Short Title:

EFFECT OF·PHENELZINE ON PHARMACOLOGY AND MÈTABOLISM OF' ETHANOL'•. ': .. " r. ' . •.- "

THE EFFECT OF PHENELZINE ON THE PHARMACOÏ,OGY AND MÈTA':BOLISM OF ETHANOL

by

DANIEL J. WADE, B. Sc.

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the, degree of Master of,Science.

Department of Pharmacology and Therapeutics, McGill University, Montreal. August, 1968.

® Daniel J. vlade Wade, Daniel James

The Effect of Phenelzine on the Pharmacology and Metabolism of Ethanol

Department of Pharmacology and Therapeutics

Master of Science

ABSTRACT

Studies were conducted to determine the effect of phenelzine on the

pharmacology and metabolism of ethanol. Pretreatment for one half hour

with phenelzine (40 mg./kg. i.p.) prolonged the mean ethanol sleeping time

in mice by more than 350%, but, unexpectedly, decreased the toxicity of

ethanol, as reflected in a change of LD 50 from a control value of 4.14g./kg.

to 5.16 g./kg. A partial explanation of these results was thought to

reside in the effect of phenelzine on the rate of ethanol degradation.

Determinations of ethanol conéentrations in whole mice over a 4 hour period indicated that phenelzine caused a 43% increase in the time required to eliminate one half the amount of ethanol initially injected.

Acetaldehyde concentrations after ethanol were found to be lower in phenelzine pretreated mice. In vitro, phenelzine inhibited the oxidative activity of yeast alcohol dehydrogenase by a competitive interaction with both DPN+ and ethanol at enzymic receptor sites. In contrast, the in- hibition of liver alcohol dehydrogenase by phenelzine was competitive with + DPN only, and non-competitive with respect to ethanol. This thesis is dedicated to my parents " . who have been unf'ailing .in' the ir encouragement and understand.ing. ACKNOWLEDGMENTS ,.'".. It bas been my unique privilege to complete this research project under the tutelage of Dr. N.R. Eade, whose instruction, advice, and when necessary, frank criticism, transformed an

initiate to the w~ys of science into something resembling a graduate student. In particular, l would like to express my appreciation to Dr. Eade for his boundless patience and fortitude during the preparation of this thesis.

l would like to exte nd my app re C! ia t ion to Dr. M. Nicke rson, chairman of the Department of Pharmacology and Therapeutics, who provided inspiration and a stimulating environment in which to work. To Mr. Kenneth Renton must go special thanks for his excellent tecbnical assistance, practical advice and, more important, for his relaxed and unfailing companionship. Credit for the photographie work shown in thisthesis belongs also to Mr. Renton. As a master of the two-fingered method, l can fully appreciate' the work and frustration involved in typing a thesis. For this reason, my sympathies, as weIl as my thanks, go to Miss Tyleen Macvean who bas done a magnificent job under the circumstances. For their conscientious efforts in proof-reading this thesis, l thank my brother Rick, Miss Eva Poucha and Dr. Harvey Rabin. This work was supported by grants to Dr. N.R. Eade from the Canadian Foundation for the Advancement of Therapeutics. INDEX Page INTRODUCTION: 1

Derivation of the Problem: 8 MONOAMINE OXIDASE: 11 Enzymes 11 Substrates 11 Physiological Role 13 Inhibi tion . 16 Molecular Mechanism of Monoamine Oxidase Inhibition 18 Pharmacological Effects of Inhibition 22

PYRIDOXAL PHOSPHATE DEPENDENT ENZYMES: 25 Cofactor 25 Enzymes 25 Inhibition 26

N-DEALKYLATION ENZYME SYSTEMS·: 30 Enzymes 30 Inhibition 30

METRODS: 36 GENERAL PROCEDURE: 36 EFFECT OF PHENELZINE ON ETHANOL SLEEPING TIME: 37 EFFECT OF PHENELZINE ON THE LD 50 OF ETHANOL: 37 EFFECT OF PHENELZINE ON THE DISAPPEARANCE OF INJECTED ETHANOL: 37 Apparatus 38 •• Standard Curve 38 Page Experimental Procedure 39 • Per Cent Recovery 43

EFFECT OF PHENELZINE ON ACETALDEHYDE CONCENTRATIONS: 43 Apparatus 45 Standard Curve 45 Color reaction 46 Per Cent Recovery 50

EFFECT OF PHENELZINE ON YEAST ALCOHOL DEHYDROGENASE (YADH) ACTIVITY: 51 General Procedure 52 Incubation Medium 52 Yeast Alcohol Dehydrogenase 52 ~-Diphosphopyridine Nucleotide (DPN+) 53 Measurement of YADH Activity 54 Standard Curve 55 - Inhibition by Phenelzine with Respect to Ethanol 55 Inhibition by Phenelzine with Respect to DPN+ 58

EFFECT OF PHENELZINE ON LIVER ALCOHOL DEHYDROGENASE (LADH) ACTIVITY: 58 General Procedure 58 Incubation Medium 58 Liver Alcohol Dehydrogenase 59 ~-Diphosphopyridine Nucleotide (DPN+) 61 Protein Determination 61 , Measurement of LADH Activity 61 ... , Page

" • Inhibition by Phenelzine with Respect to Ethanol 62 Inhibition by Phenelzine with Respect ta DPN+ 63

STATISTICS: 64

RESULTS: 65 EFFECT OF PHENELZINE ON MOUSE BEHAVIOR: 65 . . . EFFECT OF PHENELZINE ON ETHANOL SLEEPING TIME: 65

. . ~ . . .. '- EFFECT OF PHENELZINE ON THE LD 50 OF ETHANOL:, 71 . . . . EFFECT OF PHENELZINE ON THE DISAPPEARANCE OF INJECTED ETHANOL: . 74 . . EFFECT OF PHENELZINE ON ACETALDEHYDE CONCENTRATIONS: 79 EFFECT OF PHENELZINE ON YEAST ALCOHOL DEHYDROGENASE (YADH) ACTIVITY: 83 Inhibition by Phenelzine with Respect to -Ethanol 84 Inhibition by Phenelzine with Respect to DPN+ 92

EFFECT OF PHENELZINE ON LIVER ALCOHOL DEHYDROGENASE . (LADH) ACTIVITY: 99 Inhibition by Phenelzine with Respect to Ethanol 99 Inhibition by Phenelzine with Respect to DPN+ 107

DISCUSSION: EFFECT OF PHENELZINE ON ETHANOL SLEEPING TIME: 114 ._. . Increased Ethanol Concentration h14 Incr'eased Acetaldehyde Concentration 116 Potentiation of the Depressant Action of Ethanol on the Central Nervous System .. 117 Potentiation of the Depressant Action of Acetaldehyde on the Central Nervous System 120 • EFFECT OF PHENELZINE ON THE LD 50 ~F ETHANOL: 120 Page EFFECT OF PHENELZINE ON ETHANOL METABOLISM: 124

. '.' '. EFFECT OF PHENELZINE ON ACETALDEHYDE METABOLISM: 129

,SUMMARY AND CONCLUSIONS: 132

APPENDIX 1: , Drags 136

APPENDIX II: Reagents 139

BIBLIOGRAPHY: 142 1

INTRODUCTION

~edical treatment for mental depression occurring as a part of an affective psychosis has tradit10nally been based on the induction of convulsive states, either electrically (E.C.T.: electro-convulsive therapy,) or by drugs such as pentylenetetrazol (Metrazol). Drug induced convulsive states have now been almost abandoned and within the past few years the development of effective antidepressant drugs has considerably decreased the use of electroconvulsive therapy, although no form of drug treatment has yet exceeded it in effectiveness. The first of the antidepressant drugs to corne into cornrnon use was amphetamine. The development of this drug grew directly out of systematic alterat10ns of the adrena1ine molecule by Barger and Dale (1910). These studies led to the synthe sis of a number of pheny1ethy1amines which

~CH2-ÇH-NH2 OI~ CH3 Amphetamine

l9H O~ Ouc-O

Pipradol (Meratran) Methylphenidate (Ritalin) 2 • accentuated sorne of the pharmacological properties of adrenaline and attenuated others. Of this series, only a-rnethylphenylethylarnine, or amphetamine, was found to pro­ duce mood-elevating effects in man (Alles, 1927). However, this property proved to be of no value in psychotic depres­ sions and of litt le or no use in neurotic depressions. The structurally modified amphetamines such as methylphenidate (Ritalin) and pipradol (Meratran) were of no greater value in this regard than the parent compound. A more effective group of drugs ·for the treatment of depression was synthesized during the development of a series of antihistaminic compounds. The early antihistarnines were essentially ethylene diamine derivatives and the joining of two phenyl groups by a sulphide bridge in one such compound produced a highly potent and long-lasting ant1histamin1c (promethazine) with marked central depressant act1vity. Replacement of the branched chain by an N­ propylene side chain (promazine) reduced the antihistaminic act1vity markedly but accentuated the tranquilizing proper­ ties while the addition of a 2-chloro group to the pheno­ thiazine ring yielded a tranquilizing and ant1psychotic drug, chlorpromazine. This compound represented a major break­ through in the treatment of mental illness, particularly the psychoses. A further modification, the isosteric replacement of the sulphur by an ethylene bridge (Hafliger • and Schindler, 1954) produced the weak tranqu11izer 3

imipramine (Tofran11) wh1ch became the f1rst major anti- depressant drug. The demonstration that imipramine was effective in the treatment of endogenous depression was not

made until several yearsafter its synthesis. However~

once its ant1depressant activity had been establ1shed~ a major effort was made to synthesize analogues of im1pramine and to devise new methods of animal test1ng wh1ch would allow

such activity to be detected. At present~ the agents 1n

common use are imipramine~ and amitr1ptyl1ne (Elavil) and their demethylated derivatives desipramine (Pertofran) and nortriptyline (Avent yI). Another group of drugs which proved to be more effective in the treatment of depression than amphetamine were the monoamine ox1dase inhibitors (MAOI) wh1ch included both amine and hydrazine der1vat1ves. In 1948~ Burger and Yost began a search for an agent possessing long lasting amphetam1ne­ like stimulation by structurally modifying a series of sympathomimetic amines. Using the amphetamine molecule as a basis~ they succeeded 1n bonding the a-methyl group to the ~-carbon of the alkyl side -chain to produce trans-2- phenylcyclopropylamine (tranylcypromine: parnate)~ an amine monoamine oxidase inhibitor (MAOI) having a prolonged stimulatory effect on the central nervous system. The antidepressant properties of tranylcypromine were not utilized clinically until more than a decade later~ but the compound is now frequently used in the therapy of mental • ·4

Chlorpromazine (Largactil) Imipramine (Tofranil)

Amitriptyline (Elavil)

Desipramine (Pertofran) Nortriptyline (Aventyl) 5

depression despite its potential hepato-toxicity. Another amine MAOl, pargyline (Eutony1), was derived from the mod­ ification of a phenyla1ky1amine side chain to an acety1enic mOiety. However, the presence of marked hypotensive activity caused pargy1ine to be marketed as an anti­ hypertensive agent rather than an antidepressant.

~H-CH-NH2 . )C~ H H

Tranylcypromine (Parnate) Pargyline (Eutonyl)

..... The deve10pment of hydrazine and hydrazide compounds as antidepressants was stimulated by c1inica1 reports that euphoria and central nervous system stimulation occurred in tubercular patients undergoing treatment with the hydrazine. derivative 1-isonicotinyl-2-isopropyl hydrazine (iproniazid: Marsilid) (Selikoff et al, 1952). lt was subsequently shown by Ze11er (1952, 1955) that iproniazid was a MAOl capable of preventing the in vivo destruction of endogenous excitatory amines in the brain. The frequencywith which iproniazid induced undesirable symptoms of central nervous system origin eventual1y necessitated its withdrawal from anti- tubercular therapy. lnterest in this compound was renewed when experimental evidence indicated that a deficiency in the 5-HT content of the brain might be responsible for some 6

mental disorders. Wooley and Shaw (1954) and Saunders (1955) suggested that iproniazid should be used in an attempt to relieve mental depression and clinical trials werê initiated in 1956. The success of iproniazid (Loomer et al, 1957) in alleviating psychotic and neurotic depressions inspired Kline (195'S) to introduce iproniazid as a "pyschic energizer" into psychiatry. However, the threat of hepato- cellular toxicity was sufficiently great to stimulate a search for similarly acting drugs possessing less toxicity. The introduction of an isoxazolyl carbonyl group to the basic ipron­ iazid structure resulted in isocarboxazid: (Marplan), a MAO! with improved specificity of action, and improved passage across the blood-brain barrier. Another attempt to lower toxicity produced

Iproniazid (Ma~silid) Isocarboxazid (Marplan)

Nialamide (Niamid) 7

the hydrazide MAOI antidepressant nialamide (Niamid), a .' der~vative of iproniazid in which the isopropyl group was replaced with a phenylalkylamide moiety. Unfortunately, this modification caused a concomitant decrease in clinical effectiveness as weIl. In 1959, Biel et al substituted the terminal amine group of a number of sympathomimetic derivatives with a hydrazine

mo~ety in the hope of improving the affinity of these compounds for cell receptor sites and increasing their re­ sistance to enzymic degradation. When these sympathomimetic hydrazines were tested for their action on the central nervous system, their structure-activity relationships with respect to central nervous system stimulation paralleled their ability

to inhibit monoamine oxidase (MAO). The replacemento~ an amine witha hydrazine group intensified the MAO inhibitory properties of these compounds and prolonged their action. Maximum MAO inhibition and analeptic activity were obtained with drugs containing theamphetamine side chain, such as

~-phenylisopropylhydrazine (pheniprazine: catron). The

synthesis of ~-phenylethylhydrazine (phenelzine: Nardil) by the removal of the' a-methyl group of pheniprazine reduced the -"enzyme inhibitory potency ten fold. Phenelzine displayed no analeptic activity and proved to be less toxic than pheniprazine. Although reduced by a-demethylation, MAO inhibitory power was still four times more potent in phenelzine than in iproniazid. 8 •

Pheniprazine (Catron) Phenelzine (Nardil)

Pheniprazine and phenelzine resembled the a~Phetamine derivatives in structural specificity, but differed in that 1) they were more potent as MAO inhibitors in the brain, 2) they had a prolonged duration of central nervous system stimulation, 3) they did not produce depression after the stimulatory effects had worn off, and 4) they were hypo- tensive in man rather than hypertensive. The hydrazine monoamine oxidase inhibitors presently in use are phenelzine, nialamide and isocarboxazid. Pheniprazine was recently withdrawn from clinical use because of excessive toxicity, but its pot'eht MAO inhibitory properties have made it an excellent pharmacological tool in animal experiments.

