (Figure 34) to deplete catecholamine stores, show a marked decrease in the pressor response to tyramine. Acetaldehyde on the other hand, causes a fall in blood pressure. Reserpine pretreatment practically abolish es the nictitating membrane contraction to both compounds. NE infusion in both species, rabbits and cats ( see Table 26), restores the tyramine pressor response almost to levels of the control group. The membrane contraction is also enhanced. The effect of acet aldehyde in these animals does not seem to be altered by NE repleting infusions as far as vascular responses, but it does improve membrane activity. MeDOPA does not significantly alter the tyr amine responses. Similarly, DA restoration of blood pressure is variable and it seems inhibitory on the nictitating membrane con traction.. Neither DA nor MeDOPA were effective in changing the acetaldehyde depressor response in these animals.

Figure 35 illustrates in similar format the effect of tyramine and acetaldehyde in rabbits pretreated with 200 mg/Kg of disulfiram 20 hours and four hours prior to surgery. Disulfiram pretreatment markedly diminished the pressor and membrane responses produced by tyramine. Again, acetaldehyde exhibits a hypotensive activity, but the nictitating membrane reactivity is not as severely repressed as with reserpine. Norepinephrine infusion restores tyramine res ponses completely. As was the case in reserpine treated animals, the acetaldehyde response is neither lessened nor reversed substan tially by NE infusion. MeDOPA and DA were ineffective in changing 145

■—r XUXi 1 ■ r.TfiJ HtcTiTATtuc■ * T* EEEtl^n^F- ixiiiipjxf i" m e m b r a n e - i h 'r^fcp.T- H i t * -V ------r — ‘- 3 - — ---*• s § • " , JOCf - -H • . r*I ■ I •• J***"* 1 ILDQD ; i ■ P 1'-'-" vTijs^; r ' i PRESSURE -J IdU ■ SSmnt

,~ZZZ-£7ZZ'~iZ *rf-^— 4*:~v- RESPIRATIOH r * ” !" ^r'l1'i I L *.-> «■!*•• ■ *., r , ^ _J» T DEPLETING NOREPINEPHRINE DOPAMINE t AIDOMET INFUSIONS INFUSION, INFUSION INFUSION ISns'Kt I U m*/K( K r i/Ki - I J i NICTITATING a ’. ____ ■». MEMBRANE

lilt*inn HOOD PRESSURE , lilt* ItO.U--^™;* f»JA\ ----- ISmnKc 1 t f c i i ^ • v -- L“ U.

______. ______- . 1 v.vty' r * 1 !i' L zd j_ t l ...... RESPIRATION Jj %Jr- tr\ '"■**][ 3JTT - ...Z -T7X T ~ T ™ ' | * r w i r t 111 t V-*** A I RESERPINE Ty r a m i n e (t) s o o ^ K g . PRETREATED RABBITS ACETALDEHYDE (A) 3 0 m g / K g

. Figure 3^: Nictitating membrane contractions, blood pressure responses and respiration recorded in response to i.v. injections of tyramine (T) 800 ‘T'/Kg, (upper tracings) or acetaldehyde (A) 30 mg/Kg, (lower tracings) in rabbits pretreated with reserpine (5 mg/Kg. i.p.) given ^8 and 24 hours prior to the experiment. Responses were recorded before and after various drug infusions. Time line ( |___ | ) indicates 25 seconds. TABLE 2 6

RATIOS OF BLOOD HtESSURE (BP) RESPONSES AND SCALE RATINGS OF NICUTATINE MEMBRANE (KM) CONTRACTIONS AFTER TEST DOSES OF TSCRAMUJE 800 Y/Kg AND ACETALDEHIDE 30 mg/Kg TO AVERAGE OF CONTROL RESPONSES TO NOREPINEPHRINE 2 Y/Kg BEFORE AND AFTER VARIOUS ’'REPLETIKG" INFUSIONS IN FEMALE CATS

Pre-InFusion Responses "Repleting"a Post-Infusion Responses Tyramine Acetaldehyde Infusion T/ramlne Acetaldehyde Eretreatment BP NM BP NM BP NM BP NM

NE 0 .8 2 (2) 2+ 0.52 (1) 4+ Control 0.85 (2) 2+ 0.53 (2) 4+ DA 0.51 (1) 2+ 0.73 (1) 4+ MeDOPA 0.36 (l) 2+ 0.80 (i) 4+

NE 0.66 (l) 2+ -0.48 (l) 2+ Reserpine 0.29 (1) 0+ -0 .3 0 (i) 1+ DA o.4i (l) 0+ -0.75 (1) 0+ MeDOPA 0 .3 8 (l) 1+ -0.44 (l) 1+

NE 0.73 (1) 2+ -0.43 (1) 2+ Disulfiram 0.56 (1) 1+ -o.4i (3) 2+ DA 0 .5 6 (l) 2+ -0.36 (1) 2+ MeDOPA 0.39 (1) 0+ -0.43 (1) 1+

(NE) Norepinephrine Bitartrate, (DA) Dopamine HC1, (MeDOPA) alpha Methyl&opa

15 Numbers in parentheses represent the number of observations* 147

HICTIUTItlG 1 1 T MEMBRANE J---I— r._.:r. | , ■ —■--7 :::::* L.. .,1--- • | lllc . 1 i 1 |m " -itp T : :r.:. • • * 1 # ... ILOOD MD ■~/.yk r ■ PRESSURE llx in :::V:rr.rr.r.s:.vr« ~ r . KcnHt i RESPIRATION ^: I * -p n * T DOPAMINE' ' - ‘ 7 ’ AltOMET \ REPLET1HG MREPIHEPHR'HE INFUSION INFUSION INFUSION | ------| INFUSIONS H R{fl<( I.Smj/Kt MRt,K* NICTITATING I IIEMBRAHE iTTTht ) I mm in i iiooo10 PRESSURE r - % r —'■ y—a _ - .. . ! « * ■ StnHt ,M ‘V ‘!*Tv-TT"n? ......

RESPIRAT10H . 1 p . l V " *.-v -1 j y - j.,

DISULFIRAM TYRAMINE (T) B O O ^K g PRETREATED RABBITS ACETALDEHYDE (A| i30 ms /Kg.

Tlgure 35* Nictitating meiribrane contractions, blood pressure responses and respiration recorded in response to i.v. injections of tyramine (T) 800 V/Kg, (upper tracings) end acetaldehyde (A) 30 mg/Kg, (lover tracings) in rabbits pretreated with disulfiram (200 mg/Kg.) 20 hours and h hours prior to the experiment. Responses were recorded before and after various drug infusions. Time line ( «___ < ) indicates 25 seconds. 148 the pressure response of tyramine. This is in keeping with the fact that inhibition of dopamine-(3-oxidase prevents the formation of the transmitters, a-methyl-norepinephrine and norepinephrine, from MeDOPA and DA, respectively. Similarly, aldomet and dop amine failed to affect the vasodilator activity of acetaldehyde in these animals. The actions of tyramine and acetaldehyde in female cats was also examined (Table 26). The results produced by di sulfiram and reserpine pretreatment agree closely with those ob tained in rabbits.

F. Vasodepressor Activity of Acetaldehyde Since depletion of catecholamines with reserpine or disul firam causes a reversal of the acetaldehyde pressor response, the nature of this vasodilation was more closely examined. Figure 36 illustrates the results from four experiments, using disulfiram pre treated rabbits. The possible neurohumoral mechanisms involved in this depressor action have been examined by using specific antag onists. The results show that propranolol fails to block this res ponse. Therefore, dn effect on beta receptors is not involved in this effect. These results have been demonstrated in six rabbits using propranolol and in two rabbits using dichloroisoproterenol (DCl) to block beta receptors.

To eliminate the possibility of a cholinergically mediated dilitation, atropine was infused in animals treated with disulfiram. This muscarinic blocking drug failed to alter the acetaldehyde res ponse. Injection of tripelennamine caused a reversal of the vaso depressor effect to a biphasic response. The pressor activity of acetaldehyde is restored as in untreated animals, however, the dur ation of this response is very prolonged. Nevertheless, the de pressor effect is still present and persists for almost three minutes. This type of action was similarly elicited by chlorpheniramine. tr«t>tano4itac

Figure 36 : Effect of propranolol (5 mg/Kg.), atropine (2 mg/Kg*), cocaine (2 mg/Kg.) and tripelennaraine (5 mg/Kg.) Injections on the vesodepressor response to acetaldehyde (30 rag/Kg.) In rabbits pretreated with disulfiram (200 mg/Kg.). 150 Figure 36 shows that a reversal of the acetaldehyde effect can also be produced by injection of cocaine, A similar effect was obtained when guanethidine was injected into rabbits given disulfiram (Table 27). This response is not reflex in nature since it still persists in spinal animals, It should be noted that this depressor action in both disulfiram and reserpine pretreated animals is potentiated when phentolamine is used to block alpha receptors. As in the case of disulfiram, the effect produced in reserpine pretreated animals is not blocked by propranolol either,

C14-Norepinephrine Metabolism and CNS Actions of Ethanol and Acetaldehyde

A. Excretion of Total Radioactivity These results were obtained in rats, which had been injected

intravenously with l.Opc of d, l-C14-norepinephrine (C 1 4 -NE) , Carbon-14 was detected in the urine during the firstIwo hour sample period ( refer to methods, Figure 7) . Under the conditions of diu resis used, approximately two-thirds of the administered isotope (65/£) appeared in tli e urine within ten hours after injection. An

other 1 2 % appears in the urine during the succeeding 2 0 hours ( re

fer to methods, Figure 6 ). At least two phases of isotope excretion are apparent. The initial rapid phase of excretion has a rate con

stant of kj * 0.433 hour " 1 and therefore a time constant of 2.31 hrs. During this phase, the radioactivity declines by 50 °/a every 1.6 hours ( t l ). After the rapid excretion, the rate slows to a single phase of excretion having a half-life of seven hours. This slower process ex

hibits a rate constant of 0.099 hour ” 1 and a time constant of 1 0 . 1 hr. These calculations are in close agreement with those of Kopin and Gordon ( 1963) after injection of tritium labeled norepinephrine in rats. The alternate pathways for the metabolism of norepinephrine and the structures of the metabolites are shewn in Figure 37, As previously 151

TABLE 2k

COMPARISON OF THE SYMPATHOMIMETIC ACTIVITY ON BLOOD PRESSURE OF TYRAMINE AND ACETALDEHYDE IN UNTREATED ANIMALS

TREATMENT TYRAMINE ACETALDEHYDE

CONTROL RESPONSE PRESSOR PRESSOR

Oi BLOCKADE REVERSAL

/} BLOCKADE 0 0

GUANETHIDINE O

PHENYLETHYL AMINES o

COCAINE o -d h

•ADRENALECTOMY 0 0, S L IG H T ^

••SPINAL ANIMAL 0 0

•••GANGLIONIC BLOCKADE 0 0

ATROPINE

SYMBOL increase or decrease In control respones; 0 = no change In control response; * - Akobanc 1964, ** = Eade 1959, • * • = Akabane 1964. 152

no-if^v CH-ch 2 -N Hz CH^O-rf^VCH-CHp-NHp OH COMT HO-LJ OH Norepinephrine (NE) Normelonephrine (NM) MAO MAO i ' / > CH3 Onp*rCH-c^I ' , H O ^ L ; ^ o h ' h H O - V ^ OH H WAnu NE-oldehyde NM-aldehyde NADH NAD 'J / ' \ f NA° ) / / \ c ° nad ^ dh ai ^ adh NAD jfiD H r V - DH

HO-rt^|-9 H-CH2-OH HO -|< S -C H -C \ CH30-^ ^ C H -C H 2“0HCH3Mf<5s!VCH - c \ h o 4 ! ^ J o h HO OH OH h o 4 J OH h o 4 J 6 h OH 3,4 Dihydroxy- 3 , 4 Dihydroxy- 3-Methoxy-4- 3-Methoxy-4- phenylglycot mandelic odd hydroxyphenyf-ypnenyi- hydroxy t n a n - glycol (MHPG) delic acid (VMA) ______COMT J COMT

Figure 37i Metabolic pathway for the metabolism of norepinephrine. Enzymes: MAO - Monoamine Oxidase, COM2? - Catechol 0-Methyl Transferase, ADH - Alcohol Dehydrogenase, Ald-DH - Aldehyde Dehydrogenase. 153 stated, the slower excretion curve represents the release of the

1-NE-C 1 4 and follows the curve described by 3.6e“°’°99t percent of the administered dose per hour, where t equals hours after in jection. The integral of this curve is 36.4e“0,0"* percent. There fore, at zero time, 36 0/o of the administered norepinephrine may be considered to have been bound in a slowly metabolized pool.