Derivation of the Problem: Adverse, sometimes fatal reactions subsequent to the ingestion of particular foods, or the administration of certain drugs to patients undergoing therapy with MAO!, have been reported in the medical literature. The symptoms were

suggesti ve of MAO! poisoning, 01' of excessive sympathetic

discharge, and typically involved anx~ety, extreme restlessnes~ muscle spasms in the face and extremities (especially in the 9

'. hands~ hyperpyrexia, and hypotension in interactions with drugs or hypertension, accompan1èd by the possibility of intracranial bleeding,in interactions with foods. The acute hypertensive crises resulting from foods have been attributed to their sympathomimetic amine content. Tyramine, phenylethylamine and tryptamine, three pressor amines found in aged cheeses and wines and in yeast extracts (Marmite), are normally degraded by MAO in the gut and the liver before they can be distributed throughout the body. The inhibition of MAO allows these amines access to sympathet­ ically innervated vascular structures where they"exert a direct pressor effect or an indirect action mediated by the release of endogenous excitatory amines. An action of the monoamine oxidase inhibitors on microsomal N-dealkylating enzymes May be responsible for their interaction with drugs. By serving as an alternate substrate with a greater affinity for the N-dealkylating enzyme, the monoamine oxidase inhibitors could competitively inhibit the degradation of barbiturates, imipramine, pethidine and other compounds normally N-dealkylated by microsomal enzymes. The absence of alternate metabolic pathways for these drugs would resultin their accumulating in toxicconcentrations in body tissues. Drugs presently contraindicated in patients undergoing MAOI therapy because of reported interactions include peth­ idine, or meperidine (Demerol), the tri-cyclic anti- depressants and amphetamine. Central nervous system 10

4It1 depressant drugs are universally contraindicated in MAOl treated patients whether or not individual agents have been reported to interact with MAOl. Such a contrain­ dication was reasonable for drugs known to utilize liver micro.somal metabolic pathways inhibited by MAOl, but there was a deficit of experimental evidence to support such a contraindication for compounds degraded by cytoplasmic enzyme systems, as for example, ethanol. lt has been stated that the antidepressant ·MAOl prolong and intensify the central depressant effects of alcohol (Goldberg, 1964; Vigran, 1964; Shaw, 1964), although such interactions had not been substantiated experimentally. lt was therefore decided to determine if phenelzine, a hydrazine monoamine oxidase inhibitor in therapeutic use, could alter the pharmacological properties of' etharlol in vivo, and if an interaction did occur, to determine whether it could be explained on the basis of an inhibition of ethanol metabolism. The literature contained few reports of the experimental study of the effect of monoamine oxidase inhibitors on the pharmacological actions of ethanol in vivo (Smith et al, 1961) and of their effect on cytoplasmic dehydrogenase enzyme systems in vitro (Redetzki and O'Bourke, 1961; Sankar et al, 1961; Smith et al, 1961). Experimental approaches to the problem of an ethanol-phenelzine interaction had to be derived from in vivo studies done with other central nervous system depressants, such as ,fi; the barbiturates, and from in vitro work invo1ving the inhibitory Il

effect of the MAOl on enzyme systems other than the dehydrogenases. The literature was studied with the fol­ lowing goals in mind: 1) To become familiar with techniques commonly used to study drug interactions and enzyme inhibition. 2) To investigate the mechanisms by which the monoamine oxidase inhibitors blocked otherenzyme systems. 3) Ta gain an understanding of the relationships between enzymes and their substrates, and of the possible consequences of enzyme inhibition with respect to :this relationship. 4) To explore the possibility of an ethanol-phenelzine inter­ action mediated at the level of an enzyme system and at the level of the central nervous system. Experimental evidence applicable to these goals was most extensively available for three enzyme systems inhibited by MAOl; 1) monoamine oxidase, in abundant detail, 2) pyridoxal , phosphate dependent enzymes, and 3) microsomal N-dealkylating enzymes.

MONOAMINE OXIDASE: Enzymes: The term monoamine oxidase (MAO) de signa tes a series of closely related enzymes capable of oxidatively deaminating biologically active amines to their corresponding aldehyde derivatives. These enzymes are characterized by a similar sensitivity to the inhibitory action of unsubstituted acyl­ • hydrazines and "by their substrate affinity for certain 12

alkylmonoamines. and arylalkylmonoamines. MAO is primarily found in the mitochondrial fraction of numerous organs throughout the body (Blaschko, 1963) though it may exist in other particulate matter, especially in the microsomes and lysosomes (Birkhauser, 1940; Cotzias and Dale, 1951). The liver is generally the best source of MAO but the enzyme is also located in the gray matter and basal ganglia of nervous tissue (Blaschko, 1963). Investigations of MAO substrate and inhibitor activity revealed quantitative and qualitative differences in activity among oxidases of different origins. Among species, bovine liver mitochondrial MAO was blocked ten times more effectively by tranylcypromine than was rabbit or mouse liver MAO (Sarkar, et al, 1960). MAO of beef liver mitochondria was preferentially inhibited by short chained aliphatic hydrazines while rabbit liver MAO was more susceptible to the higher homologues of the N-alkylhydrazines (Zeller, 1961). Within species, liver MAO in rabbit was six times less sensitive to tranylcypromine than brain MAO. MAO from various species also differed with respect to substrate affinities. Extracts from guinea pig liver, kidney. and intestine metabolized tyramine and tryptamine equally well, but tyramine proved to be the better substrate in the corresponding organs of cats and dogs (Holtz and Buchael, 1942) • • 13

Substrates: Primary and secondary amines are degraded by MAO, although secondary amines are deaminated only if the sub­ stituent is a methyl group. Tertiary amines could be oxidized by MAO, but at a significantly slower rate. sarkar et al (1960) concluded that the most suitable substrate for MAO was a two carbon chain containing an a-hydrogen and an

a-amino group and substituted in the fo position by an aromatic ring. Naturally occurring substrates of importance are the catecholamines noradrenaline, aOdrenaline and dopamine (Blaschko, 1952; Kopin, 1964) and their ortho-methylated metabolites normetanephrine and metanephrine (Axelrod, 1959).

~-Phenylethylamines deaminated by MAO are tyramine and octopamine. The most important tryptamine derivative deaminated by MAO is 5-hydroxytryptamine (serotonin) (Blaschko, 1952), but tryptamine itself and the N-methyl derivative of 5-hydroxytryptamine, bufotenine, can also serve as substrates.

Physiological Role: The physiological role of monoamine oxidase may be the inactivation of released monoamines, or, by terminating their action, the maintenance of a concentration gradient large enough to permit the continued release of amines (Holtz and Westermann, 1965). It is now recognized, however, that • the removal of the basic amine group by MAO does not 14

necessarily constitute the only, or even the initial step • in the degradation of the monoamines. A second enzyme, catechol-ortho-methyl-transferase (CCMT), is intimately involved in catecholamine metabolism (Axelrod, 1959; Crout et al, 1961) and there is substantia1 evidence that 5- hydroxytryptamin~ (serotonin) can be conjugated in the 1iver and excreted as a glucuronide (Weissbach et al, 19S8). Whether or not COMT is as important as MAO in the normal catabo1ism of bio1ogical amines, it might represent an essential alternate pathway for monoamine degradation during MAO inhibition. This possibi1ity is substantiated by the observation that animal brain amine leve1s stabi1ize within twenty-four hours after the administration of a MAOI, despite the persistence of complete MAO inhibition for at 1east forty-eight hours (Holtz and Westermann, 1965). Shifts in metabolic pathways after a reduction in MAO activity are revealed in chromatographs of urinary extracts of aryla1ky1amines and their degradation products. Treatment with a MAO! has been corre1ated with a dim1nished urinary output of deaminated catecholamine metabo1ites such as 3, 4-dihydroxymande1ic acid, 3-methoxy-4-hydroxymande1ic acid, 3,4-dihydroxyphenylacetic acid, and 3-methoxy-4-hydroxypheny1- glycol (Rosen and Gooda1l, 1962; P1etscher et al, 1960). Converse1y, the urinary concentration of ortho-methylated metabo1ites was increased by the administration of MAOI . 14 • The infusion of C14-noradrena1ine or C -adrenaline 15

~ollowing treatment with iproniazid in man (Rosen and Goodal1, 1962), mice (Kopin et al, 1961) and cats (Kirshner et al, 1961; Hertting et al, 1961), enhanced the urinary excretion

o~ labelled normetanephrine and labe11ed metanephrine. Normetanephrine concentrations were also e1evated in the liver (Hertting et al, 1961) and brain (Carlsson, 1960; ,

Axelrod and Tomchiak, 1958) a~ter MAOI treatment.' Marked changes in the urinary excretion patterns ,of

5-hydroxytryptamin~ tyramine, tryptamine and dopamine have also been reported in anima1s treated with MAOI. Weissbach

et al -(1958) ~ound that although iproniazid and pheniprazine

had little in~luence on the rate of disappearance o~ 5- hydroxytryptamine given to mice, urinary concentrations of the deaminated Metabolite, 5-hydroxyindoleacetic acid, were diminished and the conjugation product of glucuronic acid and 5-hydroxytryptamine became the major metabo1ite. The deamination of tyramine to mandelic acid was blocked by treatment with iproniazid (Schayer, 1953; Goodall, 1959),

but the rate o~ tryptamine degradation in mice was only slowed. Indoleacetic acid remained the predominant

Metabolite o~ tryptamine (Weissbach et al, 1961). Shifts in the dopamine excretion pattern following treatment with iproniazid or pheniprazine were revealed by a reduction in the urinary concentration of p-hydroxymandelic acid ànd by the appearance of octopamine in human urine. Octopamine

' was also ~ound to be the MOSt prominent phenolic amine in •, . 16

kidney extracts 'from iproniazid treated rabbits (Kakimoto • and Armstrong, 1962). Rats adrninistered iproniazid and dopamine showed evidence of utilizing a route capable of N-acetylating and 3-methylating dopamine (Goldstein and Musacchio, 1962).

Inhibition: MAO inhibition by hydrazine and hydrazide derivatives was first discovered by Zeller et al (1952) during a series of in vitro studies on the effect of isoniazid and iproniazid on enzyme systems related to the function of the autonomie nervous system. Iproniazid concentrations of 0.1 mM in­ hibited the MAO activity of brain and liver mitochondrial preparations from seven animal species by 90 to 100%. Maximum inhibition in vitro occurred when mitochondrial preparations were incubated with iproniazid in the absence of a substrate for six to twelve minutes, the degree of inhibition reflecting the length of incubacion time up to that point. This finding was corroborated by Corne (1956) and by Corne and Graham (1957). In vitro assays of the activity of brain and liver MAO derived from male and female rats pretreated in vivo with 10 or 20 mg./kg. iproniazid demonstrated a simi1ar inhibition (Zeller et al, 1952). Later studies with dogs (Zeller et al, 1955) showed that pretreatment in vivo with 0.4 • mg./kg. of iproniazid inhibited liver MAO activity comp1etely and 17

reduced the activity of brain MAO to27% of the control value. About five days were required for .comp1ete recovery of activity, an observation that suggested that the enzyme might be destroyed by the inhibitor. Trany1cypromine, a pheny1cyc1opropy1amine derivative, marked1y reduced MAO activity in guinea pig 1iver (Shore and Cohn, 1960) and in brain and 1iver preparations from rabbits and mice (Sarkar et al, 1960). Inhibition of brain MAO in rabbits and mice remained 75% complete after four days. In sharp centrast to the long lasting in- hibitory effect of trany1cyprpmine was the short-lasting in vivo bloc·kade of mouse brain MAO by pheniprazine, a hydrazine compound known to be a potent inhibitor of rat liver MAO (Biel et al, 1959). Complete inhibition of - mouse brain MAO by pheniprazine (2 mg./kg.) required only one half hour, but MAO activity returned to 60% of the control level within eighteen heurs (Spector et al, 1960). It was speculated that the rate of MAO synthe sis in the mouse might be sufficiently rapid to replace Most of the destroyed enzyme within this period of time. Although shown to be ten times less effective than pheniprazine as an in vivo inhibitor of rat liver MAO, phenelzine proved to be about equally effective as an in- hibitor of mouse brain MAO. In experiments by Laroche and Brodie (1960), using mice treated with a dose of 20 mg./kg., l, phenelzine inhibited brain MAO completely in ten minutes. 18

Blockade was still complete after four hours. Rapid in­ • hibition of rnouse brain MAO was also observed by Chessin et al (1959). In these studies, the enzyme remained completely inactivated for at least two days and decreased activity persisted for two to three weeks.

Molecular Mechanism of Monoamine Oxidase Inhibition: Efforts to elucidate the mechanism underlying the in­ hibition of MAO were stimulated by the finding that the hydrazine MAOl and pargyline maximally inhibited MAO only after a period of preincubation with the enzyme in the absence of substrate (Zeller et al, 1952; Davison, 1957) and that this inhibition was potentiated by cyanide ions. lt was therefore probable that inhibition of MAO by the hydrazine compounds was due to active metabolites. However, tranyl­ cypromine, a primary amine, inhibited MAO maximally in vitro wiythout aerobic preincubation. The electronic properties of tranylcypromine may confer upon it an affinity for the active enzyme site without chernical alteration of its structure (Belleau and Moran, 1963; Zeller and Sarkar, 1962). Support for the hydrazine derivative as an active inhibitor was gained frorn the observation that iproniazid could be metabolized to isonicotinic acid and isopropyl­ hydrazine (Koechlin and Iliev, 1959). lsopropylhydrazine was several times more potent as an MAOl than the parent • compound. The incubation of isocarboxazid, labelled in the 19

benzene moiety, with rat liver homogenate yielded the labelled • Metabolite benzylhydrazine, a potent MAOl (Schwartz, 1961), and recent efforts by Kory and Mingioli (1964) led to '~he discovery of volatile inhibitors derived from iproniazid and benzylhydr·azine. The production of these inhibitors required oxygen and the presence of cyanide ions, but neither oxygen nor cyanide was involved in the interaction between the inhibitor and MAO. lt was hypothe'sized that hydrazine MAOl like iproniazid formed an irreversible complex with the enzyme only after initial a-dehydrogenation of the hydrazine moiety to form a substituted hydrazone (Davison, 1957; Barsky et al, 1959) which then reacted with the enzyme by addition across the double bond. Carbon et al (1958) felt that inhibition was initiated by a direct nucleophilic attack by an active hydrazine derivative on an amide mOiety. Support for the involvement of a-hydrogens was derived from the finding that the a-hydrogens of an MAO substrate May be active in binding it to the enzyme. Experiments using tyramine monodeuterated on the a-carbon, showed that the dideuterated compound was

oxidized more slowly by MAO than was cold tyra~ine (Belleau, 1960). The two forms of monodeuterated derivatives were degraded at intermediate rates. The hydrazone mechanism would account for inhibition by both hydrazine and alkylated hydrazide compounqs and possesses 20

the added attraction of mimicking the oxidative deamination of amine substrates. However, Green (1964) stated that if this hypothesis were correct, the dehydrogenation and ad­ dition reactions would have to occur simultaneously without the incipient double bond ever occurring, since it was shown that the hydrazones were not themselves inhibitors (Barsky et al, 1959). Other evidence against this mechanism was that it required a hydrogen on the a-carbon, and MAOl which are exceptions to this are· tertiary butylhydrazine and p-tolhydrazine. Both lack a-hydrogens but exhibit kinetic behavior resembling that of the arylalkylhydrazines, and probably inhibit MAO by the same mechanism as members of this group (Green, 1964). Also, maximum inhibition of MAO by the formation of hydrazones would not require preincubation, catalysis by oxygen or the presence of cyanide ions. For these reasons, another mechanism might be involved. Zeller and Sarkar (1962) postulated that the a-hydrogens of the inhibitor Molecules could Mediate their initial attraction to the active enzyme site through van der Waal's forces and hydrophobie bonding. The rupture of the a-carbon-hydrogen bonds and the subsequent development of covalent bonds could establish an irreversible, three pOint union between the active site and the inhibitor or substrate. This argument was also subject to the criticism that a-hydrogens May not be absolutely necessary to MAO inhibition. 21

Some studies have suggested that MAO is a metalloenzyme" with copper a possible prosthetic moiety (Mann" 1961; Yamada and Yasunobu" 1962). The importance of metal ions in MAO­ substrate-inhibitor interactions is emphasized by the protection afforded against the action of the long-lasting MAOI by the reversible inhibitor 8-hydroxyquinoline" a competitive chelating agent (Gorkin et al; 1966)" and by evidence that iproniazid can form chelates with copper (saunders" 1961). Eberson and Persson (1962) have shown that the hydrazine bases are uns table and that phenelzine siowly auotoxidizes in the presence of cupric ions to form free aryl or alkyl radicals by a mechanism which could conceivably occur in biological systems. Green (1964) has postulated that these free radicals could reversibly inhibit enzymes" if the oxidation occurred near enough to an active center, as for example by combining with a thiol group at this center. MAO does not appear to be a metallic 'catalysed enzyme, therefore if the hydrazines are to be oXidized, any metallic activity would probably be confined to a site on the enzyme itself, either at the active center or nearit. Copper is known to be present in certain soluble amine oxidases and recent work by Nara et al (1966) indicated that beef liver mitochondrial MAO is a copper contain1ng protein. Thé general lack of correlation between the protecting ability of the chelating agents and their ability to inhibit MAO, 22

and the fact that inhibition by these compounds is re­ latively weak, suggests that copper is not part of .the active site of the enzyme Molecule. Although the catalytic properties of copper May be necessary tothe inhibitory mechanism"the work of Mann (1961) and Yamada and Yasunobu (1962) suggested another possible mechanism involving copper. Mann, on the basis of MAO inhibition by hydrazine compounds and spectral changes in the enzyme, postulated that the catalytic mechanism of MAO is dependent on a complex, or chelate, of copper and 'pyridoxal

phosphate (PALP). Yamada and Yasnobu found that bee"f lj, v~r mit6chondrial MAO contained PALP as weIl as copper in a ratio of about 2 Cu++ ions to 1 mole of PALP. The possibility that the.cupric ion was complexed with PALPwas strengthened bythe finding that the 480 mu absorption peak of MAO de­ creased on removal of the copper while the 380 mu peak of PALP became increasingly more evident. The significance of these findings, with respect to MAO inhibition by the hydrazine derivatives, is interesting in view of the hypo­ thesis that these compounds inhibit the activity of glutamic acid decarboxylase by complexing with PALP, the cofactor for "this enzyme.

Pharmacological Effects Of Inhibition~ The inhibition of monoamine oxidase caused the accumul- ation of excessive quantities of substrate in various body 23

tissues (Maling et al, 1962; Weil-Malherbe et al, 1961; Costa et al, 1960) and in the brain (Pletscher, 1956; Dubnick et al, 1962; Sanan and Vogt, 1962). The increased concentrations of excitatory amines in the brain may be the basis for the pharmacological actions produced bythe monoamine oxidase

inhibitors, particularly thei~ antidepressant and central nervous system stimulatory effects. Of particular "interest are the roles played by elevated levels of noradrenaline and 5-hydroxytryptamine in the brain following MAOl treatment. Single doses of iproniazid (100 mg/kg.) increased nor­ adrenaline concentrations in rabbit brain two or three fold over a four to five day period without eliciting any signs of central nervous system stimulation. Over the same period of time, 5-hydroxytryptamine levels were doubled (Spector et al, 1960). Chronic administratïon of iproniazid in a daily dose of 25 mg./kg. doubled the 5-hydroxytryptamine content of the brain in two to three days with no sign of excitation. By the four th or fifth day, however, nor- adrenaline concentrations had reached a maximum greater than that elicited by a single dose, and concomitant with this graduaI increase were symptoms of CNS stimulation such as hyperactivity, vasoconstriction in the ears and mydriasis. These symptoms gradually disappeared as noradrenaline levels dropped, even when the 5-hydroxytryptamine content of the brain remained almost maximal. Doses of 50 mg./kg. of • iproniazid daily induced maximum noradrenaline concentrations 24

of a similar magnitude within three days and excitation was • again temporally related to the rise. The administration of pheniprazine (2 mg./kg.) produced results sim1lar to those obtained with iproniazid for both acute and chronic treatments (Spector et al, 1960). The effect of the MAOl on brain catecholamine con- centrations was species dependent, and in contrast to the general pattern of increased brain noradrenaline, pheni- prazine and.iproniazid were unable to induce changes in the

noradrenaline levels of cat or dog br~in (Spector et al, 1960). Maling (1962), however, was able to demonstrate a small increase in cat brain noradrenaline. Excitation was not evident in any of these animaIs, even though 5-hydroxytrypt- amine concentrations in the dog increased seven times the control value after three weeks of daily administration of

',' MAOl. Rats and mice responded in the same manner as rabbits with respect to amine levels and stimulation of the central nervous system (Spector et al, 1963; Prokop et al, 1959) The ability of the MAOl to induce excitation May not be due entirely to increased amine concentrations in the brain. A prominent side effect of the MAOl possessing the phenyl­ isopropyl moiety is an amphetamine-like stimulation which appears to be unrelated to MAO inhibition. lts onset is

rapid and its duration shor~Jusually disappearing before MAO inhibition is attained, and it is unaffected by previous inhibition of MAO by the same or another inhibitor (Holtz • and Westermann, 1965). 25

PYRIDOXAL PHOSPHATE DEPENDENT ENZY.MES: • The Cofactor: One half of the vitamin B6 in the body exists as the 5-phosphorylated form of pyridoxal, pyridoxal phosphate (PALP), the remainder being distributed as pyridoxine (PN), pyridoxamine (PM) and their 5-phosphorylated derivatives (Rabinowitz and Snell, 1948). Pyridoxamine phosphate (PMP) may also function as a cofactor (Meister et al, 1952), but pyridoxine is not generally accorded any significant physiological role. Pyridoxal phosphate and pyridoxamine phosphate are found in most tissues in accordance with the distribution of pyridoxal phosphate dependent enzymes.

The Enzymes: Pyridoxal phosphate was first shownto be a cofactor, or prosthetic group, in enzymic function by Umbreit et al (1945) and Gunsalus et al (1945), for the enzymes glutarate- pyruvate transaminase and glutarate-oxaloacetate transaminase. Experimental evidence has since indicated that both trans- amination and amine acid decarboxylation reactions are dependent on the presence of pyr1doxal phosphate for normal funct1on. Part1cular interest has been focused on L-glutamic acid decarboxylase (GAD) and on y-aminobutyric ac1d-a­ ketoglutarate transaminase with respect to the metabo11sm of y-aminobutyric acid (GABA). A L-Glutamic acid decarboxylase 1s localized almost ~~ 26

~.) exclusively in the central nervous sys.tem and primarily in

the gray matter (Roberts and Frankel~ 1951)~ raising the possibility that it bas a function in the electro- physiological activity of the brain. Roberts and Frankel also showed that GAD catalysed the essentially irreversible decarboxylation of L-glutamic acid to synthesize y-amino-

butyric acid~ thought to be an inhibitory factor in the

central nervous system (Killam~ 1957j~ or the precursor ror

possible inhibitors such as y-amino-~-hydroxybutyric acid

and carnitine (Pisano et al~ 1960).

y-Aminobutyrate-a-ketoglutarate transaminase~ found in the gray matter of the central nervous system and in other

tissues~ catalyses the reversible transamination of y-aminobutyric acid with a-ketoglutarate (Salvador and

Albers~ 1959; Roberts and Bregoff~ 1953)~ to regenerate

glutamic acid. Succinic semialdehyde~ a product of the

reaction containing the carbon chain from GABA~ is further metabolized by entering the tricarboxylic cycle at the level

of succinate (Roberts et al~ 1958).