B. Urinary Metabolites of Norepinephrine-C 1 4 Table 28 shows the pattern of urinary metabolites of C14- norepinephrine in the rat and compares the metabolites excreted in 24 hours, with those excreted during the 12 to 16 hour period. The analysis of the 24 hour urine represents about 7 5 °/0 of the administered C 1 4 -NE, whereas, the 12-16 hour urine consists of only about 4 °/6 of the administered carbon-14 ( about 80,000 - 100,000 dpm s ) . The samples were completely hydrolyzed either with acid or enzymatically (See Methods) to break glucuronide and sulfate conjugates of the various metabolites. There is a marked temporal variation in the excretion products of CI4-NE in the rat. After 24 hours, the major urinary metabolite is normetanephrine ( NM) which comprises 22 °/0 of the C1 4 excreted, after which is 3-methoxy- 4-hydroxyphenyl glycol (MHPG) making up another 19.5 °/a. A large proportion ( 14.3 % ) ot the radioactivity is also represented by unmet abolized norepinephrine ( NE ), most of which is excreted during the first few hours (Kopin and Gordon, 1963), The deaminated metab olites are excreted in equal proportion: 3, 4-dihydroxymandelic acid ( DHMA) equals 5.2 0/0, and 3, 4-dihydroxyphenyl glycol (DHPG) equals

6 . 8 /£. The other major metabolite which is deaminated and o-methylated, 3-methoxy-4-hydroxymandelic acid (vanillylmandelic acid, VMA), appears in the urine as 13.6 % of the radioactivity excreted in 24 hours. This pattern is compared with the metabolism of bound C14-NE in TABLE 28

METABOLIC FATE OF C1^-NOREPINEPHRINE IN RATS: COMPARISON OF 24-HOUR AND 12-16 HOUR METABOLIC PATTERN

Total6, Bound6 0-24 Hour 12-16 Hour

Norepinephrine 14.33 ± 1.2 4.87 ± 1.55

Normetanephrine 21.94 ± 0.75 5.49 ± 0.32

Deaminated Catechols 12.00 ± 1.06 24.14 + I.16

3.4-Dihydroxymandelic Acid 5.20 4 0.62 12.16 4 1.16

3.4-Dihydroxyphenyl glycol 6.80 4 0.81 11.98 4 0.88

1 O-Methylated, deaminated 23.13 ± 1.53 46.68 4 2.52 Metabolites

3-Methoxy-4-hydroxymandelic 13.62 ± 1.16 20.95 4 2.25 acid

3-Methoxy-4-hydroxyphenyl 19.51 ± 1.7 25.73 i 2.8 glycol

recovered in metabolite 81.4 ± 4.01 81.2 4 3.28 fractions

6 Values represent $ of total C ^ ± S.D, excreted in the urine during that time period. 155 Table 28. The metabolites present in the urine during this interval represent C14-nor epinephrine which has been bound and slowly re leased and metabolized (Whitby et al., 1961) . During this time period ( 12-16 hours), norepinephrine and normetanephrine account for a total of only 1 0 °/0 of the excreted radioactivity.

The deaminated O-methylated metabolites have increased to 47 %: VMA is 21 °/0 and MHPG is 27 In the rat, the major metabolite of endogenous norepinephrine is MHPG (Axelrod et al., 1959), unlike in the human in which the major urinary metabolite is VMA (Armstrong et al., 1957) . Similarly, eight hours after C14- norepinephrine injection the major metabolite in humans is VMA {45 °/g ) while MHPG comprises only 15 % (Davis et a l., 1967b). In our studies, MHPG was the major urinary metabolite in rats. The deaminated metabolites make up 24 °/0 of the urinary radioactivity. Both DHMA and DHPG still occur in about equal proportions. The precision of this method for analyzing the metabolites of C14-NE can be seen by examining the individual values for seven saline treated control rats ( Table 29) . In all cases the standard error of the mean is four percent or less of the mean value, except in the case of NE, in which it is about 11 Q/a. This is quite common because of the ad sorption procedure and the fact that the NE values were arrived at by subtraction. These data substantiate quite well the reproducibility of the method,

C, Effect of Various Drug Treatment on the Metabolism of Cl4-Nor epinephrine Table 30 gives a summary of the results obtained with various drug treatments on the metabolism of bound C 1 4 -norepinephrine. It can be seen that the different drug treatments had little effect on the amount of C14-NE excreted during this time period ( Figure 38). All TABLE 2 9

METABOLIC PATE OF BOUND -NOREPINEPHRINE IN CONTROL RATS8,

Experiment*5 C^-NE C^-NM C^-DHPG C^-DHMA C^-MHFG C^-VMA Total

1 5-70 5.59 1 2 . 7 9 1 0 . 7 9 28.07 1 8 . 4 6 8 1 . 4 8 2 5 . 6 8 5.13 1 1 . 7 8 1 1 . 2 0 2 8 . 4 3 2 2 . 3 3 8 4 . 5 5 3 2 . 7 8 5 . 0 7 1 0 . 8 7 1 3 . 4 2 24.92 17.80 74.86 4 7 . 3 4 5.84 1 3 . 1 7 13.70 2 1 . 0 4 2 1 . 4 9 82.58 5 3 .86 5.28 1 1 . 6 3 1 1 . 1 4 2 8 . 4 1 2 0 . 0 1 8 0 . 3 3 6 3 . 7 0 5.78 1 1 . 0 6 1 2 . 2 8 23.66 23. 6 9 8 0 . 1 7 7 4 . 9 2 5 .71 1 2 . 5 8 12.60 2 5 . 5 9 22.85 84 . 2 5

KeanC 4 . 8 7 5 .49 11.98 12.16 2 5 . 7 3 2 0 . 9 5 8 1 . 1 8

S.D. 1 . 5 5 0 . 3 2 0 . 8 8 1 . 1 6 2 . 8 0 2 . 2 5 3.28

S.E. 0 . 5 8 0 . 1 2 0 . 3 3 0 . 4 4 1 . 0 6 0 . 8 5 1 . 2 4

a Metabolites are expressed as percent of C1* In the urine collected, during the 12-16 hour period. All saline treated controls are pooled since there va3 no significant difference between animals (l,2,3) given saline orally, 2 0 ml/Xg, and animals (4, 5, 6, 7) given 3 i.p. injections of saline, 3 ml/Kg. e S.P.-standard deviation; S.E.-standard error of the mean. | TABLE 30

EFFECT OF DRUGS ON THE URINARY EXCRETION OF C1^-NOREPINEPHRINE AND ITS METABOLITES

Calcium Disulfiram Calcium Carblmide Control Eton CH^CHO Disulfiram + EtOH Carblmide + EtOH

£ of Administered Dose 3 . 9 8 3.16 3 . 3 0 4 . 1 2 3 .43 4 . 2 1 3 . 6 0 Secreted In 1 2 - 1 6 Hour Period

Norepinephrine 4 . 8 7 3 . 9 9 5 .36 5.59 5.79 4 . 7 7 3 . 5 5

Deaminated Catechols 2 4 . 1 4 18.43 19.61 1 8 . 1 0 15.85 16.25 14.79

3,4- Bihydroxyphenyl glycol 1 1 . 9 8 9 .2 7 1 1 . 4 4 1 0 . 9 5 1 1 . 3 8 1 1 . 0 1 0 . 2 2 3, 4-Idhydroxyicandellc acid 1 2 . 1 6 8 .7 1 8 .1 7 7 .15 4 . 4 7 5 . 2 5 4 . 5 7

Uometanephrine 5 .49 6.92 7 . 6 7 5.40 8 . 4 2 5.30 7.26

O-Methylated, Deaminated J*6.68 4 8 . 6 9 4 7 . 3 4 4 3 . 7 8 4 2 . 7 5 4 5 . 3 4 4 9 . 0 Metabolites

3-Methoxy-4-bydroxyphenyl 2 5 . 7 3 2 9 . 5 2 35.57 3 3 . 5 2 3 6 . 5 0 32.0 39.0 glycol 3-Methoxy-4-hydroxyirandelic 2 0 . 9 5 1 9 - 1 7 1 1 . 7 7 1 0 . 2 6 6.25 1 3 . 3 4 1 0 . 0 a d d

Total Urinary Recovered 8 1 . 1 8 7 8 . 0 1 8 0 . 7 2 7 3 . 2 4 72.81 7 1 . 6 6 74.6 In Metabolite Fractions

3 6 rats received 1 uc d, 1- norepinephrine intravenously and the urine collected 12-16 hours after ^ and analyzed for C3- metabolites. Results ere mean values expressed as percentage of total radio- tn activity excreted during the collection period. ~'J 158

AMINE METABOLITES

SALINE TREATED CONTROLS ±S.D .

^ 7 *u S 6 0 1 X 4.07% i 5* (7) n 1

b to -H 2 * lu 339 5.36 3.95 5.79 4.77 3.35 u I (6 ) (7) (6 ) (6 ) (2 ) (2)

Zi TREATMENT: EtOH CHjCHO DISULFIRAM DISULFIRAM CALCIUM CALCIUM |O i 4grr>/Kg 300irg/Kg + CARBIMIDE CAR8IMIDE PO. 9«l. P. EtOH + 9- EtOH * X M 9% (7) » I

6.92 7.67 3.40 0.42 5.30 7.26 (6 ) (7) (6 ) <6) (2 ) (2 )

Figure 38: Effect of various drug treatments on the proportion of the amines (NE and NM) recovered in the urine 12-16 hours after Injection of 1 pc of C ^ -NE i.v. a. Values in hars represent mean percentages. b . Values in parentheses equal the number of animals. c. The points in the calcium carblmide groups represent the actual values for tiro experiments. 159 all of the bar graphs overlap the control value ± S. D. On the bottom part of the graph, is shown the effects on the excretion of

CJ4-normetanephrine (C 1 4 -NM) . Ethanol caused a significant in crease in the amount of NM excreted (p < 0.05) . A sa result of 4 gm/Kg of ethanol, NM was increased 26 °f0 above the control level. The administration of acetaldehyde (300 mg/Kg) to rats caused an even greater rise in the proportion of radioactivity that was excreted as NM (48 % rise, p<0.01).

The pretreatment of rats with either of the two aldehyde de hydrogenase inhibitors, disulfiram or calcium carbimide, failed to alter the control values obtained for NM excretion. However, when ethanol was administered to these animals (pretreated) a significant increase in NM excretion was observed. Pretreatment with disulfiram and then administration of ethanol produced a 62 °/a rise in NM (p< 0.01) . In a similar manner, pretreatment with calcium carbimide and administration of ethanol increased the NM from 5,30 °/0 to 7,26 0/o . This represented a 40 ^ change from the con trol. This effect on ^JM excretion parallels the blood levels of acet aldehyde that are produced with these treatments ( refer to Table 10).

Figure 39 illustrates the effects of these compounds on the excretion of deaminated metabolites DHMA and DHPG. Ethanol re duced the proportion of dihydroxymandelic acid from 1 2 . 1 6 to 8.71 °/0 . Injection of acetaldehyde caused a slightly greater reduction to 8.17 0/o . Treatment with disulfiram or disulfiram plus ethanol pro duced a 41 and 63 e/0 reduction from control in DHMA respectively, Calcium carbimide treatment lowered this acid to 5.25 °/0 and in the presence of ethanol reduced it further to 4.57 % of the urine radio activity. There is little effect of any of these agents on the excret

ion of DHPG. This metabolite appeared as 12 /£ of the C 14 excreted in the urine. 160

DEAMINATED METABOLITES

IB- S'K^xs SALINE TREATED CONTROLS t S.O. 14- 01 X 12.16%, ( 12 (7) r v ^ :■'*•.•'; » <«*. *■.##.; / < W V-n < 10 + 6 r i h

6 4 " ■Jm a 4H o i 8,71 B.I7 7.15 4.47 9.25 4.57 41 (6 ) 17) (6 ) ♦ 2 (6) (2) (2) s« £ EI0H CHjCHO DISULFIRAM DISULFIRAM CALCIUM CALCIUM TREATMENT! 4gm/Kg 300mg/Kg + CARBIMIDE CARBIMIDE BO. 3 k IP Eton + b_ o EtOH

I6 -1 0I *•1 u i

«j

8 9.72 11.0 10.22 S (2) V o

Figure 39* Effect of various drug treatments on the proportion of the deaminated metabolites of NE (DHMA and DHPG) recovered in, the urine 12-16 hours after injection of 1 uc of C^-NE i.v. a. Values in bars represent mean percentages. b. Values in parentheses equal the number of animals. c. The points in the calcium carbimide groups represent the actual values for tuo experiments. Examination of the effect of these drugs on the O-methylated, deaminated metabolites shows a pronounced effect ( Figure 40) . Ethanol in the rat failed to alter significantly the proportion of these two major metabolites in the urine. However, administration of acetaldehyde lowered the amount of VMA from 20.95 % to 11.77 0/o of the total urine activity. This 41 0/o reduction in the acid metab olite was accompanied by a concomitant and equivalent rise in the amount of MHPG excreted (40 °/0 increase) . Disulfiram caused VMA to fall to 10,26 9/0 while the glycol increase from control levels of 25.7 e/0 to 33,6 °/a , Administration of ethanol to disulfiram pre treated rats markedly potentiated this shift in metabolism. Under these conditions there was a 70°/ reduction in VMA from control and a 42 °/0 rise in the glycol metabolite. Calcium carbimide by it self caused a similar shift from the oxidative metabolite (VMA ) to the reductive product (MHPG). When ethanol was administered to animals given calcium carbimide, the alteration was much more pronounced. Table 31 summarizes these effects, illustrates the results more emphatically by using relative ratios. It can be seen that administration of acetaldehyde, disulfiram, calcium carbimide, or either of the last two agents in the presence of ethanol caused a significant reduction in CI4-VMA and a concomitant increase in

C 1 4 -MHPG. Ethanol was ineffective in causing this shift.