Inhibition: The function of pyridoxal phosphate in the maintenance of normal nervous activity became the object of increased interest consequent to the finding of Park et al (1952)

that pyridoxine prevented hydrazide induced conVulsions~ a ~ result confirmed and extended to other members of the vitamin 27

B6 complex by Jenney and Lee (1951,). The discovery of increased renal excretion of xanthurenic aCid, indicative of vitamin B6 deficiency, and vitamin B6 compounds by patients treated with isoniazid, led Biehl and Vilter (1954) to postulate the formation of a pyridoxal hydrazone. This view was shared by Williams and Abdulian (1956) who· observed large quantities of pyridoxal hydrazone in the urine of dogs treated with hydrazide compounds. These authors suggested that hydrazines and hydrazides might cause convulsions by inhibiting a vitamin B6 dependent enzyme system. Roberts (1956) had previously suggested that y-amino­ butyric acid might play a role in the regulation of physiological activity in the brain. Killam and Bain (1957) extensively investigated the effect of convulsant hydrazides on GABA metabolism and correlated isoniazid and semicarbazide induced seizure activity in mice with a striking decrease in levels of brain y-aminobutyric acid and with the inhibition of L-glutamic acid decarboxylase in vivo. The addition of pyridoxal phosphate increased GAD activity at aIl stages of the inhibition during the progression of the convulsions. The involvement of a hydrazone as the probable inhibitor, as suggested originally by Biehl and Vilter (1954), was emphasized by chromatographie evidence for the formation of pyridoxal and pyridoxal phosphate hydrazones in isoniazid .' convulsed mice (Bain and Williams, 1960). The administ- 2,8

ration of pyridoxal hydrazones~ rather than the parent

hydrazines or hydrazides~ proved them to be more potent inhibitors of L-glutamic acid decarboxylase (McCormick and

Snell~ 1959; McCormick et al~ 1960; Dubnick et al, 1960). Roberts (1960) concluded from the experimental evidence available that free, inactive coenzyme derivatives were formed by the reaction of hydrazines or hydrazides with pyridoxal phosphate bound to the apoenzyme. Sub­ sequent to the dissociation of the complex from the apo­

enzyme~ the inactive derivative might compete with free pyridoxal phosphate for the active site on the apoenzyme. This hypothesis could be an explanation for potentiation of seizure activity and for inhibition of L-glutamic acid decarboxylase when pyridoxal or pyridoxal phosphate were injecte'd simultaneously with hydrazine derivatives

(Dubnick et al, 1960; Medina~ 1963)~ and for the antidotal effects of pyridoxine and pyridoxamine. Presumably, the aldehyde cofactors complexed with the hydrazines to form more hydrazones which inhibited L-glutamic acid decar­ boxylase in the manner described by Roberts; pyridoxine and

pyridoxamine~ both lacking the aldehyde group~ were in­ capable of hydrazone formation and continued to function as cofactors. There is evidence, however, of a seizure mechanism other than direct inhibition of L-glutamic acid decar- .' boxylase. Bain and Williams (1960) gave isoniazid and 29

pyridoxal to mice and found that during the subsequent seizures the pyridoxal phosphatelevel of the brain dropped to 26% of the control, but the pyridoxine content increased by 400%. McCormick and Snell (1959) pointed out that several hydrazides and hydrazones may inhibit pyridoxal kinase in brain and liver one hundred to one thousand times more than they inhibit L-glutamic acid decarboxylase. In their experiments, hydrazine derivatives inhibited the kinase more than the decarboxylase in vivo and the dihydrazone of pyridoxal markedly decreased brain and liver kinase activity without affecting the decar­ boxylase. These findings raised the possibility that the seizure-GABA correlations may be related to inhibition of the kinase rather than the decarboxylase. Dubnick et al (1960) concurred with this view 'in their statement that inhibition of decarboxylase enzymes observed in vivo with phenelzine was due to inactivation of pyridoxal kinase by a hydrazone complex of phenelzine and pyridoxal. The results obtained by Medina (1963) using symetrical dimethylhydrazine (SDMH) provide evidence for a seizure mechanism which does not involve formation of a hydrazone complex or, possibly, even inhibition of L-glutamic acid decarboxylase. SDMH, unable to form hydrazones because of substitution on both active groups of the hydrazine molecule, was as potent a convulsant as hydrazine, monomethylhydrazine and ~symetrical dimethylhydrazine, but had little or no effect 30

on L-glutamic ac).d decarboxylase activity. However~ y-aminobutyrate-a-ketoglutarate transaminase activity was reduced by 75%. Both pyridoxine and pyridoxal phosphate were effective antagonists of the convulsant action of SDMH.

N-DEALKYLATION ENZYME SYSTEMS: The Enzyme s: Liver microsomal enzyme systems are concerned with the oxidation of a number of compounds through a variety of met­

abolie pathways~ including side chain oxidation (Cooper and

Brodie~ 1955)~ ether cleavage (Axelrod~ 1956)~ deamination;

(Axelrod~ 1955)~ dealkylation (La Du et al~ 1955) and hydr-

oxylation (Mitoma et al~ 1956). The 0 b se rva tion :.: that the

degradation of drugs via these pathway~ was dependent on the

presence of TPNH and oxygen (Gillette et al~ 1957)~ and that a number of drugs were mutually inhibitory with respect to

their metabolism by microsomal enzymes~ led to the speculation that some factor was common to each of these enzyme systems.

As an extension of this hypothesis~ Rubin et al (1964) postulated that the mutual inhibition observed for a series of drugs N-dealkylated in the microsomes was due to the existence of a single N-dealkylating enzyme. An invest- igation into the validity of the hypothesis of Rubin et al (1964) was conducted primarily by a group headed by

G.J. Mannering~ using the compound 2-diethylaminoethyl-2~2- diphenylvalerate HCl (SKF-525A)~ a general m~crosomal enzyme 31

inhibitor. The results of these studies have provided a reasonable explanation for the contraindication of a number of drugs in patients using monoamine oxidase inhibitors as will be discussed.

Inhtbition: SKF-525A and ten congeners were shown to be N-dealkylated by the liver microsomal enzyme system and aIl proved to be competitive inhibitors of the N-demethylation of ethylmorphine (Anders and Manne·ring., 1966). PreviquslY., it was felt that SKF-525A inhibited the metabolism of drugs by"altering membrane permeability (Brodie, 1956; Brodie.,:1962) or by uncoupling the TPNH oxidizing system associated with the microsomal enzymes (Netter., 1962). The results of Anders and Mannering (1966) encouraged these authors to postulate triat SKF-525A:. and its congeners. inhibited the oxidation of ethylmorphine by com­ bining with the N-demethylase enzyme as alternate substrates., and that other drugs which utilized the same microsomal enzymes responsible for the oxidation of SKF-525A would also be in­ hibited competitively. Subsequent studies revealed that the N-dealkylation of SKF-525A involved N-deethylation to 2-etnylaminoethyl-2.,2-diphenylvalerate HGl (SKF-8742) which compound was further N-deethylated to a primary amine analog of' SKF-525A., aminoe·thyl-2.,2-diphenylvalerate HGl (Anders et al., 1966). Both metabolites were competitive inhibitors ~) of' the N-demethylation of' ethylmorphine., with Kifs equivalent 32

to that of SKF-525A (Anders et al, 1966), suggesting that they • required thesame enzyme system for deactivation as did the parent compound. SKF-525A and its metabolites were shown by Stitzel et al (1966) to prolong hexobarbital sleeping time and to inhibit the oxidative dealkylation of hexobarbital in the intact rat and in isolated, perfused rat liver. SKF-525A was the most potent agent of the three in prolonging sleeping time and producing inhibition, as would be anticipated if it and each successive metabolite was capable of occupying the N­ dealkylating enzyme responsible for inactivating hexobarbital. The alternate substrate hypothesis was also shown to be valid for enzyme systems other than those concerned with N­ dealkylation. For example the mechanism by which SKF-525A inhibited procaine esterase was elucidated by Netter (1959) who showed that the esterase enzyme was competitively inhibited because SKF-525A was also an ester. In view of the above hypothesis, the results of Fouts and Brodie (1956) obtained a de cade before have been subjectèd to a new interpretation. In their study comparing iproniazid and SKF-525A, both compounds prolonged barbiturate sleeping time in mice to the same extent and both were equally ef- fective in blocking the dealkylation of aminopyrine, monomethyl-4-aminoantipyrine" and the hydroxylation of acetanilid in rabbit liver preparations. SKF-525A was more efficient, 4ItJ however, as an inhibitor of the N-dealkylation of hexobarbital 33 and the deamination of amphetamine. The fact that the inhibitory patterns of iproniazid and SKF-525A were similar

sûggested a mechanism of action shared in comm~n,an argument supported by the fact that iproniazid could be dealkylated by liver microsomes to acetone and isoniazid (Koechlin and Iliev, 1959). The difference' between the abilities of iproniazid and SKF-525A to block hexobarbital metabolism would then be dependent on the relative affinities of the three compounds for a common'enzyme; the Km values were: 3.64 x 10-5 M for SKF-525A; 8.22 x 10-3 M for iproniazid (Anders and Mannering, 1966) and 1.2 x 10-3 M for hexobarbital (Rubin et al, 1964). The alternate substrate hypothesis would also explain the finding that a time interval of one to two hours between the administration of iproniazid and hexobarbital reduced or eliminated potentiation of hexobarbital hypnosis (Laroche and Brodie, 1960). Iproniazid was probably metabolized sufficiently within that time by the enzyme common to it and hexobarbital so that the concentration remaining at two hours was in- effective. The ability of phenelzine and nialamide to prolong barbiturate sleeping time (Laroche and Brodie, 1960) was also lost with increasing pretreatment time with iproniazid, perhaps for the same reason. The administration of pethidine (meperidine: Demerol) to humans (Reid and Jones, 1962; Craig, 1962) and animals (Brownlee and Williams, 1963) pretreated.with phenelzine has produced hyperpyrexia, hyperactivity and death. The problem 34

of the interaction between phenelzine and pethidine has been '.' studied in vitro with rat and rabbit liver microsomal fractions by Clark (1967) and the results are compatible with the hypo- the sis of alternate substrate inhibition. Phenelzine blocked the N-demethylation of pethidine competitively and the in­ hibition could be reversed by increasing the concentration of pethidine. The Km value for pethidine with the pethidine N-demethylase system from rat liver microsomes (0.35 ± 0.14 x 10-3 M: Clark~ 1967) was of the same order as the Km values for other drugs undergoing oxidative metabolism in this system. The Km for phenelzine (1.1 x 10-5 M to 15.2 x 10-5 M: Clark~ 1967) would make it a relatively potent inhibitor of pethidine metabolism. No evidence was found by Clark for an action by phenelzine on the associated TPNH oxidase system, for non­ specifie effects on microsomal membranes or for complex formation between phenelzine and the substrate. In one interesting clinical case (Cocks and Fassmore-Reive,

1962)~ a patient undergoing therapy with phenelzine became comatose after being given 100 mg. of a combination of pethidine and levallorphan. Nalorphine, a narcotic an- tagonist metabolized by microsomal enzymes, was administered to alleviate the condition, but worsened the coma instead. Seemingly inexplicable, this effect might be interpreted according to Mannering's theory of inhibition by alternate substrates • •, ""' Another group of drugs interacting with the monoamine 35

oxidase inhibitors in man (Jarecki, 1963; Saunders, 1965) and in anima1s (Himwich and Peterson, 1961; Love1ess and Maxwell, 1965) and demethy1ated by the 1iver microsoma1 enzyme system (Gillette et al, 1961) are the tri-cyc1ic anti­ depressants, particularly imipramine and amitriptyline. Himwich and Peterson (1961) stated that the toxic effects observed with the simultaneous use of an MAOI and imipramine might resu1t from b10ckade of the metabo1ic pathway of an imipramine derivative. Assuming that imipramine N-demethy1ase and pethidine N-demethy1ase are the same enzyme, as suggested by the finding that phene1zine blocked the degradation of both pethidine and imipramine with a simi1ar Ki value (Clark, 1967), then the "accumu1ated substance" postu1ated by Himwich and Peterson (1961) might very weIl be imipramine itse1f. 36

METHODS

GENERAL PROCEDURE: The preparation of drugs and reagents used in the following methods is described in detail in Appendix l (drugs) and APpendix II (reagents). AnimaIs used for in vivo experiments were male swiss mice weighing between 20 and 30 g. obtained from the canadian Breeding Laboratories, LaPrairie, P.Q., Canada. They were fed a standard diet of Purina mouse chow and allowed free access to water. During experiments,the mice were kept in individual cages to minimize the pos­ sible effects of aggregation toxicity and had access to food and water only in experiments lasting longer than four hours. The room temperature was maintained at 23 0 C. The animaIs were randomly divided into two groups and each group received one of two acute treatments. The mice referred to as controls were pretreated with 0.9% sodium chloride solution, 10 ml./kg. intraperitoneally (i.p.). Experimental animaIs were pretreated with phenelzine sulphate solution, 40 mg./kg., i.p. This dose was shown by Renton (1967) to be sub-lethal when given alone. AlI animaIs received an i.p. injection of ethanol solution after one half hour pretreatment. Unless spec­ ified otherwise, 3.5 g./kg. of ethanol was administered, a dose which was hypnotic but not lethal when given alone. 37

EFFECT OF PHENELZINE ON ETHANOL SLEEPING TIME: Mice were treated with 0.9% sodium chloride solution, or phenelzine sulphate solution, and 40% ethanol as de­ scribed in the section on general procedure. The animaIs were placed on their backs when they lost their righting reflex and the tirne noted. Sleeping tirne was defined as the time interval required for a rnouse to right itself frorn this supine position. The end pOint was the time at which a mouse had cornpletely righted itself. In sorne mice, the total body concentration of ethanol was determined at the end point.

EFFECT OF PHENELZlNE ON THE LD 50 OF ETHANOL: One half hour after the standard pretreatrnent, control and experirnental rnice were given an injection of ethanol within a dose range of 3.75 g.Jkg. to 6.5 g.Jkg. Ten mice were allotted to each dose interval and the number of mice dying in a given interval over a twenty-four hour period were counted and the data analysed as described in the results.

EFFECT OF PHENELZINE ON THE DISAPPEARANCE OF INJECTED ETHANOL: The assay for ethanol in the total mouse was derived frorn that used by Aull and McCord (1964) to determine the concentration of ethanol in blood. Because sufficient quantities of blood could not be obtained from the mouse, 38 .l the procedure was modified so that the ethanol content of­ the total mouse could be assayed. Appara tus: - The distilling flask was a _125 ml. Erlenmeyer flask attached to the inlet tube of a 50 ml. Kjeldahl bulb with a rubber stopper. A 100 cm. U-shaped outlet tube connected the bulb to a Leibig condenser, the delivery tube of which was long enough to reach the bottom of a 50 ml. graduated cylinder. Standard Curve: The colorimetrie determination of ethanol involved the stoichiometric reduction of 1.0 ml. of potassium dichromate solution (cr6+ to cr3+) by each 0.5 mg. quantity of ethanol added. The disappearance of the potassium di chromate was measured by the progresstve decrease of the optical density (on) of the reaction mixture. For convenience in preparing the standard curve, the amounts of ethanol to be measured up to 2.5 mg. were substituted with chvomic nitrate sol­ ution (Cr3+) and 1.0 ml. of this solutionwas used for every 0.5 mg. of ethanol. The volumes of chromic nitrate were made up to 5 ml. in a 50 ml. graduated cylinder with potassium dichromate solution (cr6+), as detailed in Table 1. The contents of each cylinder were diluted with dis­ tilled water to 30 ml., cooled to room temperature with running water, then further diluted with distilled water to exactly 50 ml. The solutions were well shaken and portions 39

Cy1inder Chromic Potassium Ethanol • No. Nitrate Dichromate Equivalent ml. ml. mg. 1 (B1ank) 0 5 0.0 2 1 4 0.5 3 2 3 1.0 4 3 2 1.5 5 4 1 2.0 6 5 0 2.5'

Table 1. Volumes or solutions used for the preparation of the Standard Curve.

transrerred to a glass cuvette with a 1ight path of 0.5 cm. and read against disti11ed water at 400 mu in a Unicam SP-500 spectrophotometer. The optica1 density for the water b1ank was subtracted from the OD ror each sample and the corrected values subtracted from the corrected OD for

potassium dichromate a1one. Th~ values obtained by the latter ca1cu1ation are tabu1ated in Table 2 as the means and standard errors for three determinations. When p10tted against the corresponding ethano1 equiva1ents, othe re1ationship was 1inear over the range of ethano1 con­ centrations used (Fig. 1).

Experimental Procedure: Mice were treated as described under genera1 procedure. At time interva1s ranging from zero to twe1ve hours, mice -. •

ETHANOL CONCENTRATION (mg. 150 ml.)

0.5 1.0 1.5 2.0 2.5

1 0.227 0.460 0.674 0.833 1.009

2 0.190 0.387 0.593 0.776 0.945

3 0.243 0.478 0.673 0.889 1.042

MEAN +

STANDARD ERROR 0.220 + 0.016 0.442 + 0.028 0.646 + 0.027 0.833 + 0.033 0.999 + 0.028

Table 2: Coordinate values for the standard curve relating optical density to ethanol concentration.

o~ 41

1.0

0.9 .

0.8

0.7

> l- 0.6 V) Z w Cl 0.5

«....1 U 0.4 1- a.. 0 0.3

0.2

0.1

/

o 0.5 1.0 1.5 2.0 2.5 3.0 ETHANOL. EQUIVALENT CONCENTRATION (mg/50 ml)

Fig. 1: Standard curve re1ating optica1 density to ethano1 equiva1ent concentration. from control and experimental groups were killed by a • sharp blow on the head. Individual animaIs were im­ mediately homogenized for forty-five seconds in a Waring blender in five volumes of sodium sulphate-sodium'tungstate protein precipitating solution; one volume was equivalent to the gram weight of a mous'e in milliliters. Six ml. of homogenate were added to a distilling flask c9ntaining 10 ml. of distilled water and 5 ml. of sodium sulphate­ sUlphuric acid reagent. The contents of the flask were

gently heated and the distillate collected in 5 ml~ of potassium dichromate solution contained in a 50 ml. graduated cylinder. Frothing of the flask contents was observed occasionally and minimized by lowering t,he flame. The 'tip of the delivery tube from the condenser was kept belpw the surface of the dichromate solution ,at aIl timesduring the distillation. Where large amounts of ethnol were present in the homogenate, the total reduction of the dichromate was prevented by the addition to the distillate during distillation, of up to 5 ml,. of dichromate solution. The final volume of the distillate, approximately 25 ml.,was diluted with distilled water to 50 ml. and a sample assayed in a Unicam SP-500 spectrophotometer at400 mu, using a glass cuvette with a light path of 0.5 cm. The optical densities were corrected as described in the section on the standard curve and the standard curve used to convert these . • calculated values to the corresponding quantities of ethanol. 43

, Corrections were made for extra dichromate solution added • and for dilutions made during homogenization to give the total number of milligrams of ethanol recovered. This

value was conv~rted to per cent recovery by the formula:

%Recovery = total mg. of ethanol recovered x 100% total mg. of ethanol injected

Per cent Recovery: The efficiency of the recovery procedure was tested by adding 3.5 mg. of ethanol per gram of mouse to an un­ treated mouse homogenate and recovering the ethanol in the standard manner. The efficiency, in per cent' recovery, was calculated as:

% Recovery = total mg. of ethanol' recovered x 100% total mg. of ethanol added to the homogenate

The calculated per cent recoveries are shown in Table 3A.

The mean per cent recovery was 90 + 0~9% (S.E.).

EFFECT OF PHENELZINE ON ACETALDEHYDE CONCENTRATIONS: The assay for acetaldehyde in the total mouse was derived from the method described by stotz (1943). Because this technique proved unsuitable for the determination of the small concentrations of acetaldehyde found in the mouse, it was modified to increase the quantity of acetaldehyde 44

PER CENT RECOVERY

A B

1 86 82

2 92 89

3 92 96

4 92 85

5 88

6 92

MEAN +

STANDARDERROR 90 + 1 88 + 3

Table 3: Percentage recovery of ethanol and acetaldehyde.

A. EfficiencY'of recovery of ethanol

from whole mouse homogenate.

B. Efficiency of recovery of acetaldehyde

from whole mouse homogenate •

• recovered from the homogenate so that it could be detected by the colorimetric assay.

APparatus: A micro-distillation apparatus was designed in this . . .

laboratory and constructed by Scientific Glassblowing Inc. 3

Montrea1 3 P.Q.J canada. It consisted of a 100 ml. pea:ç-- shaped distilling flask connected by an asymetrical U-shaped tube to a micro-.condenser fitted with a delivery tube capable of reachihg the bottom of a 25 ml. graduated cylinder. AlI connections were of ground glass because acetaldehydewas found to dissolve large quantities of 1nterfering substances from rUbber·stoppers. A large cylinder'of light Metal was used as a wind screen for the Bunsen burner to ensure a steadYJ low flame. Excessive heating caused violent boiling and contamination of the distillateJ and varying heat tended to draw the distillate up the delivery tube into the distilling flask.

Standard Curve: The quantitative determination of acetaldehyde was based on the pOlymerization of acetaldehyde to paraldehydeJ a liquid trimerJ induced by the addition of concentrated sulphuric acid. The reaction was highly exothermic and care had to be taken to main tain the temperature of the • reaction mixture below the boiling point of acetaldehyde 46 -.,-., \.. ! A chemical reaction between paraldehyde and p-hydroxybiphenyl produced a purple color measurable at

540 ~ in a spectrophotometer. Equivalent weights of acetaldehyde and paraldehyde yielded equal color inten- sities (stotz, 1943). Because of its higher boiling point and the comparative ease with which it could be handled, paraldehyde was used instead of acetaldehyde for the deter­ mination of the standard curve relating acetaldehyde con- centration to optical density. Paraldehyde concentrations

of 0 to 10~g./ml. were assayed for their yield of color intensity.

Color Reaction: One ml. of paraldehyde solution was added to 0.05 ml. of copper sulphate solution contained in a 12 ml. test tube; for the assay of unknown amounts of acetaldehyde, l ml. of acetaldehyde-sodium bisulphite solution was added instead of paraldehyde to the copper sulphate solution. The tubes were kept in an ice bath and removed only to mix the contents with a Vortex electric mixer after each new addition. Eight ml. of concentrated sulphuric acid (S.G. 1.84) were added very slowly to each tube while the contents were shaken gently. P-Hydroxybiphenyl solution {0.2 ml.) was pipetted into each tube and the color allowed ta develop for one hour at room temperature. Samples were

assayed for acetaldehyde at 540 ~ in a Unicam SP-500 / .. spectrophotometer, using a glass cuvette with a 0.5 cm. light •r· - 47

path. The optical density for an acid blank composedof all the solutions except paraldehyde (or acetaldehyde during the experimental procedure) was subtracted from the optical density for each sample by adjusting the spectro- photometer to zero OD for the blank. The results are summarized in Table 4. The corrected optical densities were plotted against the acetaldehyde equivalents of the paraldehyde concentrations used and the relationship found to be linear over the range tested (Fig. 2).

Experimental Procedure: Two mice from the control group and two mice from the experimental group were killed at appropriate time intervals by a sharp blow on the head;two mice were used for each determination in an attempt to decrease the large mouse to mouse variation found in preliminary work. The two sim- ilarly treated mice were homogenized together in a waring blender at top speed for one minute. The protein pre- cipitating solutions used during the homogenization were as follows: sulphuric acid, 1 volume; sodium tungstate, l volume; distilled water, 2 volumes. One volume was equi valent to the to.tal gram welght of the two mice in milliliters. To minimize the loss of acetaldehyde at room o 0-' temperature, aIl solutions were kept at 2 .0/5 C and the o glass container of the Waring blender cooled to 4 C with ice cold water before each homogenization. Eighty ml. of :..• • .'

ACETALDEHYDE CONCENTRATION' (llg/m1.)

0.5 1.0 1.5 2.0 2.5 5.0 8.0 10.0

1 0.034 0.064 0.109 0.115 0.146 0.331 0.507 0.574

2 ,0.·034 0.059 0.089 0.112 0.145 0.339 0.500 0.593

3 0 •. 034 '0.055 0.089 0.114 . 0.146 0.358 0.503 0.665

4 0.032 0.056 0.097 0.118 0.143

5 0.039 0.059 0.091 0.166 ' 0.145

- MEAN + 0.035 0.058 0.09,5 0.115 0~145 0.287 0.447 0.538 + + +' + + + + + STANDARD ERROR 0.003 0.001 0.004 0.001 0.0 0.008 0~002 0.013

"J

Table 4~ Coordinate values for the standard curve relating optica1 density to aceta1dehyde concentr~tion.

co-1=" 49

0.9

0.8

0.7

0.6 ..-> in Z w 0.5 0 ...... (C( 0.4 U ~ Q.. 0 0.3

0.2

0.1

1 234 5 678 9 10 11 ACETALDEHYDE EQUIVALENT CONCENTRATION (p.g/ml)

.. ~, .

Fig. 2: Standard curve relating optical density to acetaldehyde· equivalent concentration. 50 homogenate were distributed equally betweentwo cold, stoppered polypropylene centrifuge tubes and centrifuged • for five to six minutes at 3300 r.p.m. in an International model UV centrifuge. Bucket containers for the tubes were refrigerated at -lOoe until used. The temperatures of the

supernatants were checked occasionally at the conclusio~ of the centrifugation and found to be withinan acceptable range (less than 200 e). Forty ml. of supernatant were" decanted into50 ml., graduated, stoppered centrifuge tubes - . . . and refrigerated at 40 e for up to four hours while the remainder of the homogenizations were done.The 40 ml. of supernatant were then distilled over a low flame from a Bunsen burner and the distillate collected in 1 ml. of sodium bisulphite solution contained in a 25 ml. graduated cylinder. The cylinder was kept in an ice bath during the distillation. The total volume after distillation was exactly 3.0 ml. The tip of the condenser delivery tube was kept below the surface of the bisulphite solution at aIl times during the distillation. The ac.:-!taldehyde-sodium was refrigerated without freezing for up to one and a half hours prior to assay by the colorimetric method described for the

standard curv~.

per cent Recovery: Redistilled acetaldehyde (500 mg.) was weighed out • using a 1 ml. tuberculin syringe and glassware previously 51

o kept at a temperature of -10 C. The 500 mg. of acetalde- • hyde were diluted with cold disti11ed water to provide a solution of 125 JJ8./ml. and the concentration verified by the previously described colorimetrie assay. A sufficient quantity of this solution was added to untreated mouse

homogenate to a final concentration of 0.3 ~g./ml. Acetaldehyde was recovered by the procedure described above and the per cent recovery calculated as:

% Recovery = amount of acetaldehyde determined x 100% amount added to the homogenate

The percent recoveries are shown in Table 3B. The Mean for four determinations was 88 + 3.0 % (S.E.). Per cent recovery calculated for the original method was 97% (stotz, 1943) .

EFFECT OF PHENELZINE ON YEAST ALCOHOL DEHYDROGENASE (YADH) ACTIVITY: The oxidation of ethanol to acetaldehyde by yeast alcohol dehydrogenase and its quantitative determination by

measuring the production of reduced ~-diphosphopyridine

nucleotide (DPNH) at a wavelength of 340 m~has frequently been used as an assay method for ethanol. The rate of ethanol oxidation also provided an indication of the re- • lative activity of the enzyme . 52

General Procedure: • -Incubation Medium: The încubation medium used for the determination of YADH activity was a buffered solution consisting of 0.1 M sodium pyrophosphatej 0.05 M glycine to stabilize the enzyme preparation, and 0.15 M semicarbazide hydroch10ride to bind aceta1dehyde formed, allowing the normally reversible reaction to go to completion. Sodium pyrophosphate (44.606 g.) and glycine (3.754 g.) were added to 500 ml. of distilled water. The dissolution of the pyrophosphate required the assistance of a hot water bath and repeated shaking. Semicarbazide hydrochloride (16.67 g., Sigma Chemical Co., st. Louis, Mo., U.S.A.) was dissolved in about 200 ml. of distilled water, neutralized with 2N sodium hydroxide and then added to the pyrophosphate- glycine solution. The medium was adjusted to pH 8.5 with 2N sodium hydroxide and made up to a final volume of one liter with distilled water. The pH of this medium was measured with a PHmeter before use each time and readjusted to 8.5 with 2N sodium hydroxide when necessary.