D. Correlation with Blood Levels of Acetaldehyde This shift in metabolism of norepinephrine from an oxidative route to a reductive one is well correlated with the amount of acet aldehyde present. In the rat, ethanol ( 4 g/Kg ) produces blood levels of acetaldehyde of 1,17 pg/nl and 2,65 |Jg/4nl at one and two hours respectively after alcohol administration ( refer to Table 10). Experiments were also performed in which rats were given three doses of acetaldehyde ( 300 mg/Kg, i. p. ) at half hour intervals and 162

0 - METHYLATED DEAMINATED METABOLITES

E 3 SALINE TREATED CONTROLS +S.D.

5*20.95% - * f * ' ‘ v" a' ink S T f * % ^ r '- * i c 2 0 - •.r A 'is°A * i •<-V ...... *■■■’ * • *T ^ * * ■ 4 • v * n w v w > i .vAT.-A-.'.-.»>vX.‘-:-r' i * JO- A i 10.17 II.77 I025 6,25 0.34 (0 . 0 (6 ) (7) (6 ) (6 ) (2) (2) s P a♦* 2. ° o EI0H CH3CH0 DISULFIRAM 0ISULFIRAJ4 CALCIUM CALCIUM TREATMENT: 4gm/Kg SOOmg/Kg + CARBIMIDE CARBIMIDE R0. S k I.R EtOH + EtOH

4 0 - i rih r i * 3 0 - ■«»*'*r+*+t WA**>v X*25.73%- 1T> \ i±Z

20 -

20.52 3557 3352 3550 320 39.0 (6 ) (7) (6 ) (6 ) (2) (2 ) to-

*o O'

Figure 1*0: Effect of various drug treatments on the proportion of the O-ir.ethylated deaminated metabolites of NE (VM/l and MHPG) recovered In the urine 12-16 hours after Injection of 1 fie of C^- N E l.v, a. Values in bars represent mean percentages. b. Values in parentheses equal the number of animals. c. The points in the calcium carbimide groups represent the actual values for two experiments. TABLE 3 1

EFFECT OF VARIOUS TREATMENTS OH THE RELATIVE AMOUMTS OF VMA AMD MHPG AFTER ADMINISTRATION OF C^- -MOREPIMEFHRIME IK RATS

Vffl-... XlOO 2ffiL_xi00 Treatincnt Dose n - VMA.+HGPG T» VMA.+MHPG T>

Saline-control — 7 hh.O$ - 55.l£ -

Ethanol h gm/Kg p.o. 6 3 9 - ^ M.S. 60 .6 $ M.S.

Acetaldehyde 300 mg/Kg l.p. 7 2^.9# p<0.001 75.1# p<0.001 3X

Disulfiram 200 rng/Kg p.o. 6 23. 5# p<0.01 76.5^ p<0.01 2X

Disulfiram 200 mg/Kg p.o. 6 l4.6# p<0.001 8 5 . ^ p<0.001 + 2X Ethanol U gm/Kg p.o. Calcium 50 mg/Kg l.p. 2 29.4£ p<0.01 70.6^ p<0.01 Carbimide 2X

Calcium 50 mg/Kg l.p. 2 20.^ p<0.001 79.6* p<0.001 Carbimide 2X + Ethanol ^ gm/Kg p.o. 164 the blood levels monitored ( Table 32 ) . Examination of these blood levels shows at least 5-10 fold greater level of acetaldehyde in these animals as compared with ethanol treated rats. Comparison of the blood levels show that administration of these amounts of acetalde hyde produced levels that were comparable to those produced when ethanol is given to disulfiram pretreated rats. Table 32 shows that this regimen of acetaldehyde produces levels of acetaldehyde from 9.71 to 19.69 pg/ml. It will be recalled that levels after ethanol and disulfiram ranged from 14,63 to 19. 64 fJg/ml ( see Table 10) . An other important observation shown in Table 32, is the m vivo pro duction of ethanol when large amounts of acetaldehyde are injected. The levels of ethanol parallel the acetaldehyde levels at the various times. In vivo formation of ethanol from acetaldehyde demonstrates the reversibility of alcohol dehydrogenase in the organism.

E. Effects of Ethanol and Acetaldehyde on the Central Ner vous System Experiments were performed to compare the relative potencies of ethanol and acetaldehyde in producing a sleeping state and in causing toxicity in mice. The curves represent mean values of actual ob served effects in five to ten animals per group. The EDso's and LD50's were experimentally determined in all cases except for ethanol hypnosis which was estimated from the curve. These data are depicted in Figure 41. Panel a shows the hypnotic actions of acetaldehyde. A dose of 275 mg/Kg produces hypnosis in 50 °/0 of the animals. Five mice were administered this dose and the blood level determined two and one-half minutes after the induction of hypnosis. The mean blood level occurring in animals which only fell asleep was 108[ig/-nl. This is in close agreement with the level of 100 (Jg/nl, observed by Mac Leod { 1950) to produce severe intoxication in mice. 165

TABLE 32

ACETALDEHYDE AMD ETHANOL BLOOD LEVELS FROM PROTEIN-FREE FILTRATES AFTER INJECTION OF ACETALDEHYDE (300 n®/Kg i.p.) IN RATSa

Time in m/nH ± S.E, Minutes Dose Ethanol Acetaldehyde

0 300 mg/Kg i,p. ■*

15 A5.olf ± 9.21 10.52 ± 2.20

30 300 mg/Kg i.p. ------

^5 62,08 ± 6.32 19.69 ± 3.97

60 300 mg/Kg i.p. —

120 8,83 ± 1.78 9*71 ± O .98

a n=10 or 12 rats for each time period. 166

Acetoldehyde 100 I 100

s 7 5 / 75 / Cfl K 50 i 50 i i £ 2 5 J 2 5 /

^ 1 ____ 1.., 0 / 1 200 250 300 350 400 500 600 D o s e , m g / k g ip Dose ,m g/kg ip o. Hypnosis ED-50-275m g/kg b, M ortality L D -5 0 -5 0 0 m g/kg Blood level-108 y/ml Blood level-2 2 1 yfml

E th o n o l E th a n o l 100 1 100

2 75 1 75 - /

o> a go if 50 d c $ a 2 5 / 25 /

1 1 0 i 2 .5 3 3.5 8 10 Dose,gm/kgip Dose ,gm /kgip c. Hypnosis ED-50-275 g/kg d. Mortality LD-50-8.0g/kg Blood level-3.4 mg/mt Blood level-6.7 mg/ml

Figure 4l: Dose-response relationships for the hypnotic and toxic effects of acetaldehyde and ethanol after i.p. injection In mice {n=5 to 10 animals per dose level). 167 Administration of this dose of acetaldehyde produced a hyp notic state for a duration of about' five and one-half minutes in animals that were affected, and the induction of sleep took about one and one-half minutes after injection { Table 33). Similar data for ethanol is shown in panel c of Figure 41, The approximate ED 50 for ethanol in these mice was 2,75 g/Kg. These mice slept an aver age of 7.3 (n = 6 ) minutes and exhibited a blood ethanol concentrat ion of 3,4 m g/nl at two and one half minutes after sleep was initiated. Comparison of the relative potencies of ethanol and acetaldehyde can be made in two ways: by comparing the dose administered or by comparing the blood levels that produced a hypnotic state. By com paring the doses, acetaldehyde appears to be about ten times as potent a hypnotic as is ethanol, since the dose required to produce sleep is about one-tenth that of ethanol. By examination of the blood level data, acetaldehyde is about 35 times as potent as ethanol, as a hypnotic agent. This comparison is a more valid one since it eliminater^the variables of differences in concentrations of the sub stances administered, and differences in absorption which are in- i herent in the comparison of dosage alope.

The pharmacologic actions of acetaldehyde in mice as related to dosage are summarized in Table 33, A sedative effect exhibited by a lack of activity begins to appear at a dose of about 200 mg/Kg. This is accompanied also by a slight ataxia. Increasing the dose to 250 mg/Kg, produces ataxia, increased respiration and hypnosis in 16 of the animals which sleep about three minutes. The duration of hypnosis is directly related to the administered dose. Injection of 300-350 mg/Kg produced sleep which lasted 7.8 and 10.6 minutes respectively, and it occurred within one to two minutes after injection, A dose of 400 mg produced severe intoxication, gasping and sleep with a mean duration of 12.1 minutep. Fifty percent mortality was produced 168

TABLE 33

RELATIONSHIP OF DOSE TO INTOXICATING AND TOXIC EFFECTS PRODUCED BY ACETALDEHYDE BY IHTRAPERITONEAL INJECTION IN MICE

Dose Sleeping-time mg/Kg n Condition of Animal ± S.D, Minutes

200 6 Lack of Activity, sedation, awake, to to « slight ataxia.

250 8 Increased rate of respiration, ataxia, 3.0 ± 0.5 some Bleep.

275 10 ED-50 for hypnotic activity of 5.5 ± 0.6 acetaldehyde, motor Incoordination in one minute, induction 1^ minutes. < 300 6 Ataxic one minute, sleep in 1^ minutes. 7 .8 ± 0 .1*

1*00 6 All sleep, gasping, severe intoxication. 12,1 ± 1.2

500 8 LD-50, 50$ mortality within 15 minutes 23.1* ± 2.1 after injection, others sleep with gasping.

600 5 All die within 12 minutes after injection mm m probably from respiratory depression. 169 by 500 mg/Kg, whereas the other animals slept an average of 23.4 minutes. One hundred percent mortality was produced by 600 mg/Kg. All animals expired within 12 minutes after injection.

The CNS effects of ethanol were also examined. A dose of 2 g/Kg i.p. produced only ataxia, whereas 3 g/Kg produced hypnosis in 9 0 % of the animals with a mean sleeping time of 9 . 6 minutes. Injection of 3.5 mg/Kg produced hypnosis in all animals lasting

approximately 25 minutes. A dose of 6 g/Kg induced sleep within

two minutes which lasted 75 minutes. The LD S 0 of 8.0 g/Kg produced hypnosis lasting 90 minutes to 126 minutes in about half of the animals, while this dose was fatal within 6.5 minutes to the remainder. Ad ministration of 10 g/Kg produced 100 % mortality which occurred within two minutes after i. p. injection.

In Figure 41, the dose-response relationship of the toxic

effects of acetaldehyde and ethanol are also depicted. The LD S 0 for acetaldehyde is 500 mg/Kg and therefore has a therapeutic index of 1.85. Half of the animals died within 15 minutes when given 500 mg/Kg Two and one-half minutes after induction of hypnosis with this dose

produced a blood level of 221 pg/ml. The LD 5 0 for ethanol was

8 g/Kg by this route with a mean blood level of 6.7 mg/ml. The

therapeutic index for ethanol in these mice is therefore 1 .9 1 , al most the same as for acetaldehyde. Comparison of the relative toxicities of these agents shows that acetaldehyde is about eight times more toxic than ethanol as measured by the ratio of their LDgo's ( 8000 mg/Kg/500 mg/Kg) . When the relative blood levels are compared in this way, acetaldehyde seems to be about 30 times as toxic as ethanol. Thus, the relative ability of acetaldehyde compared to ethanol in producing both hypnosis and mortality is approximately the same. 170 F. Effect of Norepinephrine and its Metabolites on Ethanol Hypnosis In order to study the effect of norepinephrine (NE) on the hypnosis produced by ethanol; a sublethal, hypnotic dose of 4.5 g/Kg was administered to mice intraperitoneally. Norepinephrine or its metabolites were administered 2 0 minutes after the induction of sleep by ethanol. Table 34 illustrated that i. p. injection of large doses of NE fail to produce any CNS effects in these mice. There was no demonstrable sedative effect of this dose of NE. Injection of 4,5 g/Kg of ethanol produced hypnosis with a mean duration of 43.6 minutes. Injection of 0,06 mM/Kg ( 10.1 mg/Kg) of NE after ethanol produced a 130 °/0 increase in the duration of sleep to 100.5 minutes. This effect of NE was dose related, increasing the dose to 0.12 mM/Kg caused a concomitant increase in the sleeping time to 158.2 minutes (260 °/0 increase) . Blood levels of ethanol were examined at 60 and 9 0 minutes after the administration of ethanol (Table 34) , This was done, because of the fact that the aldehyde intermediate of NE is reduced by an alcohol dehydrogenase enzyme, and could conceivabl*y compete for the enzyme and elevate ethanol levels. The levels of ethanol, however, were not significantly al tered by the administration of this catecholamine. Since NE does not effectively cross the blood-brain barrier, it was conceivable that one of its metabolites could be responsible for this action, The five metabolites of NE were injected i. p. at doses of 0.12 mM/Kg in a similar manner as was NE. The results obtained are shown in Table 35. Ethanol induced hypnosis, which lasted 44.8 minutes. The injection of the metabolites of NE failed to alter significantly the duration of this ethanol-induced comatose state.

The effect of pretreatment with two different monoamine oxidase inhibitors was also examined (Table 36) . Tranylcypromine ( Parnate**, 10 mg/Kg) and iproniazid (Marsilid**, 2 5 mg/Kg) were 1

TABLE 3 4

NOREPINEPHRINE (HE) POTENTIATION OF ETHANOL (EtOH) HYPNOSIS IN MICE .

EtOH Blood Level Sleeping-Time r M ± S.D. . Treatment Bose n ± S.D. na 60 min 90 min

Saline + 0 .5 ml HE 0 .1 2 mM/Kg 5 0

EtOH + 4 .5 gm/Kg Saline 0 .5 m l 2 1 4 3 .6 ± 6 .2 0 - 4 ,0 3 8 ± 9 8 4,462 ± 197

EtOH + 4 .5 gm/Kg HE 0 .0 6 mM/Kg 2 0 100.5 ± 10.34 p < 0 .0 0 1 4,215 ± 271 4 ,3 0 0 ± 1 4 4

EtOH + 4 .5 gm/Kg HE 0 .1 2 mM/Kg 1 8 158.2 ± 8.04 p<0.001 4,045 ± 448 4 ,4 3 6 ± 1 5 8

a p obtained by comparing with the sleeping time for EtOH + saline control. No significant difference in alcohol blood level among groups.

I TABLE 35

EFFECT OF METABOLITES OF HE ON THE DURATION OF ETHANOL (EtOH) INDUCED HYPNOSIS IN MICE

Sleeping Treatment®*^ n Time ± S.D.

EtOH + Saline 12 1*1*.8 ± 7.2

EtOH 4 NM 8 U6.9 ± 11.7

EtOH + DHMA 8 1*0.6 ± 8.3

EtOH 4 VMA 10 1*1.2 ± 5.2

EtOH 4 DHPG lh 52.1* ± 7.H

EtOH 4 MHPG i 15 1*2.1* ± 8.1

a Ethanol was administered i.p, at a dose of 1*.5 gm/Kg. The metabolites of norepinephrine were given at a dose of 0,12 mM/Kg, 20 minutes after ethanol which was about midway In the sleep period.

^ See Figure 37 for abbreviations. 173

TABLE 36

EFFECT OF MONOAMINE OXIDASE INHIBITORS ON THE HYPNOSIS PRODUCED BY ETHANOL (EtOH) ALONE OR IN COMBINATION WITH NOREPINEPHRINE (NE)

Sleeping $ Increase Treatment® n Time ± S.D. ± S.D.

EtOH 9 6l.h ± 19.1 m m m

Tranylcypromine 9 121,7 ± 8.3 98 ± lty + EtOH

Iproniazid 9 103.1 ± 6,k 76 ± 11$ + EtOH

EtOH + NE 10 156.lt + 12.8 155 ± 21#

Tranylcypromine i 9 166.5 ± 15.6 170 ± 25# + EtOH + NE

Iproniazid 10 1^7.3 ± 8.7 Iko i lk$ + EtOH + NE

a Tranylcypromine (Parnate 10 mg/Kg) and Iproniazid (Marsilid 25 mg/l^) vere administered i.p. 20 hours and 2 hours prior to ethanol (*t.5 e^/Kg# I.p.). Norepinephrine was given l.p. at a dose of 0.06 mM/Kg 20 minutes after ethanol administration. 174 given i. p. at 20 and 2 hours before injection of ethanol, This was done to see if an increase in the endogenous stores of NE also pro longed the ethanol sleeping time. Neither agent significantly al tered ethanol blood levels. Ethanol induced a coma in mice which lasted 61.44 minutes. Pretreatment with tranylcypromine poten tiated this hypnotic state by prolonging the duration of sleep to 121,7 minutes ( almost doubled) . Iproniazid pretreatment produced a similar effect (108.1 minutes). As shown previously, adminis tration of NE potentiated the ethanol hypnosis, and in this case pro duced a 155 °/0 increase in the duration ( 156,4 minutes) . Pretreat ment with tranylcypromine or iproniazid and then administration of ethanol and NE did not produce a further prolongation of the sleeping time. That is, the simultaneous increase in endogenous and exogenous NE did not produce a synergistic effect in prolonging the hypnotic action of ethanol. Instead, the duration of coma was statistically the same as with ethanol and NE alone,

I DISCUSSION

A fundamental examination of the pharmacological proper ties of acetaldehyde using several test systems has been the major aim of this investigation. Based on the evidence presented, it is apparent that acetaldehyde can induce: ( 1 ) a release of catechol amines, both in vivo and in vitro, ( 2 ) an alteration in the metab olism of administered CI4-norepinephrine ( C 1 4 -NE) , (3) sym pathomimetic activation of the cardiovascular system and the nictitating membrane in cats and rabbits, and (4) a depressant and hypnotic state in mice. These observations have been correl ated with the levels of acetaldehyde which can be produced in animals and man after ethanol administration. The discussion of these re sults will be organized according to the order in which the results have been presented.

i Ethanol and Acetaldehyde Levels The observation that acetaldehyde can be produced when eth anol is added to precipitated whole blood (Barker, 1941; Stotz, 1942; MacLeod, 1950; Truitt, 1969) has led to this re-evaluation of acet aldehyde blood levels in various species. The phenomenon of pro duction of acetaldehyde was examined in various species in relation to the gas chromatographic procedure which we have used for analysis of ethanol and acetaldehyde (modification of the method of Duritz and Truitt, 1964). The method involves equilibration of blood at 55°C for 15 minutes in order to reach complete liquid to air equilibration of these volatiles. Under these conditions, the blood from cow, monkey or man produces a substantial amount of acet aldehyde, when ethanol is added to the precipitated whole blood. When the blood of mouse, rat , rabbit or dog are analyzed in this same manner after 15 minutes of equilibration, no significant pro duction of acetaldehyde is seen. Therefore, the method ( Duritz and Truitt, 1964) as originally published may be satisfactory for blood analysis for these latter species. However, when longer equilibration times are used, the blood of all species will produce amounts of acetaldehyde sufficient to contribute a substantial artifact to reported values. The original procedure requires, therefore, a strict adherence to this time schedule. All values reported in this investigation have utilized a protein-free filtrate (Truitt, 1969) of blood, which has been found to obviate this in herent problem.

In order to attribute any of the human responses of ethanol to its metabolite, acetaldehyde, it was necessary to re-evaluate the levels of acetaldehyde produced after ethanol administration. Four human subjects weref examined for ethanol and acetaldehyde levels after administration of 0.75 gm/Kg of ethanol. The subjects were classified as social drinkers, and received ethanol after fasting overnight. The ethanol levels appeared to peak at 30 to 60 minutes with levels ranging from 88.0 to 132.5mg °/0 . The post absorptive levels of ethanol demonstrated the well documented ( reviewed by Kalant, 1962; von Wartburg, 1966) linear decline at an average rate of 12.9 mg °/0 per hour in these subjects. This rate of alcohol dis appearance is in close agreement with previous findings of Truitt

(unpublished results) who found a rate of about 1 2 mg °/0 per hour during this same time period. The acetaldehyde levels in these same subjects were quite low, peak values ranging from 0,1 to 0,55 Hg/nl. The acetaldehyde level appears to oscillate in a given subject, 177 a phenomenon observed previously by Ridge ( 1963) in both blood and brain. The failure to find a constant level in blood after alcohol is difficult to explain, since one would expect this if the rate of formation and degradation were constant. The absolute levels found in this study are in close agreement with those of Lundquist and Wolthers ( 1958), who found peak levels around 0.3 fjg/nl. Yet, examination of literature reports illustrates a hundred-fold range in reported values from 0.3 to 30 |Jg/nl ( see Table l) . Therefore, this limited study of human data, has served to delineate a general range of values of acetaldehyde for a reference point in evaluating its pharmacologic effects.

In rats, the blood levels of ethanol reached about 190 mg °/ at both one and two hours after administration of 4 g/Kg of 20 °/a ethanol by gavage. The acetaldehyde level in these animals rose from 1.17 (Jg/nl at one hour to 2.65 Hg/nl at 2 hours. These levels are two to five times higher than levels attained in humans. However, the dos age of alcohol given rats was four times greater than that adminis tered to the four human subjects. Administration of disulfiram, an aldehyde dehydrogenase inhibitor, produced a seven to twelve fold increase in acetaldehyde levels after alcohol. This rise in acet aldehyde after disulfiram is well documented { see Table 1), but has been questioned quite recently (Wagner, 1957; Casier and Polet, 1958) . A similar five- to fifteen-fold increase in acetaldehyde was also demonstrated to occur in rabbits after i. v. infusion of ethanol when pretreated with disulfiram ( see Figure 25) .

An apparently unique finding of these same studies is that ethanol levels are also elevated after disulfiram (Table 10). In early studies concerning the mechanism of disulfiram, several workers, after noting the elevated acetaldehyde levels, looked for an effect on alcohol oxidation. It was reasoned that possibly disul- 178 firam increased the rate of ethanol oxidation. In these reports ( Hald et a l., 1948, 1949a; Loomis,. 1950; Newman and Petzold, 1951) investigators failed to find increased ethanol oxidation. One study even said that if anything, ethanol oxidation was slowed (Newman and Petzold, 1951), while others dismissed the data by saying disulfiram showed no effect of enhancing alcohol oxidation.

However, based on the enzymatic mechanism of alcohol de hydrogenase, one would expect that disulfiram might well alter alcohol oxidation ( Theorell, 1967) . Studies with the purified en zyme from horse liver (Theorell, 1967; von Wartburg and Papen- berg, 1 9 6 6 ) have demonstrated that it is reversible, and that the equilibrium favors the reduction of acetaldehyde to ethanol. Asa m atter of fact, the reaction in this direction is often used to assay the activity of the enzyme in liver. It is also known that ethanol oxidation can proceed only when the NADH and acetaldehyde have been displaced from the active site on the enzyme. Thus, it would seem logical that large amounts of acetaldehyde produced after di sulfiram could occupy the active site and be converted to ethanol, while ethanol competes for the reverse process. This type of push- pull mechanism could retard the oxidation of ethanol and explain the elevated levels. Koe and workers (personal communication) have observed similar increases in ethanol as a result of other aldehyde dehydrogenase inhibitors.