Yeast Alcohol Dehydrogenase (YADH): (Sigma Chemical Co., st. Louis, Mo., U.S.A.). Twice recrystallized YABH preparation containing 100 mg. of protein per 340 mg. of o preparation, was stored in a glass bottle at -10 C. The enzyme was reconstituted when required with cold 0.01 M • phosphate buffer (eight parts of 0.01 M monobasic potassium 53

phosphate and 2 parts 0.01 M dibasic sodium phosphate), pH 7.6, and kept in an ice bath during use. The concentration of the reconstituted enzyme solution used in the experi­

mental procedure was 10~. protein/ml. This solution retained its activity for at least two and a half hours after preparation. To determine if the YADH, as supplied by Sigma Chemical Co., had deteriorated in transit, the micromolar unit

activity of the enzym~ was assayed by the following Methode YADH (0.1 ml., prepared as above) was added to a reaction mixture containing 2.7 ml. of 0.05 M sodium pyrophosphate

buffer, pH 8.8 (without glycine or semicarbazide hy~rochloride);

0.1 ml. of 18 x 10-3 M ~-diphosphopyridine nucleotide (DPN+, prepared as described below) and 0.1 ml. of 3 Methanol. The rate of oxidation of ethanol was measured by determining the rate of formation of DPNH at 340 m~. The micromolar unit activity of the enzyme (one micromolar unit is that amount of enzyme which can reduce one micromole of DPN+ per minute at 25 0 C and pH 8.8) was calculated using an equation supplied by the Sigma Chemical Co.: Activity (micromolar units) = change in OD per minute x 3 6.22 x mg. of protein in reaction

~-Diphosphopyridine Nucleotide (DPN+): (Sigma Chemical Co., St. Lousi, Mo., U.S.A.). A lyophilized prep­ aration of 98% DPN+ (anhydrous molecular weight, 663.5), w'as 54

' o + ~• stored in a glass bottle at -la c. DPN was reconstituted when required in cold dist1l1ed water and concentrations

expressed in terms or molar~ty. This preparation retained its activity for at least two and a half hours after preparation.

Measurement or YADH Activity: The changes in the DPNH content of control and reaction mixtures (see experi­ mental procedure) during the first minute of the reaction

were measured at 340 m~ in a Beckman DB-G spectrophotometer using a quartz cuvette with a light path of 1 cm. The changes in optical density were converted to velocities by the formula:

Veloci'cy (~M DPNH/mg. protein/min.) = change in aD per minute x 3 6.12 x mg. of protein in reaction

where 3 represents the volume of the reaction mixture and 6.12 the slope or the standard curve relating optical density to ethanol concentration (see standard curve). The possible loss of enzyme activity dur1ng an assay series was checked bycomparing the reaction velocities of similar reaction

mixtures J one assayed at the start of the series and the other at the end; no loss of activity was observed in any series. 55

standard Curve: An excess of YADH (2 mg. in 0.1 ml.) was added to reaction mixtures contalDing 1.8 ml. of sodium pyrophosphate buffer (0.1 M, pH 8.5); 0.1 ml. of 18. x 10-3 M DPN+; 1.0 ml. of ethanol in concentrations of 0.0225, 0.05, 0.10, 0.20, or O. 4 0 x·-10.-3 M. The final' volumes were 3 ml. The reaction mixtures were incubated for one hour at room temperature to allow the complete oxidation of ethanol and - then assayed for DPNH content by measuring their optical

density at 340 ~ against a blank solution containing the same - elements with the exception of substrate. The spectro- - . photometer was previously adjusted to zero OD'by reading the blank against itself. The optical densities tabulated in Table 5 were directly related to the original ethanol!

molarities in 3 ml. of reaction mixture; it was assumed that the ethanol had been completely oxidized to acetaldehyde, with the concomitant production of equimolar amounts of DPNH. Therefore, the standard curve related optical den- sities to the final concentrations of DPNH in the 3 ml. of reaction mixture, and was linear over the range of con- centrations tested (Fig. 3).

INHIBITION BY PHENELZINE WITH RESPECT TO ETHANOL:

One ~g. (protein) of YADH in 0.1 ml. was added to reaction mixtures containing 2.6 ml. of' medium; 0.1 ml. of 18 x 10-3 M DPN+ solutiqn; 0.1 ml. of ethanol solution in concentrations .. of 0.15, 0.30, 0.60, 1.50, 3.00 or 6.00 M; 0.1 ml. of •

DPNH CONCENTRATION IN THE MEDIUM (10-5 M)

O.~3 1 1.66 3.33 6.66 13.33

1 0.057 0.112 0.224 0.426 0.828

2 0.062 0.115 0.225 0.427 0.825

3 0.055 0.108 0.224 0.426 0.824

4 0.052 0.106 0.222 0.429 0.830

5 0.053 0.107 0.220 0.427 0.825

6 0.052 0.108 0.217 0.420 0.820

7 0.053 0.106 0.217 0.421 0.820

8 0.052 . 0.100 0.220. 0.427 0.824·

9 0.049 0.107 0.217 0.424 0.825

10 0.050 0.107 0.216 0.428 0.824

MEAN + STANDARD ERROR· 0.536 + 0.0 0.108 + 0.0 0.219 + 0.0 0.426 + 0.0 0.825 + 0.0

Table 5: Coordinate values for the standard curve re1ating.optica1 density to reduced.diphosphopyr~dine

nuc1eotide (DPNH) concentration. \Jl 0\ 57

0.9

0.8

0.7

0.6 > 1- ~ 0.5 w Cl -' 0.4 < ~ 1- Q.. o 0.3

0.2

0.1

o 2 4 6 8 10 . 12 14 CONCENTRA TlON OF DPNH . (10-5 Ml

Fig. 3: standard curve relating optical density to the concentration of reduced diphosphopyridine nucleotide (DPNH) • 58

phenelzine sulphatesolution in concentrations of 0.06, 0.30, or 0.60 M. The final· concentrations in 3 ml. of reaction mixture were 5, 10, 20, 50, 100 and 200 x 10-3 M for ethanol and 2, 10 and .20 x 10-3 M for phenelzine sulphate. Control systems,contained 0.1 ml. of distilled water instead of phenelzine sulphate solution.

" Inhibition'by Phenelzine with Respect to DPN+: One: jJ.g. (protein) of YADH in 0.1 ml. was added to reaction mixtures containing 2.6 ml. of medium;. 0.1 ml. of ,15 M ethanol; 0.1 ml. of DPN+ solution in.concefltrations of 1.5, 3.0, 6.0, 15.0 or 30.0 x 10-3 M; 0.1 ml. of phenelzine sulphate solution· in concentration's of' 0.06, 0.30 or 0.60 M. The final concentrations in 3 ml. were 0.05, 0.10, 0.20, 0.50, 1.0 and 2.0 x 10-3 M for DPN+, and 2, 10 and 20 x 10-3 M for phenelzine sulphate. Control systems contained 0.1 ml. of distilled water instead of phenelzine sulphate solution.

EFFECT OF PHENELZINE ON LIVER ALCOHOL DEHYDROGENASE (LADH) ACTIVITY: General Procedure: Incubation Medium: The incubation medium used for the de te rmination. of inhibition of LADH activity by phenelzine was a buffered solution consisting of 0.1 M sodium phosphate, pH 7.4; 0.05 M glycine; 0.15 M semi- ca.rbazide hydrochloride; 24 x 10-3 M nicotinamide. The 59

medium was prepared as ~ollows. Monobasic potassium • phosphate (68.05 g./L.) and dibasic sodium phosphate (89.05 g./L.) were made up separatel~ as 0.5 M solutions in distilled water. Glycine (..3.754 g.) and nicotinamide (1.832., Sigma Chemical Co., st. Louis Mo., U.S.A.) were ..

dissolved in a solution o~ 160 ml. o~ 0.5 M potassium

phosphate and 40 ml. o~ 0.5 M sodium phosphate. Nicotin­ amide was added to the medium to inhibit enzymes which inactivate DPN+. Semicarbazide hydrochloride (16.670 g.)

was dissqlved in 100 ml. o~ distilledwater and the strongly

acid solution (PH 1.9) neutrali~ed with 2N sodium hydroxide

be~ore it was added to the phosphate-glycine solution. The pH of the medium was adjusted to 7.4 with 2N sodium hydroxide and the volume made up to one liter with distilled water. The pH of the medium was measured with a pH meter before use each time and readjusted when necessary to pH 7.4 with 2N sodium hydroxide.

Liver Alcohol Dehydrogenase: The soluble enzyme fraction of mouse liver, containing LADH, was prepared by

the ~ollowing procedure. Livers ~rom five mice were removed and washed in cold 0.15 M potassium chloride, dried on

absorbent paper and weighed. Three volumes o~ cold 0.1 M

phosphate bu~fer pH 7.4, containing 0.05 M glycine to stabilize the enzyme preparation, were added to the livers, ,fi and the mixture homogenized in a hand-driven glass homo- 6~

genizer. One volume was equivalent to the total gram weight • or the li vers in milliliters. Two 10 ml. aliquots or homogenate were centriruged ror ten minutes at 30,000 g in a rerrigerated International centriruge and the supernatants combined. Three 5 ml. aliquots were taken from the com- bined supernatant and centrifuged for onehour at 100,000 g in a refrigerated Spinco ultracentrifuge. The final supernatant was devoid or most particulate matter and contained the soluble enzyme fraction. Quantities of supernatant sufficient ror future needs could not be

refrigerated because preliminary experiments ~howed that liver alcohol dehydrogenase activity rapidly declined when o the supernatants were frozen and stored at -10 C for more than three hours. Lyophilization (freeze-drying) of the enzyme preparation did, however, permit its storage for extended periods of time without significant loss of activity. To prepare a freeze-dried enzyme preparation containing LADH, 100,000 g supernatants from rour groups containing rive mice were pooled and 4 ml. aliquots lyophilized simultaneously in a freeze-drying apparatus (Precision Scientific Co., Chicago, U.S.A.). The freeze-dried soluble protein fraction o was stored at -10 C and, when required, reconstituted to its original concentration in the supernatant with distilled water. After storage for two weeks, the alcohol dehydrogenase activity of enzyme preparations treated in this manner had not decreased. 61

~-Diphosphopyridine Nucleotide (DPN+): There was .' insu~~icient DPN+ in the supernatantto permit the oxidation

o~ ethanol by LADH in vitro, necessitating the addition o~ 0.1 ml. o~ DPN+ solution to the "incubation medium. The DPN+ solution was prepared asdescr1bed ~or the assay o~ yeast alcohol dehydrogenase activity.

Protein Determination: The total protein content

o~ individual reaction mixtures was determined by the biuret

method o~ Kabat and Meyer (1967).

Measurement o~ LADH Activity: The rate at which ethanol was oxidized by LADH in control and experimental

reaction systems was determined by measuring the rate o~

~ormation o~ DPNH at 340 ~ in a Beckman DB-G spectrophoto­

meter, using a quartz cuvette with a light path o~ l cm. The changes in the optical densities were recorded on paper by a Beckman ten inch pen recorder. Unlike the maximum

reaction rate observed almost immediately a~ter the ad­ dition of yeast alcohol dehydrogenase, there was a striking lack of activity after the addition of LADH to control and experimental reaction systems. The reaction rate increased slowly until a constant and maximum velocity was achieved at approximately four minutes. Because this velocity re-

mained linear for about twenty minutes, aIl measurements o~ changes in optical densities were made between six and ten '\ 62

minutes after the addition of LADH. The changes in optical

• density per minute were convertedto velocities (~M DPNH/mg. protein/min.) by the formula:

Velocity = change in OD per minute x 3 6.12 x mg. prote in in the reaction'

standard Curve:. 'The standard curve prepared for the assay of yeast alcohol dehydrogenase activity (Fig. 3) was used for the conversion of optical density to DPNH con­ centrations and the slope of this regressiori used to determine velocities.

Inhibition by Phenelzine with Respect to Ethanol:

The inhibition of LADH activity by phenelzine with respect to substrate was determined by two procedures. Procedure 1: Reconstituted LADH preparation in a volume of 0.3 ml. was added to a reaction mixture containing; 2.4 ml. of medium; 0.1 ml. of 30 x 10-3 M DPN+; 0.1 ml. of ethanol in concentrations of 15, 60, 75 or 150 x 10-3 M; 0.1 ml. of 3.0 x 10-3 M phenelzine sulphate. Control and experimental reaction systems were distinct from each other, the controls containing 0.1 ml. of distilled water instead of phenelzine sulphate solution. The final concentrations in 3 ml. were; 0.5, 1.0, 2.5 and 5.0 x 10-3 M for ethanol; 0.10 x 10-3 M for phenelzine sulphate. Enzyme activities were measured between six and ten • minutes arter initiation or the reaction and the linear change in optical density over the rour minutes converted to the mean change over one minute. Procedure 2: This procedure was devised to reduce the total amount or enzyme preparation required ror an assay series. Reconstituted LADH preparation in a volume or 0.3 ml. was added'to reaction mixtures similar to those used above, with the exception that inhibitor had not been added. The reaction rates in these systems between six and eight minutes were taken as the control activities. Phenelzine sulphate 3 solution, in concentrations or 3.0 or 6.0 x 10- M, WàS·" added to these reaction mixtures at the eight minute interval and the activity measured until ten minutes. The mean change in optical density per minute was calculated ror the two minute control interval and ror the two minute interval during which phenelzine was present in the reaction mixture.