In any case, the finding of such low levels of acetaldehyde in the blood of human subjects and relatively low levels in rats after ethanol, led to the examination of tissue concentrations of acetalde hyde, The reasoning that local tissue concentrations,by localized production rather than by transport, might be more pertinent to the the actions of acetaldehyde prompted this evaluation. Preliminary data ( see Table 11) showed an accumulation of acetaldehyde in several tissues as compared with blood. Liver was extremely high, presumably because it is the major site of formation . The kidney also accumulated acetaldehyde as did the small intestine. The brain was seen to accumulate 25-60 °/0 more acetaldehyde than blood. This greater concentration in nervous tissue was felt to be a very important observation. Other tissues showed much lower concentrations than did blood. Like the blood, tissue levels were much higher at two hours compared with the one hour samples. The ethanol concentrations were essentially the same in all tissues at both one and two hours. Ethanol did not accumulate in any of the tissues, that is, the tissueiblood ratio was less than 1 . 0 in all cases. The transport of ethanol into adipose tissue and skeletal muscle was considerably less than the other tissues examined,

When rats were pretreated with disulfiram both the ethanol and acetaldehyde tissue levels were elevated. The concentration of ethanol more or less paralleled that of the blood, again with the exception of fat and skeletal muscle. The production and distri bution of acetaldehydp in the tissues was markedly altered by disul firam. Liver, kidney, small intestine and brain were still the highest. However, the same relative proportions did not occur in the tissues as they had without disulfiram treatment. Compared with the blood levels, most tissues were quite a bit lower. Only the liver exhibited a tissue:blood ratio greater than one. This alteration in the relative distribution of acetaldehyde implies that the tissue concentrations are altered by factors other than just the blood acet aldehyde. It is most probable that the relative inhibition of aldehyde dehydrogenase in each tissue may reflect the increase in acetaldehyde as much or more than the blood level does. In general, tissue con centrations were no more than tripled by disulfiram pretreatment, whereas blood levels rose seven to twelve fold. 180 The significance of the tissue concentrations of acetaldehyde, while encouraging, are doubtful. The analysis of tissue standards resulted in the discovery that acetaldehyde was produced in precip itated tissue homogenates, when ethanol was added ( see Table 13). This production of acetaldehyde by tissues was similar to that produced by whole blood, but it occurred much more rapidly. The phenomenon was shown not to be due to the precipitant interacting with the tissue or with ethanol. It occurred regardless of the type of precipitant used, and was equilivant to the values found when ethanol was administered to rats.

This production of acetaldehyde poses a serious problem in trying to analyze tissues for acetaldehyde. One very serious quest ion which arises, is the possibility that the acetaldehyde produced by the recently discovered microsomal ethanol oxidizing system (MEOS, Lieber and DeCarli, 1968) might well be artifactual pro duction. The samples which were sent to our laboratory for anal ysis were precipitated with TCA and contained ethanol. The subcellular material may well produce acetaldehyde by a non-enzymatic method. That this is possible, is evidenced by the fact that the amount of acetaldehyde produced by precipitated tissues is propor tional to the added ethanol concentration and increases with time of equilibration. These are criteria which have been used in examin ing MEOS. However, to verify this possibility, experiments with resuspended liver microsomal pellets need to be performed. Re suspension of microsomes, precipitation of protein, and then addition of ethanol should answer this question.

Since the head space gas analysis by gas chromatography was not sensitive enough to detect levels of ethanol below 1 0 0 pg/g of tissue, it was incapable of determining whether the acetaldehyde produced was formed from ethanol. Experiments with blood using trideuterated ethanol indicated that ethanol was not the source of the acetaldehyde (Truitt, 1969). Experiments using ascorbic acid as an antioxident, and EDTA or BAL to prevent nonspecific metallo- oxidation failed to inhibit acetaldehyde production in tissues. Since tissue and blood measurements of acetaldehyde pose problems due to artifactual production, other measurements were investigated. One method which proved to be quite useful involves the formation of a subcutaneous air pocket on the back of rats, and sampling from this air space which is in equilibration with the blood (Lester and Greenberg, 1950) . Values arrived at by this method were a bit higher than those obtained by analysis of a protein-free filtrate of blood, Urine analysis is quite useful for comparison of one animal to the next, however, the volatility of acetaldehyde requires that the sample be immediately sealed for analysis. Measurement of the levels expired into the air in a glass metabolism chamber were also made. Acetaldehyde was injected into the femoral vein, and the four methods of measuring acetaldehyde were compared ( see Table 17). Blood, expired air, and subcutaneous air pocket m ea surements demonstrate the rapid half-life of acetaldehyde. By all three procedures, the acetaldehyde was completely metabolized between 5-10 minutes after injection. Acetaldehyde, however, could still be detected in the urine after an hour. Administration of ethanol to untreated and disulfiram pretreated animals allowed com parison of blood, urine and air levels of ethanol and acetaldehyde. In these experiments acetaldehyde and ethanol were again both elevated by disulfiram pretreatment.

A closer examination of air measurement in a sealed metab olism chamber was made. Monitoring the chamber air at various time intervals was found to be a very effective way of measuring acetaldehyde. Injection of 30 mg/Kg of acetaldehyde into rats and then measuring the air samples revealed a two phase process 182 (see Figure 11). Initially, acetaldehyde in the air rose and peaked at five minutes, which coincides with the time when the blood level has reached almost zero. After this the acetaldehyde declined in the chamber presumably due to reabsorption by the lung and sub sequent metabolism.

Administration of ethanol allowed its measurement in expired air. No acetaldehyde was detected. Pretreatment with disulfiram and then alcohol permitted the detection of quite large amounts of acetaldehyde. The rate of the ethanol excretion by the lung was much more rapid in these animals, again indicating higher plasma levels. Acetaldehyde excretion paralleled very closely that of ethanol so that it seems to be formed directly from ethanol and excreted. The maximum excretion of acetaldehyde after ethanol (4 gm/Kg) and disulfiram was very close to that excreted when acetaldehyde was injected alone ( 30 mg/Kg) .

The measurement of acetaldehyde in air proved to be very easy and' reproducible. The method avoids the problems previously men tioned with blood and tissues, and alleviates the volatility problem encountered with urinary measurements. This procedure should have general applicability for measurement of ethanol and acet aldehyde levels in small animals, the major advantages being rap idity and lack of sample preparation.

In Vitro and In Vivo Catecholamine Release

The ability of acetaldehyde to interact with catecholamine stores has been examined in isolated left atria of guinea pigs and in whole animal experiments using rabbits and cats. Electrical and mechanical measurements were simultaneously measured in response to norepinephrine, tyramine or acetaldehyde. 183 Norepinephrine produced changes predominantly in two para m eters of the membrane potentials of the left atria of guinea pigs. These were an increase in the velocity of the action potential de polarization and an increase in the action potential area. These changes occurred simultaneously with the positive inotropic effects of NE, which were demonstrated by an increase in contractile ten sion and the rate of development of tension. These effects are sim ilar to those produced by epinephrine {Webb and Hollander, 1956; Furchgott et al. , I960) . These same characteristic responses were also produced by the typical indirect-acting sympathomimetic amine, tyr amine. The responses of the atrium to tyramine could be almost eliminated by reserpine pretreatment.

This investigation of the sympathomimetic action of acet aldehyde on isolated atria of guinea pigs confirms its peripheral action and supports its classification as an indirect sympathomi metic agent. Acetaldehyde produced positive inotropic effects in concentrations ranging from 0.3 to lOmM. The positive inotropic response induced by acetaldehyde increased in a dose-related manner. This same dose range of acetaldehyde caused progressive increases in the action potential area and rate of rise of the action potential. Therefore, the actions of acetaldehyde on the electrical and mech anical parameters of isolated left atria of guinea pigs were strik ingly similar to those produced by NE and tyramine on the same preparation. The action of acetaldehyde most closely resembles that of tyramine because of the longer time required to produce its maximum inotropic effect, and because of the elimination of its inotropic response by reserpine pretreatment.

Kumar and Sheth ( 1962 ) demonstrated that acetaldehyde caused positive inotropic and chronotropic responses in isolated paired rabbit atria at concentrations ranging from 100-800 pg/nl (2.27 - 184 17.2 mM) . In a similar manner acetaldehyde produced positive inotropic and chronotropic effects by perfusion through the sinus

node artery of the dog in concentrations of 1 0 0 and 1 0 0 0 fJg/ml (James and Bear, 1967, 1968). In our experiments, pretreat ment with reserpine inhibited the positive inotropic responses pro duced by acetaldehyde (0.3 - 10mM) which is consistent with the observations of the above investigators. Reserpine treatment also prevented the changes in the monophasic action potential produced by these concentrations of acetaldehyde.

However, higher concentrations of acetaldehyde, lOmM (at three minutes) and 30 mM (440 and 132 0 (Jg/nl respectively) pro duced negative inotropic effects on the isolated left atria. These findings are inconsistent with the two reports indicated above. The apparent discrepancy may be due in one case (Kumar and Sheth, 1962) to the time of measurement of the response. As has been demonstrated, the biphasic effect of a lOmM concentration of acet aldehyde is a time-related phenomenon. These investigators may have reported only the positive inotropic responses produced by higher concentrations of acetaldehyde (400-800 pg/lnl) . In the second report (James and Bear, 1967) , the deviation from our results may be due to the rapid transit of acetaldehyde ( 2 ml of 1 0 0 0 \ig/m\ at ten minute intervals) past the nodal area and into the general cir culation without sufficient time of contact to induce a depressant action. They mentioned that if the interval of injections were shortened, acetaldehyde would produce sinus arrest and periodic rhythm which is consistent with the above explanation.

The negative inotropic effects and the prolonged action poten tials produced by acetaldehyde at higher concentrations were not prevented by reserpine pretreatment. These responses therefore do not represent catecholamine-mediated effects of acetaldehyde, 185 but most probably direct effects of acetaldehyde on myocardial cells. This depressant action of high doses of acetaldehyde ( 10- 30mM = 440-1320 |jg/nl)is similar to that produced by formalde hyde in equivalent concentrations (James and Bear, 1967, 1968). They showed that formaldehyde produced significant depression on the heart at a concentration of 1000 |Jg/4nl. Likewise, the increase in the AF-area at these higher concentrations of acetaldehyde is like that produced by formaldehyde on cardiac potentials ( Fozzard and Dominguez, 1968) , It seems highly probable that the entire homologous series of aldehydes have a common action on cardiac function, while only a select few also possess sympathomimetic activity (Hitchcock, 1947) . James and Bear ( 1968) have examined the structure-action relationship of various aldehydes on the ECG after inlra-nodal infusion. Their analysis was in agreement with this hypothesis.

The actions of acetaldehyde can be blocked by beta-adrenergic blocking drugs. We have shown here that the positive inotropic eff ects of acetaldehyde are inhibited by propranolol, in what may be a noncompetitive manner. Similarly, propranolol blocked the negative inotropic effects of higher concentrations of acetaldehyde. The positive inotropic actions of tyramine are also apparently non- competitively inhibited by propranolol in the isolated atria of guinea pigs (Benfey and Varma, 1966), and also spontaneously beating right atria of rabbits (Thompson and West, 1968) . Thus, there is another similarity between the actions of tyramine and that of acet aldehyde. However, the non-competitive antagonism of propranolol against acetaldehyde and tyramine effects should not be taken liter ally in the biochemical sense, as the mechanism by which these agents interact with beta-blocking drugs. It seems most likely that this kinetic analysis results simply from the fact that there is a maximum rate of NE release which is less than the release rate required to overcome the effects of propranolol concentrations used in these experiments. This seems a reasonable explanation since chronotropic effects of added norepinephrine were competitively- inhibited by propranolol, but apparent non-competitive kinetics were seen where propranolol blocked the positive chronotropic response induced by electrical stimulation (Thompson and West, 1968). Additionally, an action of propranolol pre-synaptically to inhibit NE release may enter into this phenomenon ( Foo et al. , 1968). In a derived calculation of the data of Benfey and Varma

( 1 9 6 6 ), the positive inotropic responses to tyramine were inhibited by propranolol at a pD/ = 6,32. The positive inotropic action of acetaldehyde was inhibited by propranolol at pD2^ = 6 .8 . This sim ilarity implies a similar site of action of these two drugs. Never theless, this does not mean to imply that their mechanisms of re lease of norepinephrine are the same. On the contrary, there are several distinct differences between the actions of tyramine and acetaldehyde (Eade, 1959; Akabane et al., 1964a; See Results).

An inhibition of the negative inotropic effects of acetaldehyde was also produced by propranolol. The depressant effect of acet aldehyde on atria does not seem to be a catecholamine-mediated response, since it persisted in reserpine pretreated animals. Pro pranolol may be inhibiting this response by a secondary action. This action has been termed antifibrillatory (Benfey and Varma, 1966) or local anesthetic action( Levy, 1967, 1968) of propranolol, and appears to be unrelated to its beta-blocking properties. If this secondary action of propranolol is a membrane effect, it is most likely that acetaldehyde has a depressant action on myocardial tissue in higher concentrations. Alternatively, it may be postulated that acetaldehyde may have a direct action on cardiac beta-receptors. 187 Propranolol, by blocking the receptors may protect these receptors from the effects of high concentrations of acetaldehyde.

Acetaldehyde altered the decay pattern of plasma tritium after the injection of 7-H 3 -norepinephrine. This two carbon aldehyde produced a sharp rise in the plasma tritium which peaked at one minute and rapidly declined. The time course of this effect was quite parallel with the time course of blood pressure effects ( see Figure 33) and with the ephemeral existance of the aldehyde in animals after injection alone ( refer to Figure 24) . A similar effect of tyramine on plasma catecholamines has recently been demonstrated ( Harvey et al., 1967) . The rapid rise in plasma tritium occurred in both cats and rabbits. Infusion of 2 gm/Kg of ethanol caused a similar alteration.in the plasma decay pattern but the magnitude of the response was much less. Ethanol produced an 8 rise in plasma tritium which was prolonged over the duration of the experi ment. The time course of this effect and the degree of response is in agreement with the continuous production of acetaldehyde and the relative amount produced by i. v. infusion of ethanol (2 gm/Kg) . These increases of the tritium in plasma was not due to volume changes since injection of equivalent volumes of saline did not alter the decay pattern.

Pretreatment of rabbits with disulfiram, and then infusion of ethanol produced a marked rise in the tritium in plasma. The rise was equivalent to that produced by acetaldehyde injection, while the duration of the response was prolonged. The acetaldehyde levels in these animals were increased from 4.5 to 9 fold when disulfiram was used. The potentiation of the response produced by ethanol in rabbits pretreated with disulfiram was three to four fold greater than that produced by ethanol in untreated animals. Further evi dence for the release of H3-norepinephrine by acetaldehyde was 188 obtained by examining the urine of these same animals. Acet aldehyde produced a marked rise in the tritium appearing in the urine.

The validity of this method as a test for a release of radio active norepinephrine wasTtested. It is reasonable that if the in jected H3-norepinephrine (is^taken up into nerve endings, it should be able to be demonstrated, BVLlateral carotid occlusion, which causes a generalized reflex actiivation of the sympathetic nervous system also produced rises in plasma tritium that were equivalent to those produced by acetaldehyde, A similar effect in urine was also noted. Therefore, the method which we have developed here may have general applicability in the screening of agents which are in direct-acting sympathetic agents. Hertting et al ( 1961) used a . similar method to see the effect of various agents in blocking uptake of injected H 3 -norepinephrine. They also used whole animals and monitored plasma tritium. 7-H3-norepinephrine was injected at various times after other agents had been injected and the relative rate of initial decline in tritium was used as a measure of neuronal uptake blockade. Our method as utilized here to see a releasing action of acetaldehyde is exactly the converse, and used mainly to demonstrate neuronal release of the H 3 -norepinephrine. The method is easier and less cumbersome than that of Kopin and Gordon ( 1962) which requires examination of the urine after about ten hours of prior injection of the radiolabeled transmitter. Their method was utilized in this study for examination of changes in catecholamine metabolism. Therefore, monitoring of plasma levels may be more representative of what is occurring at sympathetic nerve endings and may give a more precise time course of these events. Utili zation of plasma measurements only required prior equilibration 189 for an hour and needs only minimal surgery. Thus, this test system offers the advantages of rapidity and ease for testing chemorelease of the adrenergic transmitter by drugs.

The most reasonable conclusions which can be reached from these studies is that acetaldehyde is an indirect acting sympatho mimetic agent, based on the data obtained from isolated atria of guinea pigs and plasma tritium measurements in cats and rabbits. Acetaldehyde produced responses on isolated atria which were sim ilar to those produced by norepinephrine and tyramine. Also, the releasing phenomenon on H3-norepinephrine in whole animals was similar to that produced by sympathetic nerve activation elicited by bilateral carotid occlusion. These data indicate that acetaldehyde is capable of producing cardiac and possibly other effects by a peripheral action mediated by release of nerve ending norepinephrine.

Furthermore, many effects heretofore attributed to ethanol which have latencies in response and are sympathomimetic in nature may be explained by this action of acetaldehyde on catecholamine stores. For example, the stress-like urinary excretion of catechol amines and their metabolites after ethanol administration (Kling- man and Goodall, 1957; Anton, 1965; Mendelson et al. , 1969) may be due to this metabolite. Similarly, other sympathomimetic actions produced by ethanol or acetaldehyde such as the accumulat ion of hepatic triglycerides ( Truitt et al., 1966), mobilization of free fatty acids (Truitt, unpublished data), activation of cardiac lipoprotein lipase (Mallov and Cerra, 1967), and the hyperglycemic effect (Kohei, 1967a; 1967b) all seem to be mediated by the actions of acetaldehyde on norepinephrine stores.

In the case of the cardiovascular effects of ethanol, the older belief that alcohol was a stimulant might have some foundation in the fact that its major metabolite, acetaldehyde, does cause positive 190 inotropic effects. Several more recent studies of the effect of ethanol on the cardiovascular system indicate that alcohol may serve as a sympathetic activator (Kissin et al,, 1959; Docter and Perkins, 1961). Similarly, the role of acetaldehyde in alcoholic cardiomyo pathies (reviewed by Truitt, 1966), deserves further investigation. Cardiomyopathy due to alcohol consumption has recently been re cognized as a substantial contribution to causes of obscure heart failure (anonymous, 1967). Explanations of the pathogenesis of cardiovascular defects in alcoholics as being due to malnutrition, vitamin insufficiency, and bacterial infection are not necessarily correct in a majority of alcoholic patients (Burch and Walsh, i 9 6 0 ). Thus continuous production of acetaldehyde may be causally related to these cardiac problems.

The most interesting question which these experiments pre sent, is why a two carbon aldehyde, which is structurally unrelated to catecholamines, should cause a release of amines. This action of tyramine is understood on a displacement and affinity basis for the binding site of norepinephrine, but this type of reasoning of the acetaldehyde releasing mechanism is not easily rationalized. It was suggested that acetaldehyde by forming a Schiff base with nor epinephrine causes its release (Romano et al., 1954; Beck et al., 1968) . However, there is no good evidence for this. Another suggestion (G. Cohen, personal communication), is the formation of a tetrahydroisoquinoline by cyclization of the intermediate Schiff base of norepinephrine and acetaldehyde which he has isolated by perfusion of isolated bovine adrenals with acetaldehyde. These suggestions would require further investigation before such conclus ions can be reached. However, an interaction with the amine is likely rather than with the binding site, since a tachyphylaxis on blood pressure does not seem to be produced to rapid and consecutive acetaldehyde injections in cats and rabbits, Therefore, an action 191 such as acetylation of binding sites to displace norepinephrine would not seem to be the mechanism involved. Interaction with the amine to reduce its binding affinity would allow synthesis to replace the released amine and tachyphylaxis would not be expected. Further investigation of the release of amines by acetaldehyde needs to be made before the mechanism involved can be elucidated.

Disulfiram-Alcohol Reaction

The pattern of sympathomimetic activity on blood pressure shown by acetaldehyde differs in several respects from tyramine. In untreated control rabbits and cats the two drugs behave qualit atively in a similar manner but differ quantitatively in the release of norepinephrine and the influence of norepinephrine, methyldopate and dopamine as repleting infusions, However, in reserpine and disulfiram pretreated animals, acetaldehyde displays a vasodepres sor action whereas the tyramine action is only reduced in ampli tude. Norepinephrine re-infusion partially restored the pressor effect of tyramine in these animals, while the response to acet aldehyde was essentially unchanged. Responses of the nictitating membrane were more similar between the two catecholamine re leasing agents and in some cases, the recovery of contraction to acetaldehyde was obtained more readily with some reinfusions of norepinephrine.

The differences in the blood pressure response and similarit ies of the nictitating membrane contraction between tyramine and acetaldehyde can best be explained by assuming a single action for tyramine and a multiple effect produced by acetaldehyde. Akabane and associates ( 1964a) noted sim ilar differences between the two drugs in spinal-sectioned cats with and without reserpine pretreat ment. They noted, as shown in this study in the reserpine-pretreated animal, that the vasodepressor action of acetaldehyde is increased by norepinephrine re-infusion. As many others have shown, the vasopressor action of acetaldehyde is blocked by alpha receptor adrenergic blocking drugs. In a subsequent study (Akabane et al., 1965), the adrenal action of acetaldehyde was said to be a direct action on medullary cells independent of an acetylcholine trigger. Eade { 1959) also noted the difference that the tyramine response could be restored by norepinephrine but acetaldehyde could not be reversed. Cocaine, guanethidine, chlorpheniramine, and triplennamine all were found to potentiate the pressor responses to acetaldehyde as has been found by others. This constitutes a marked difference in the activity of acetaldehyde and tyramine. All of these drugs have been shown to attenuate the blood pressure responses to tyramine. One possible basis for this difference is that tyramine, being a phenethylamine, is subject to uptake blockade by these drugs as is the normal transmitter, norepinephrine. All of the agents mentioned have been shown to block neuronal uptake of norepinephrine (Isaac and Goth, 1967), and thus would interfere also with the action of tyramine. However, because of its structure, acetaldehyde should not need specialized transport into the neuron. Acetaldehyde probably enters by simple diffusion along lipoid chan nels. Therefore, in the presence of these drugs acetaldehyde would still release norepinephrine from adrenergic nerve nedings, but the action of norepinephrine on adrenergic receptors would be poten tiated due to prevention of its reuptake mechanism by these agents. Therefore, these differences between tyramine and acetaldehyde seem to be explanatory on this basis.

The effect of.disulfiram pretreatment on the acetaldehyde blood pressure response was first noted by Christensen ( 1951) . He suggested that it was similar to epinephrine reversal, but this 193 was denied by Feingold ( 1952, 1954). Still, it was shown that the vasopressor action of acetaldehyde could be reversed by alpha- adrenergic blockade with phentolamine. Test of the beta-adrenergic receptor blocking drug, propranolol, in this study also confirms that the fall in pressure is not an action of acetaldehyde on this vaso dilator receptor. Neither was there observed any changes in the vascular responses to norepinephrine or epinephrine by disulfiram pretreatment. Hypoglycemic sulfonylurea drugs such as tolbutamide produce a change sim ilar to disulfiram in the acetaldehyde blood pressure action, and this action is not reversed by atropine or glucose ( Truitt et a l., 1952). In this study, atropine was not effective in blocking the vasodilation produced by acetaldehyde in disulfiram treated rabbits, so that cholinergic dilation does not seem to be involved. The suggestion that histamine release may be in volved, seems doubtful. Perm an ( 1952) found that neither atropine nor antihistamines were capable of preventing the hypotension in rabbits produced by ethanol after disulfiram pretreatment. Here it was shown that the vasodilation by acetaldehyde could be initially reversed by tripelennamine, however, a prolonged secondary vaso- dilitation persisted and was not abolished by this antihistamine. This effect is probably due to potentiation of catecholamines by re uptake blockade by this agent (Isaac and Goth, 1957) . This con clusion is suggested because cocaine, a familiar norepinephrine reuptake blocking drug, also reverses the acetaldehyde effect. As a matter of fact, it has been shown that antihistaminic drugs are effective in relieving some symptoms of the disulfiram-alcohol reaction ( Lester et al., 1952). There is no experimental evidence that serotonin release is involved, but since in the presence of reserpine, acetaldehyde is still a depressor, this suggestion seems untenable. 194 Akabane and associates ( 1964a) noted cardiac slowing and electrocardiographic alterations characteristic of cardiac depress ion during acetaldehyde action in the disulfiram treated cat. Marked bradycardia was noted with acetaldehyde injection in di sulfiram or reserpine treated cats and rabbits, but atropine, while preventing the slowing, deepened the vasodepressor response. These observations suggest that direct cardiac depression by acet aldehyde supervenes when norepinephrine release is blocked by either reserpine or disulfiram as an explanation of the intensified fall in pressure. In vitro results also showed cardiac depression with high concentrations of acetaldehyde.

Acetaldehyde has been demonstrated to be vasopressor (Perman, 1962; Eade, 1959; Akabane et al., 1964a) when injected into untreated animals. This adrenergic constrictor action is in- consistant with the flushing of the face, dilitation of the scleral blood vessels and hypotension seen in alcoholic patients under treat ment with disulfiram after consuming small amounts of alcohol, and it can not be explained solely by the accumulation of acetalde hyde which occurs ( Forney and Harger, 1965). This apparent disparity led Wagner ( 1957) to suggest that other substances, which are measured non-specifically by the chemical but not enzymatic determination of acetaldehyde might be responsible for the syndrome. More recently, Casier and Merlevede ( 1962) attributed the reaction to a hypothetical combination of disulfiram with alcohol. That acetaldehyde itslef could produce a depressor action in the presence of disulfiram was adequately shown by P e r

man ( 1 9 6 2 ), but he disregarded the data and attributed the hypo tension of the disulfiram-alcohol reaction to some other metabolite.