Inhibition by Phenelzine with Respect to DPN+: The inhibition or LADH by phenelzine with respect to the coenzyme was determined by the protocol described under Procedure 2 in the preceding section. Reconstituted LADH preparation in a volume or 0.3 ml. was added to reaction mixtures containing; 2.4 ml. or medium; 0.1 ml. or 0.45 M ethanol; 0.1 ml. or DPN+ solution in concentrations or 1.5, 3.0, 7.5 or 15.0 x 10-3 M. Phenelzine sulphat:e solution in 63

Enzyme activities were measured petween six and ten • minutes after initiation of the reaction and the linear change in optical density over the four minutes converted tothe mean change over one minute. Procedure 2: This procedure was devised to reduce the total amount of enzyme preparation required for an assay series. Reconstituted LADH preparation in a volume of 0.3 ml. was added'to reaction mixtures similar to those used above, with the exception that inhibitor had not been added. The reaction rates in these systems between six and eight minutes were taken as the control activities. Phenelzine sulphate so lution, in concentrations of 3.0 or 6.0 x 10-3 M, was:- added to these reaction mixtures at the eight minute interval and the activity measured until ten minutes. The mean change in optical density per minute was calculated for the two minute control interval and for the two minute interval during which phenelzine was present in the reaction mixture.

Inhibition by Phenelzine with Respect to DPN+: The inhibition of LADH by phenelzine with respect to the coenzyme was determined by the protocol described under Procedure 2 in the preceding section. Reconstituted LADH preparation in a volume of 0.3 ml. was added to reaction mixtures containing; 2.4 ml. of medium; 0.1 ml. of 0.45 M ethanolj 0.1 ml. of DPN+ solution in concentrations of 1.5, 3.0, 7.5 or 15.0 x 10-3 M. Phenelzine sulphat:e solution in 64

--- , • concentrations of 3.0 or 6.0 x 10-3 M was added t~ the reaction mixture eight minutes later, after the control activity had been measured. Activity in the presence of inhibitor was measured until the ten minute interval and the mean changes per minute in optical density calculated for each two minute interval.

STATISTICS: Standard errors, and regression lines fitted by the method of least squares,were calculated by the methods of Steele and Torrie (1960). Unless specified otherw1se, aIl measures of variability shown in this report are the standard error of the mean. The difference.· between rneans and between regressions were tested for statistical significance withthe appropriate Student's t-tests. Where necessary, details of the statistical procedures and/or mathematics employed have been described in the results. AlI mathematical operations were programmed on magnetic cards and performed by an Olivetti-Underwood Programma 101 desk computer. 65

. -- ,, • RESULTS THE EFFECT OF PHENELZINE ON MOUSE BEHAVIOR: One half hour after an intraperitoneal injection of phenelzine, physical and behavioral changes were apparent in individually caged mice. Exophthalmos and the Straub tail response (pronounced ·spasm:j of the muscles around the tail so that the tails of the mice are raised in a charaeteristic s-shaped formj Straub, 19l1) were observed in some mice and exploratory and grooming behavior was markedly diminished in Most. The appearance of mice on the floor of the cage resembled the "start relex" observed in dogs treated with morphine. "The animals were braced as if' to spring into action but maintained this position without completing the moti on. " (Forney et al, 1963). The mice would also remain totally immobile for minutes while standing on their hindlegs with forelegs stretched up the walls of the cages or while hanging suspended on the wall. These states of immobility were punctuated by bursts of' hyperactivity in response to sudden noises nearby, movements of' the cage or blowing on the mouse. Righting reflex was not lost and no convulsions or deaths occurred during the one half' hour of pretreatment with phenelzine.

EFFECT OF PHENELZINE ON ETHANOL SLEEPING TIME: The problem of a pharmacological interaction between phenelzine and ethanol was approached by determining the 66

effect of phenelzine pretreatment on the length of ethanol • induced sleep. Control and experimental mice were treated as described in the section on methods and the data obtained has been tabulated in Fig. 4 as the number of mice awakening 1 within successive ten minute intervals after the loss of righting reflex. Fifty of seventy-five control mice re- gained their righting reflex within thirty minutes and aIl but nine had recovered within one hour. The mean sleeping time for seventy-five mice in the control group was 27 ± 2 minutes. A righting reflex was exhibited by only seven of seventy-two phenelzine pretreated mice with1n the same one ha If hour period and forty-:-:five mice remained asleep at one hour. The potentiating effect of phenelzine was further illustrated by a marked prolongation of a maximum individual duration of hypnos1s from a control value of 96 minutes to 385 minutes in a phenelzine pretreated mouse. The mean ethanol sleeping time for seventy-two phenelzine pretreated mice was 98 + 7 minutes. This mean was significantly dif-

ferent from the control mean at P< 0.001. However J the validity of this comparison is questionable because of the bias accorded the experimental mean by the small number of very long sleeping times wh1ch were included. A more lucid demonstration of the effect of phenelzine pretreatment on ethanol induced sleeping times was obta1ned by plotting the per cent m1ce asleep within a given ten minute interval against t1me (Fig. 5). This method revealed 67

24 ...

22 ~

~20 :!(. , ~ 18 w - Z 16 w 14 ~ ~ 12 ~10 - - CONTR Ol ~ 8 ~ ~ 6 C> 4 ,,"""r- Z Z 2 ~ w !It! nn ~ ~ 10 PHENELZINE w 8 U PRETREATED ~ 6 o 4 d 2 Z

o 30 60 90 120 180 240 300 360 420 SLEEPING TIME (Minutes)

Fig. 4: The e~~ect o~ phenelzine pretreatment on the length

o~ ethanol induced sleep: a histogram illustrating

the number o~ mice awakening during any given ten

minute interval a~ter the loss o~ righting re~lex, 68

100

80

Q. 60 w w ~ IJ) ~ w u :E 40 I~

20

60 TlME (m inutes) /

Fig. 5: The effect of phenelzine pretreatment on the length of ethanol induced sleep: a plot illustrating the percentage number of mice asleep during any given ten minute interval after the loss of righting reflex. 69

.....,

. ) •.~ ! that the time of awakening observed in both groups followed a curvilinear pattern and clearly demonstrated the tendency

~or experimental mice to sleep for longer periods of time. The possibility existed that the increased sleeping times observed in mice pretreated with phenelzine might have resulted from potentiation of the depressant effect of ethanol on the central nervous system, i.e., a given ethanol con­ centration in the mouse would have induced a.deeper sleep than it could have in the absence of phenelzine. If this were the case, experimental mice should remain asleep at body ethanol concentrations lower than the control "threshold"

value, below which aIl control mice are awake. This hypo-'~ thesis was tested by determining the total ethanol content of six control and nine phenelzine pretreated mice upon their awakening. The method for the assay of ethanol in the total mouse is described in the section on methods. A summary of sleeping times and ethanol concentrations in these mice is shown in Table 6. The lowest concentration of ethanol found in control mice upon awakening was 2.98 g.Jkg. and the Mean for these six mice was 3.20 + 0.04 g.Jkg. Ethanol concentrations in phenelzine pretreatedmice fell as low as 1.89 g.Jkg. before the righting reflex was regained and the mean for these nine mice was 2.47 ± 0.14 g.Jkg. The difference between control and experimental means was significant at P

GROUP SLEEPING TIME ETHANOL CONCENTRATION

minutes % RECOVERY g./kg.

9 96 3.35 30 94 3.29 "38 92 3.22 CONTROLS 46 89 3.12 46 93 3.25 56 85 2.98

37 80 3.12 41 78 3.04 116 64 2.49 PHENELZINE 131 69 2.70 PRETREATED 161 65 2.51 190 62 2.41 220 49 1.89 311 54 2.10 385 51 1.99

Table 6: The effect of phene1zine pretreatment on body con- centrations of ethano1 in mice awakening from ethano1 induced sleep.

Ethanol concentrations upon awakening in control and

phene1zine pretreated mice are shown as per cent recovery

and as calcu1ated equivalent in g./kg. The values are • not means and therefore have no .standard errors. 71

EFFECT OF PHENELZlNE ON THE LD 50 OF ETHANOL: The prolongation of ethanol induced sleeping time in mice by phenelzine pretreatment suggested that the depressant effects of ethanol had been potentiated or prolonged. 'The acute toxicity of ethanol was studied in control and experi­ mental mice to determine if phenelzine a1tered the lethal action of ethanol. The percentage mortality obtained with a given dose of ethanol was a measure of the number of mice with a tolerance to the 'lethal effect of ethano1 less than the amount administered. The effect',;'of phenelzine on this tolerance was analysed by comparing the LD 50 values for ethanol in control and experimental mice. The LD 50 is the calculated dose of ethanol required to kill 50% of the animaIs. The treatment procedures are described in the section on methods. The mice were observed for a twenty-four hour period after treatment and the number of animaIs dying in each dose interval during this time were counted and expressed as per cent dead. Table 7 contains a summary of the ethanol dose intervals and the corresponding per cent dead. A plot of the per cent dead in control and experimental groups against the log-dose of ethanol produced the sigmoid-like curves representative of this type of study (Fig. 6). Contrary to expectation based,largely on the effect of phenelzine on,the sleeping time, a decrease in ethanol toxicity occurred after phenelzine pretreatment. This was indicated by a shift of the log-dose response curve for .~~ .-'~ . • ~.

GROUP LOG DOSE DEAn /TESTED . PER CENT DEAD PROBIT RESPONSE %

3.75 0.574 o / 10 0 4.25 0.628 . 5 / 10 50 5.000 4.50 0.653 7 / 10 70 5.524 4.75 0.677 7 / 10 70 5.524 5.00 0.699 8/ 10 80 5.842 CONTROLS 5.25 0.720 9 / 10 90 6.282 5.75 0.760 9 / 10 90 6.282 6.00 0.778 10 /10 100 4.00 0.602 o / 10 0 4.25 0.628 1 / 10 10 3.718 4.50 0.653 1 / 10 10 3.718 4.75 0.677 4 / 10 40 4.747 PHENELZINE 5.00 0.699 4 / 10 40 4.747 PRETREATED 5.25 0.720 5 / 10 50 5.000 5.50 0.740 6 / 10 60 5.253 5.75 .0.760 8 / 10 80 5.842 6.00 0.778 7 / 8 87 6.126 6.50 0.813 10 / 10 100

------~~_ .. ---~-_._---~_. -

Table 7: The effect of phene1zine pretreatment ori the morta1ity rate in mice at varying dosage

1eve1s of ethano1.

The morta1ity rate among control and pherie1zine pretreatedmice is expressed as

per cent dead and as a probabi1ity unit of per cent death (probit). The probit

values were derived from tables in the paper pub1ished by Litchfie1d and Wi1coxin ~ro (1949). There is no probit value for zero or 100% response. 73

100

80

60

CI) l: CONTROL

0·5 0·6 0'7 0·8 LOG DOSE ETHANOL

/

Fig. 6: The effect of phenelzine pretreatment on the LD 50 of ethanol:- a plot of per cent response against the log dose of ethanol. 74 • ethanol to the right. The slcpes of the curve for control mice and the curve for phenalzine pretreated mice were not noticeably different, suggesting no change had occurred in the mechanism of toxicity. The LD 50's were determined from Fig. 6 by obtaining. the log-doses of ethanol corresponding to the ordinates for a 50% response on control and experimental curves. The antilogs of these values were 4.25 g./kg. for the control group and 5.16 g./kg. for phenelzine pretreated animaIs. More accurate estimates of the LD 50's, taking into account experimental variation, were calculated by the.method of Litchfield and Wilcoxin (1949) by plotting probability units of per cent response (probit response) against the log-dose of ethanol (Fig. 7). The LD 50's and confidence intervals derived by the probit response method were 4.14 + 0.43 g./kg. for controls and 5.16 + 0.25 g./kg. for experimental animaIs. The results obtained by the two methods were almost identical.