The demonstration of disulfiram as ^n inhibitor of dopamine- beta-hydroxylase in the synthesis of norepinephrine (Goldstein et al., 195 1964; Musacchio et al., 1964, 1966b) has provided us with a new insight into the probable mechanism of the disulfiram-alcohol reaction. Inhibition by disulfiram makes this the rate limiting step and decreases the NE content of cardiovascular organs (Musacchio et a l., 1966b), adrenals ( Fuller and Snoddy, 1968), spleen, iris, heart and nictitating membrane ( Thoenen et al., 1966), and brain (Goldstein and Nakajima, 1967) . This decrease can be repleted and the vasopressor action to tyramine restored by an in fusion of norepinephrine, but not by dopamine or methyldopate

(Musacchio et al. , 1 9 6 6 a).

The experimental findings in cats and rabbits using blood pressure and nictitating membrane contractions are consistent with a catecholamine releasing action of acetaldehyde, This effect of acetaldehyde is somewhat sim ilar to tyramine in control animals. In reserpine and disulfiram pretreated animals, a vasodepressor action of acetaldehyde is revealed that is not overcome by replenish ment of neurotransmitter stores and appears to be caused by a direct depressant effect of acetaldehyde on the heart and vasculature. From these results it is possible to postulate a more tenable ex planation for the heretofore unexplained disulfiram-alcohol reaction. This hypothesis describes four essential components which are necessary for the anti-alcohol_syndrome produced by disulfiram:

( 1 ) disulfiram inhibits aldehyde dehydrogenase causing increased levels of acetaldehyde, ( 2 ) increased levels of acetaldehyde cause release of norepinephrine evoking a transient vasoconstrictive action, (3) disulfiram also blocks dopamine-beta-oxidase which lowers the norepinephrine content of tissues and prevents re synthesis of norepinephrine, (4) when norepinephrine is depleted, acetaldehyde acts directly on the heart and smooth muscle of blood vessels causing the characteristic hypotension, cardiac depression and other dysphoric effects. 196 C14-Norepinephrine Metabolism and Hypnosis

Administration of C14-nor epinephrine (C 1 4 -NE)to rats and following the excretion of radioactivity for 30 hours revealed at least two rates { see Figure 7), the slower of which represents most closely the metabolism of endogenous norepinephrine (Kopin and Gordon, 1963) . Analysis of the slower process reveals a half- life of about seven hours, a rate constant of 0 , 0 9 9 hour"1, and a time constant of about ten hours which is in close agreement with the results found by Kopin and Gordon ( 1963) in rats after admin istration of H 3 -NE. At twelve hours, essentially only C 1 4 from this slower process is being excreted. This slowly excreted radio activity represents C14-NE which has mixed with endogenous stores of this adrenergic neurotransmitter, and was released and metab olized, Neither ethanol, acetaldehyde, disulfiram, calcium carbimide, nor the latter two agents in combination with ethanol sig nificantly changed NE excretion. Ethanol significantly increased normetanephrine ( NM) excretion. Acetaldehyde injections produced an even greater effect. Normetanephrine excretion was unaffected by either disulfiram or calcium carbimide. However, when ethanol was administered after pretreatment with these agents the response was greatly potentiated. These findings are in agreement with the conclusion that acetaldehyde is an indirect sympathomimetic.

The various drug treatments did not alter dihydroxyphenyl glycol (DHFG) excretion. However, dihydroxymandelic acid was significantly lowered by all treatments indicating an inhibition of the oxidation of norepinephrine aldehyde to its corresponding acid. In the rat, ethanol failed to alter significantly the proportion of the two major O-methylated dcaminated metabolites. However, administration of acetaldehyde lowered the amount of vanillylman-

delic acid (C 1 4 -VMA) from 20.9 /£ to 11.8 % of total urine 197

radioactivity (see Figure 40). This represented a 41 */a reduction in the acid metabolite and an equivalent 40 °/0 rise in the excretion of 3-methoxy-4-hydroxyphenyl glycol (MHPG). Disulfiram pre treatment caused the excretion of VMA to fallardMHPG to increase. Administration of ethanol to disulfiram pretreated rats markedly potentiated this shift in metabolism. Under these conditions there was a 70 % decrease in VMA and a 42 °/0 rise in the glycol. Cal cium carbimide by itself caused a similar change from an oxidat ive route to a reductive pathway. When ethanol was administered to calcium carbimide pretreated animals, this alteration was much more pronounced ( see Table 31) . The relative shift in the major metabolites of norepinephrine metabolism to a reductive route was well correlated with the amount of acetaldehyde present. Admin istration of ethanol produced low levels of acetaldehyde of 1.17 and 2.65 pg/nl at one and two hours after dosage respectively ( refer to Table 10) . P rior treatment with disulfiram resulted in a 7-10 fold increase in the average acetaldehyde levels after 4 g/Kg. of ethanol. The circulating acetaldehyde levels obtained by direct injection of acetaldehyde were comparable to those achieved by administration of ethanol to disulfiram treated rats, and produced an equivalent shift in NE metabolism.

The lack of an effect of ethanol alone on NE metabolism in rats is contradictory to several human studies. Smith, Gitlow and

co-workers ( I 9 6 0 ) have previously shown decreases in the ratio of VMA to MHPG when ethanol was given after the administration of 7-H3-norepinephrine in man. This same laboratory (Bertani et al,

1 9 6 9 ) recently presented evidence that this also occurs in the central nervous system of humans. Davis and co-workers ( 1967b, 1967c) have shown substantial decreases in both endogenous and C14-VMA with concomitant increases in endogenous and radioactive MHPG after small doses of ethanol. Both of these laboratories have also 198 shown comparable results after disulfiram treatment in humans. The lack of an effect of ethanol in rats was fortunate, and strongly implicates acetaldehyde as mediating the shift in the metabolic fate of this amine.

The speculation has been made that the alteration of NE metabolism by ethanol in man might be attributed to a change in hepatic pyridine nucleotide levels as a result of ethanol oxidation (Davis et a l., 1967b; Feldstein et a l., 1967) when ethanol and acetaldehyde are metabolized there is a decrease of NAD in liver and a net production of NADH ( Buettner.et al., 1961). This ex cess of reduced coenzymc would favor the reduction of the aldehyde intermediate of various amines instead of oxidation which requires NAD-linked aldehyde dehydrogenase. The increase in NADH/NAD ratio as a result of ethanol oxidation has been repeatedly demon strated to occur in rat liver ( reviewed by Feldstein et a l., 1967) . Despite the known shift in coenzyme in the rat, very large doses of ethanol did not produce a shift in the metabolism of C14-NE in this species. Yet, administration of acetaldehyde, disulfiram, calcium carbimide or these latter two agents in the presence of ethanol caused a significant fall in CW-VMA with a simultaneous diversion of the intermediate aldehyde to C 1 4 -MHPG.

It is concluded from this study, that the genesis of this alteration in amine metabolism by ethanol is mediated by its inter mediate metabolite, acetaldehyde. The mechanism of this effect is most probably competitive substrate inhibition between acetalde hyde and the aldehyde intermediate of the amine for the active site on aldehyde dehydrogenase. That this mechanism is involved in the shift in amine metabolism is shown by the altered pattern when disulfiram or calcium carbimide, two potent aldehyde dehydrogenase inhibitors, were administered to rats. One other possibility is that the generation of NADH from oxidation of acetaldehyde to acetate, and not from ethanol oxidation, might be responsible for this activity. For instance, the local area within the cell for generat ion of the reduced pyridine nucleotide by acetaldehyde oxidation might be in the same subcellular compartment that would reduce the intermediate aldehyde of the amine. This would explain why acet aldehyde was effective and not ethanol. However, this unitary idea in the genesis of NADH for enhanced reduction of the amine can not be true. In the experiments with disulfiram and calcium carbimide, which inhibit oxidation of acetaldehyde, administration of ethanol caused a further change in the acidjglycol ratio. These experi ments show that without generation of NADH, acetaldehyde can still promote a change in C14-norepinephrine metabolism.

* i The significance of these alterations in amine metabolism to the pharmacologic effects of ethanol have been hypothesized. One suggestion ( Kvcder et al., 1962 ) has been that the biogenic alcohols may play a role in the behavioral effects elicited by ethanol. Feld stein et al ( 1967), stated that the structural resemblance of the alcohol metabolites of these amines to ethanol might modify eth- anols central effects. Rosenfeld ( I960) put fort the hypothesis that the action of ethanol and possibly other hypnotic substances might be due, in part, to the alteration of endogenous amine metabolism, particularly in the brain. He demonstrated that serotonin and other biogenic amines will prolong ethanol narcosis. A similar effect on hexobarbital hypnosis by serotonin has been demonstrated {Mahler and Humoller, 1964 ). However, recent findings ( Tyce et al,, 1968; Davis et al,, personal communications) have failed to show alterations in vivo of serotonin and norepinephrine metabolism in the brain of the rat. These findings must be re peated in a species that shows an effect with ethanol. A sa matter 200 of fact, recent evidence ( Bertani et al., 1969 ) indicated that nor epinephrine in the central nervous system of man is altered by ethanol.

Another postulate has been that the intermediate aldehyde derivatives of serotonin or norepinephrine formed after ethanol ingestion might have an effect on the central nervous system ( Feld stein et al., 1964; Davis et al., 1967a, 1967b). Indeed, after ad ministration of serotonin-C 14 to rats pretreated with disulfiram, there were increased levels of the neutral serotonin-C 14 metab olites, 5 hydroxytryptophol and/or 5-hydroxyindole acetaldehyde in brain (David et al. , personal communication) and also in rat liver after addition of acetaldehyde in vitro ( Lahti and Majchrowicz, 1967) . Whether the intermediate aldehydes in the metabolism of biogenic amines have pharmacologic activity is a matter for further investigation. However, some evidence has accumulated to suggest that they might. Keglevic et al ( 1968) has demonstrated a marked protein binding of serotonin aldehyde. Alivisatos et al ( 1966) found the radioactive aldehyde associated with the acid-insoluble material obtained from mitochondrial preparations of rat brain and guinea pig liver and suggested a Schiff base formation with protein. Bar- ondes ( 1962) established that indoleacetaldehyde and 5-hydroxyindoleacetaldehyde stimulate the oxidation of glucose by beef an terior pituitary gland. Sabelli et al ( 1968) elicited potentiation of optical evoked potentials in cat brain by local application of these intermediate aldehydes. More recently this same group (Giardina

et a l., 1 9 6 9 ) demonstrated sleep induction in newly hatched chicks by indoleacetaldehyde, However Udenfriend's laboratory (Ren- son et al,, 1964) showed that indoleacetaldehyde and 5-hydroxyindoleacetaldehyde were completely inactive on isolated smooth muscle preparations and had no modifying effect on the activity of their corresponding amines, 201 An attempt was made to examine these various hypotheses for a role of norepinephrine and its metabolites in ethanol's central effects. The potentiation of the central depressant effects of ethanol in mice was used as a test system using a sublethal hyp notic dose of ethanol (4.5 g/Kg, i. p. ). F irst it was confirmed that acetaldehyde possesses hypnotic and toxic activities of its own ( Figure 41 )in agreement with the findings of MacLeod ( 1950). Acetaldehyde is about 30 times more potent than ethanol in these activities. However, the concentrations of acetaldehyde producing intoxication are much higher than those occurring after ethanol. The low concentrations of acetaldehyde which occur in ethanol in toxicated animals discourages the hypothesis that acetaldehyde pro duces the depressant effects of ethanol. However, the concentrat ions which do occur after ethanol might well modify the actions of ethanol. Injection of norepinephrine i. p. into mice did not pro duce any symptoms of sedation or hypnosis. The administration of ethanol induced a hypnotic state in mice, with a loss of righting reflex lasting 43,6 minutes. Injection of norepinephrine about mid way in the sleeping-time of mice produced by ethanol, demonstrated a dose related potentiation of the sleeping-time ( 130 % and 260 a/0 increase). This effect occurred without altering the ethanol blood levels. The administration of the five metabolites of nor epinephrine during the sleep period failed to significantly change the duration of ethanol induced hypnosis in mice { see Table 35) . These results would suggest that the metabolites of this biogenic amine, in particular the alcohol metabolites which were shown to accumulate, are of little consequence to the central action of ethanol. However, it may well be that the metabolites failed to penetrate into the CNS because of their polar nature. Yet, if ethanol allowed the passage of norepinephrine through the blood brain barrier, a sim ilar effect on these metabolites would be expected. The hypothesis that the intermediate aldehydes might be important for the depressant effects of ethanol was also examined. Pretreatment with monoamine oxidase inhibitors (MAOl), tranyl cypromine and iproniazid, was used to examine the role of n o r epinephrine aldehyde. MAO inhibition should prevent the effects of administered NE if the aldehyde contributed to the potentiation of ethanol sleep by this amine. Similarly, if the accumulation of the aldehyde in vivo underlies the CNS depression of ethanol, one would expect reversal or some inhibition of the hypnotic state pro duced by ethanol when NE metabolism was inhibited. However, inhibition of MAO potentiated the sleeping time produced by ethanol. This implies that a build up of the amine itself in the central ner vous system may play a part or at least enhance the central action of ethanol. Tranylcypromine and iproniazid produced respectively a 9 8 % and 76°/ increase in the ethanol induced hypnosis. Admin istration of ethanol and NE to pretreated mice showed no further potentiation in sleeping time than did NE administration alone. These results would suggest that the accumulation of NE at certain receptor sites in the CNS can produce a sleeping or hypnotic state. Possibly, the combined effect of increasing endogenous NE by MAOI and administration of the amine did not produce any prolonged accumulation at these receptors than did NE administration alone. All receptors may have been occupied already when 0.06 mM/Kg of NE was administered in these ethanol treated mice. The rate of metabolism of the administered NE may control the duration of sleep prolongation. Therefore, the intermediate aldehyde of NE does not seem to participate in ethanol's CNS effects either.

Thus, these results have led us to propose two primary hypo theses concerning the role of amines in the central action of ethanol. It is the belief of this investigator that the amine releasing action of acetaldehyde may be best related to the central stimulant and 203 hallucinatory effects of ethanol. This concluclusion is based on the vast body of knowledge which is accumulating concerning the changes in norepinephrine and various effective states ( reviewed by Iversen, 1967; and Schildkraut and Kety, 1967) . All of the effects of acet aldehyde parallel quite closely those produced by the classical cen tral stimulant, d-amphetamine. Amphetamine, which exhibits both peripheral and central actions, acts in the periphery by re leasing norepinephrine from postganglionic sympathetic nerve end ings ( Burn and Hand, 1958) and thereby increases the concentration of transmitters available at post-junctional receptors (Hertling et a l., 1961) . This action has similarly been shown for acetaldehyde ( sec Results ) ,

It has been suggested that this same mechanism is responsible for the striking excitatory effects of amphetamine in the central nervous system (Glowinski and Baldessarini, 1966). Steady state concentrations of norepinephrine, but not of dopamine, are reduced in brain by large doses of amphetamine (Vogt, 1954; Moore and Lariviere, 1963) . A similar effect in brain stem NE has been shown for ethanol (Gursey et al., 1959; Gursey and Olson, I960) and has been demonstrated to be due to its intermediate metabolite acetaldehyde (Duritz and Truitt, 1966). Although the evidence has suggested that the central stimulant actions of amphetamine are causally related to theability of this drug to release brain norepi nephrine, the actual release of this amine from neurons in intact brain by amphetamine has only recently been demonstrated (Carr and Moore, 1969). These investigators injected H3-norepinephrine into the left lateral cerebral ventricle of cats and perfused the lateral and third ventricles with an artificial cerebrospinal fluid, The addition of d-amphetamine to the perfusion fluid caused a significant increase in the concentration of H3-norepinephrine in the effluent. 204 Direct evidence with the perfused brain using acetaldehyde needs to be investigated. However, evidence presented here ( see results) have shown a similar action of acetaldehyde in the periphery on

A H -norepinephrine. All of these similarities between acetaldehyde and amphetamine are quite striking. Based on these comparisons, it seems reasonable that the stimulant and euphoric effects of al cohol produced in man may well be mediated by acetaldehyde. And, it may be the amine releasing properties of acetaldehyde, the pri mary metabolite of ethanol, that is responsible for these behavioral effects. The local release of norepinephrine in certain areas within the central nervous system could contribute to affective behavior elicited by ethanol ingestion,

This hypothesis is also consistent with recent neurophysiolog- ical evidence concerning the effects of ethanol and acetaldehyde. When alcohol is administered to man or animals, the cortical fre quencies in the electroencephologram ( EEG) decrease progressively as intoxication develops. Alcohol is also known to produce a dis tinct dissociation between the electrical activity of the neocortex and that of the limbic system. The most distinct dissociation occurs when the alcohol concentration in the blood increases to over 300mg Therefore, the postulate has been made that such a dissociating effect of alcohol on the cerebral cortex may explain some behavioral effects of acute alcohol intoxication, including euphoria. This apparant stimulant effect of alcohol has been explained by sustained activity of the limbic system under marked diminution of the control mechanisms of the neocortex.

Recent findings obtained by Akabane et al ( 1964c), failed to induce a dissociation of the EEG patterns between the neocortex and the hippocampus in cats at lower blood concentrations of alcohol known to produce euphoria. Instead, at low levels of alcohol 205 intoxication, there was evidence of activation of the neocortex. Holmberg and Martens { 1955) also found that the slowing of the EEG was preceeded by increases in EEG frequency and in some cases a great deal of fast activity appeared in man.

An examination of the effects of acetaldehyde on the activity of lower brain stem and of the higher cerebral structures in cats has been made by Ogata ( 1964) . He observed a pronounced enhance ment of electrical activity of the brain which was not abolished by brain stem transection at the level of the rostral pons and was not due to a rise of arterial blood pressure produced by acetaldehyde inject ion. He proposed that acetaldehyde formed in the course of alcohol . oxidation may play a role in eliciting the short-lasting cortical activation of the fast EEG during alcohol intoxication. Similarly, sympathomimetic amines, ephedrine, norepinephrine, epinephrine and amphetamine produced EEG activation which is also abolished after decerebration at the intercollicular level. Therefore, it was suggested that acetaldehyde might act on catecholamine storage sites in the arousal system of the ascending reticular activating system to produce EEG activation. Cerebrocortical activation and reversal of sedation by acetaldehyde and certain biogenic amines and by reaction-products of acetaldehyde and biogenic amines in rabbits has also recently been reported (Beck et al., 1968) .

Therefore, the general effects of acetaldehyde and ethanol appear to be mutually antagonistic. Akabane ( I960) observed several antagonistic effects between these two agents, He found that the m or tality and toxicity to acetaldehyde was suppressed by ethanol. The respiratory stimulation and elevation of blood pressure produced by acetaldehyde were also altered by alcohol. He suggested that the effects of alcohol are the central paralizing, whereas those of acet aldehyde may be central stimulating. Our results indicate similar 206 antagonisms on other systems, Acetaldehyde produced positive inotropic effects on isolated guinea pig atria, whereas ethanol is depressant on isolated heart-preparations. Similarly, the en hanced respiration, elevated blood pressure, and increased heart rate produced by acetaldehyde injections are indicative of stimulat ing effects. Alcohol depresses all of these systems. Therefore, it seems reasonable that the euphoric, hallucinatory and central stimulant actions occuring in certain phases of alcohol intoxication might be due to its intermediate metabolite, acetaldehyde. It may be that the relative blood levels of acetaldehyde and ethanol are im portant in producing central stimulant effects. For example, acet aldehyde concentrations are high when the ethanol blood level curve is still rising and at this time euphoria is usually seen. Similarly, acetaldehyde levels are increased relative to alcohol concentrations during alcohol withdrawal, a situation in which hallucinatory symp toms are produced particularly in alcoholics. Therefore, at these times, the effects of acetaldehyde may predominate.

Attempts to understand the central depressant effect of eth anol have been the subject of numerous investigations ( reviewed by Jarnlfelt, 1961; Wallgren, 1967; and Isreal et al., 1965; 1967). The whole area of the mechanism by which drugs produce behavioral de pression and even anesthesia has been studied more than any area of CNS pharmacology. Still, no adequate explanation or hypothesis has been put forth which is wholly acceptable to all. Our results certainly are not conclusive proof of the mechanism, but do suggest a possible role of norepinephrine in ethanol produced hypnosis and intoxication. The evidence presented fiere seems to refute the idea that the alcohol metabolites of norepinephrine or the intermediate aldehydes play any role in this effect of ethanol. The best contradict ion to these hypotheses is that ethanol failed to alter the metabolism of C14-norepinephrine in the rat, but it certainly does produce an intoxi cating syndrome in this species, However, a potentiating action of 207 NE itself has been found. These findings are an extension of earlier results by Friedman (1942) and Rosenfeld ( I960) , Since injection of NE after ethanol produces a potentiation of ethanol hypnosis, it may be that the increase of NE within certain areas of the brain could contribute to intoxication and depression. This idea is sup ported by the fact that MAO inhibition, which results in increases of NE in the CNS also prolonged ethanol hypnosis. Again, the amine releasing ability of acetaldehyde might contribute to this action of ethanol. Release of NE by acetaldehyde in the brain stem ( Duritz and Truitt, 1966) or even in the periphery as demonstrated by our results could account for this effect. This conclusion is based on the fact that peripheral administration of the amine during ethanol hypnosis was capable of prolonging the sleep state. Since NE does not normally penetrate the blood brain barrier, the effect of alcohol in increasing the permeability to several substances (Lee, 1962) may account for the remarkable action of NE in this respect. The release of NE in the periphery by acetaldehyde when ethanol is pre sent may present a rushing of this amine into certain areas of the brain. Of course, this idea needs further investigation. Still, our results in mice are consistent with such an idea.

Ultimately, our findings have refuted the several postulates which have been made concerning the significance of the alterations in amine metabolism which occur in man after ethanol. The meaning of ethanol induced shifts in the degradative pathways of certain bio genic amines remains obscure. Yet, the results obtained in rats in our experiments are the most conclusive evidence for the basic tenet and overall hypothesis of this thesis. In these experiments, it has been possible for the first time to separate out an action of acet- aldehydc alone, from effects which are produced by ethanol also. It was fortunate that the use of this species ( rat) permitted the demon stration of an effect, previously attributed to ethanol to be due solely to its metabolite, acetaldehyde. Therefore, the general premise on which all of this work was based has been demonstrated; that is that acetaldehyde, the primary metabolite of ethanol is assuredly important in some actions of ethanol in the organism. SUMMARY

There are several conclusions which can be made concerning the levels of acetaldehyde, the pharmacologic actions of acetaldehyde and the importance of this metabolite in the actions and toxic ity of ethanol:

1. The levels of acetaldehyde which are achieved after ethanol administration are lower than those which have been previously reported. This is mainly due to an artifactual production of acet aldehyde in precipitated whole blood when ethanol is added.

2. The relative circulating concentrations of acetaldehyde which are metabolically produced by man, rabbits and rats are approxi mately equal in all of these species and are proprotional to the dose of alcohol.

3. Acetaldehyde does seem to accumulate in brain tissue. How ever, this conclusion requires more precise evaluation because of the non-enzymatic, artifactual production of acetaldehyde which occurs in tissues, including brain, when ethanol is present. This artifact places in question the recent finding that there is a m icro somal ethanol oxidizing system which forms acetaldehyde by en zymatic conversion of ethanol in liver.

4. The elevation of acetaldehyde levels after ethanol by disulfiram pretreatm ent has been once again confinned in this study, in con tradiction to several recent reports and doubts.

209 210 5. Relatively low concentrations of acetaldehyde are able to induce a release of H3-NE in vivo and also in isolated left atria of guinea pigs, Acetaldehyde acts as an indirect adrenergic agent similar in many respects to the classical catecholamine releasing drug, tyramine.

6 . This indirect adrenergic effect of acetaldehyde could be respon sible for several sympathomimetic actions produced by ethanol, which have also been demonstrated in many cases to be elicited by acetaldehyde alone.

7. Similarly, the increased urinary excretion of amines reported to occur with ethanol in various species can be accounted for from the pharmacological effect of acetaldehyde.

8 . A more tenable hypothesis for the disulfiram-alcohol reaction has been proposed. This investigation has demonstrated that the pha rmacologic effects of acetaldehyde combined with the alteration by disulfiram of the cardiovascular effects of this aldehyde adequate ly explain the symptoms which are produced when ethanol is admin istered in the presence of disulfiram. The postulation of other non specific substances being responsible for this syndrome is unneces sary.

9. The mechanism of alterations in biogenic amine metabolism which are produced in man by ethanol have been shown to be due to acetaldehyde directly. This effect is most probably accomplished by competitive inhibition of acetaldehyde with the aldehyde inter mediate of norepinephrine for the active sitt on aldehyde dehydro genase. This pharmacological effect is the first action of ethanol which has been solely and unequivocally shown to be due to acet aldehyde alone. 211 10. A hypothesis is presented, based on all accumulated evidence for the action of acetaldehyde on norepinephrine stores, that cen tral catecholamine release by acetaldehyde could account for the stimulant and euphoric effects of ethanol.

11. Furthermore, evidence is presented that the catecholamine releasing effect of acetaldehyde could contribute to the central nervous system depression of ethanol.

12. Finally, the ultimate hypothesis that acetaldehyde is respon sible for at least one effect of ethanol and may contribute to the many pharmacologic actions produced by ethanol in the organism is supported by this investigation. BIBLIOGRAPHY

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