EFFECT OF PHENELZINE ON THE DISAPPEARANCE OF INJECTED ETHANOL: The hydrazine monoamine oxidase inhibitors can inhibit the hepatic metabolism of a number of drugs (Laroche and Brodie, 1960j Kato et al, 1962; Clark, 1967). An inhibition of the oxidation of ethanol in the liver would be a possible explanation for the observed prolongation of ethanol induced sleep by phenelzine. This hyp·othesis was initially tested 75

------. ------~------I

7

• 6

PHENELZINE CI) :t PRETREATED 1- ~ &LI Q 5 ~

LI.. 0 0

l- ca 0 ~ Go 4

0.60 0.65 0.70 0.75 0.80 LOG DOSE ETHANOL

Fig. 7: The effect of phenelzine pretreatment on the LD 50 of ethanol: -a plot of probit response against the log dose of ethanol. 76

by determining the e~~ect o~ phenelzine pretreatment on the • rate at which injected ethanol disappeared ~rom the total mouse. For the purposes o~ these experiments, the dis-

appearance o~ ethanol was assumed to be due entirely to metabolic processes. Control and experimental mice, treated in the usual manner, were killed at predetermined time intervals and

assayed ~or their total body content o~ ethanol as described

in the section on methods. A summary o~ the time intervals

and the related mean per cent recoveries o~ ethanol is shown

~or both groups in Table. 8. A plot o~ per c~nt recovery o~

ethanol against time (Fig. 8) demonstrated that the rate o~

degradation was linear ~or both groups, but there were in­ dications that at ethanol concentrations below 0.35 g./kg. (10% recovery) an exponential pattern might have prevailed.

However, the method used ~or quantitating ethanol concent­ rations in the total mouse proved to be too insensitive to provide reliable estimates in this range.

Rates o~ ethanol metabolism, in g./kg./hour, were

equivalent to the slopes o~ the calculated regression lines in Fig. 8. The change in concentration between one hour

and ~our hours a~ter the administration o~ ethanol was

determined ~rom the graph, in g./kg., and converted to the . rate o~ change in total body ethanol in g./kg./hour. Ethariol

metabolism was reduced by phenelzine pretreatment ~rom 0.55 ~ Fë?' g./kg./hour to 0.38 g./kg./hour. A statistical comparison IVA

GROUP TIME AFrER THE INJECTION OF ETHANOL

o min. 15 min. 30 min. 1 hr. 2 hr. 3 hr. 4 hr. 6 hr 12 hr.

% Recovery 100 + 1 96 + 2 91 + 3 83 + 2 72 + 5 52 + 2 41 + 2 4 + 1 3 + 0 CONTROL g./kg. 3.50 3.35 3.18 2.90 2.52 1.82 1.43 0.14 0.10

PHENELZINE % Recovery 100 + 1 97 + 2 94 + 2 88 + 2 77 + 2 74 + 2 63 + 5 28 + 3 4 + 1 PRETREATED g./kg. 3.50 3.39 3.29 3.08 2.69 2.59 2.20 0.98 0.14 .-

Table 8: The effect of phene1zine pretr~atment on the in vivo disappearance of injected ethanol.

The amount of ethanol rema:ining in control and phenelzine pretreated'mice at various

time intervals after its injection is shown as a pe.r cent recovery value and as a

calculated equiva1ent in g./kg. The values given in g./kg. are not means and therefore

have no standard errors. -.;J -.;J 78 '.

0""" z ~ % tu &&.; 60 0 PHENELZINE t lU ~ ,~ 40 1 ~ i " <-11

1 20 1

1 2 3 4 5 6 liME (HfS.)

. ..-",.-

Fig. 8: The ef'f'ect of' phenelzine'pretreatment on the dis- ,- appearance of' ethanol in vivo. 79

of the regression slopes showed that the difference was • significant at P< 0.01.

EFFECT OF PHENELZINE ON ACETALDEHYDE CONCENTRATIONS: The results of the previous experiment. showed that phenelzine interfered with the metabolism of ethanol. To further elucidate the metabolic step or steps involved, the effect of phenelzine pretreatment on acetaldehyde concentrat­ ions was studied in control and experimental mice treated in the usual manner. A decrease in acetaldehyde concentration would be construed as reflecting an inhibition of the oxidation of ethanol to acetaldehyde, and an increase as a reflection of inhibition of the oxidation of acetaldehyde to acetic acid. The relevant metabolic pathways involved are detailed in Fig. 9. AnimaIs were assayed for total body content of acetaldehyde at 0, 15, 30, 60 and 120 minutes after receiving ethanol. The detailed procedure is described in the section on methods. The results obtained have been tabulated in Table 9 and graphically represented in Fig. 10. The results demonstrate that twenty minutes after the admin­ istration of ethanol, acetaldehyde concentrations had in­ creased in the control mice to a maximum of 0.26 rng.% and then declined almost linearly to 0.17 mg.% at two hours. Phenelzine pretreatment decreased the maximum rise at twenty

minutes to 0.20 mg.% and, subsequent to a slight decre~se to .4. 0.17 mg.% between thirty and forty-five minutes, maintained 80

METABOLISM Of ETHANOL

ETHANOL DPN

Alcohol Dehydrogenase DPN·H + H+

ACETALDEHYDE

+ H+ ACETIC ACID

ACETYL CoA

Fig. 9: The metabolism of ethanol. •

GROUP TIME AFrER THE INJECTION OF ETHANOL (minutes)

0 15 30 60 120

CONTROL 0.108 + 0.01 0.25 + 0.02 0.25 + 0.02 0.23+ 0.02 0.17 + 0.01

PHENELZINE 0.13 + 0.01 0.20 + 0.02 0.17 +0.01' 0.17 +0.02 0.19 + 0.01 PRETREATED

Table 9: The effect of pherie1zine pretreatment on aceta1dehyde concentration in mice

after injection of ethano1.

Acetaldehyde concentration, in mg. %, is shawn for control and phene1zine

pretreated mice.

Cl) ~ 82

0-26

0-22

~- C) 0-18 E - PHENELZINE w Cl PRETREATED >- J: w Cl ~ 0-14

1-« w U«

0-10

0-5 1-0 1-5 2-0 2-5 TlME (Hrs.)

/

Fig. 10: The effect of phene1zine pretreatment on acetaldehyd~ concentrations in mice given ethano1. 83

.~ this concentration within limits over the remainder of the two hour interval during which the experiment was conducted. The slope of the experimental curve at the two hour interval indicates a tendency towards increasing acetaldehyde con­

centrations at times beyond two hours~ but this was not confirmed experimentally. A statistically significant difference between acetaldehyde concentrations in control and phenelzine pretreated mice occurred at thirty minutes only. •

, EFFECT OF PHENELZlNE ON YEAST ALCOHOL DEHYDROGENASE (YADH) ACTIVITY: The alterations in time-concentration patterns for ethanol and acetaldehyde led to the speculation that phenelzine was" interfering with the degradation of ethanol at the level of alcohol dehydrogenase (see Fig. 9). To determine the precise

mechanism of action~ a reaction system as free of as Many variables as possible was necessary. This was achieved by using purified yeast alcohol dehydrogenase (YADH) in an in vitro system. In the presence of a constant amount of enzyme, variables determining the reaction rate were the relative

concentrations of substrate (ethanol)~ coenzyme ,B-diphospho­ pyridine nucleotide (DPN),+ and inhibitor (phenelzine). The experiments were designed to determine the inhj.bitory effect . . of phenelzine with respect to both substrate and coenzyme. ~ This.was accomplished by measuring the control activity of 84

'. the enzyme in the presence of' varying concentrations of' either substrate or coenzyme and comparing the control value to the enzyme activity in a similar system containing phenelzine. Enzyme activity was def'ined as the velocity at which ethanol was oxidized in the f'irst minute of the reaction, and it was measured by assaying, as described in the methods, the rate of' f'ormation of' reduced DPN+ (DPNH).

The velocity was expressed in ~M DPNH/mg. protein/minute.

Inhibition by Phenelzine with Respect to Ethanol: The data obtained for the inhibitory eff'ect of' phenel­ zine with respect to substrate is summarized in Table 10. Details of the procedure are described in the section on . . --- + methods. When concentrations of' YADH (1 ~ g. ), DPN (0.6 x 10-3 M) and ethanol (5 x 10-3 M) were held constant, a 2 x 10-3 M concentration of phenelzine in the medium pro­ duced 28% inhibition of' the reaction (Fig. Il). The degree of' inhibition was increased to 56% and 82% with 10 x 10-3 M and 20 x 10-3 M concentrations of' phenelzine respectively. Increasing the concentration of' ethanol diminished the inhibitory eff'ect in each instance. The kinetic nature of the inhibition was determined by analysing the data by the method of' Lineweaver and Burk (1934) and the results are shown in Fig. 12 as a plot of reciprocals of reaction velocities (l/v) against reciprocals of substrate • concentrations (1/[8]). The inhibition was determined to be lf\ co

ETHANOL CONCENTRATION IN THE MEDIUM (10-3 M)

5 10 20 50 100 200

CONTROL 56.7 + 5.7 70.1+ 3.9 89.6 + 2.9 116.9 + 7.3 136.1 + 5.8 144.3 + 8.4 PHENELZlNE- 2mM 4104 + 2.4 57.6 + 2.7 79.9 + 2.0 104.5 + 1.9 125.2 + 4.8 145.9 + 11.5

CONTROL 56.7+ 5.7 70.1 + 3.9 89.6 + 2.9 116.9 + 7.3 136.1 + 518 144.3 + 8.4 PHENELZINE - 10 mM 25.5 + 1.0 41.4 + 1. 7 62.3 + 1.9 90.7 + 2.2 90.6 + 17.9 122.9 + 4.8

CONTROL 56.7 + 5.7 70.1 + 3.9 89.6 + 2.9 116.9 + 7.3 136.1 + 5.8 144.3 + 8.4 PHENELZINE - 20 mM 9.9 + 0.3 21.5 + 1.3 38.2 + 1.1 61.1 + 1.3 82.6 + 3.1 90.4 + 1.6

Table 10: The effect of phene1zine concentration on the in vitro activity of yeast a1coho1

dehydrogenase, with respect to substrate (ethano1) concentration.

The activity of control and phenelzine inhibited reaction systems is expressed as

the velo city at which DPN!i was formed, in llM DPNH/mg. protein/min. Ethanol and -:.

phene1zine concentrations were shown in the table.

CP \J1 . '--'~ •. . • 86

100

> ,-t- ~ t- 80 ~ J: C ~ U- 0 60 ~ Z 0 t- ca J: Z PHENELZINE 40 ~ 20 mM

20 . 10 mM

2 mM 0~------5~~10~-2~0~--~50~~.1~00~·~2~00~----­ 3 LOG CONCENTRATION ETHANOL (10- Ml

Fig. 11: The per cent inhibition o~ yeast alcohol dehydro­ genase activity, with respect to substrate (ethanol) concentration, by 2, 10, and 20 mM phenelzine. 87

l 100 20mM

90 ."

80 3 _1 X· 10- V 70

60

50

40 10mM

30 2mM

20 ...... ::CONJRO.L ...... ~ ,...... •...... •. .~!!'" ~...-.:::-;-....'-

o 20 40 60 80 100 200 1 ~_~-1 [ETHANOL] > ,

Fig. 12: The competitive inhibition of yeast a1coho1 dehydro­

genase activity~ with respect to substrate (ethano1)

concentration~ by 2, 10 and 20 mM phenelzine:

I~ Lineweaver-Burk plot. ~fJ'·, ,. 88

'.' competi ti ve or non-competi ti ve by comparison or the calculated Km values (Km is the substrate concentration required to drive the reaction at half maximum velocity) and maximum velocities (Vmax) ror control and inhibited systems. A decrease in Vmax alone indicated non-competitive inhibition; a ,increase in Km alone was indicative or competitive inhibition. The following equations were used to calculate the Km and Vmax:

Vmax = lia Km =Vrnax x b

where "a" is the y-intercept and lib" the slope of the regression. For compar1son purposes, the data were analysed by the method of Woolf (as describedby D1xon and Webb, 1958) and the results shown in Fig. 13 as a plot of the ratio or substrate con­

centration to velocity (~]/v) against substrate concentration. The Km and Vrnax values were çalculated using the following equations:

Vrnax = lib

Km = Vmax x a

The values for Km and Vmax obtained by the Lineweaver - Burk and Woolf methods are summarized in Tables Il and 12 respectively. Phenelzine increased the Km values for ethanol up to ten times the control figure without significant change in the Vmax, indicating competitive inhibition of yeast alcohol i., 89 24

22 20 mM

20

18

10 mM 16 [ETHANOL] x 10~ 2 mM V 14 CONTROL

. 12

10

8

6

o 20 40 60 80 100 200

[ETHANOL] x 10 -3 M

Fig. 13: The competitive inhibition of yeast alcohol dehydro­ genase activity, with respect to substrate (ethanol) concentration, by 2, 10 and 20 mM phenelzine: Woolf plot. • •

CONSTANT (a) SLOPE (b) Vmax (uM/mg./min.) Km (10-3 M)

CONTROL 7.50 0.058 133 ' 7.7 PHENELZINE - 2mM 7.59 0.088 132 11.6

CONTROL 7.50 0.058 133 7.7 PHENELZINE - 10 mM 7.86 0.159 127 20.2

CONTROL 7.50 0.058 133 7.7 PHENELZINE - 20 mM 6.09 0.464 164 76.2

Table Il: The effect of pnene1zine concentration on the Km v~lue for ethano1, and on the Vmax,

in reaction systems using yeast a1coho1 dehydrogenase: Lineweaver-Burk method.

The values for Km and Vmax were ca1cu1ated by substituting the values shown for the constants and slopes in equations which are detai1ed in the texte

\0 o • •

CONSTANT (a) SLOPE (b) Vmax (uM/mg./min.) Km (10-3 M) 1

CONTROL 80.9 6.65 150 12.2 PHENELZINE - 2mM 119.3 6.58 152 18.1

CONTROL 80.9 6.65- 150 12.2 PHENELZINE - 10 mM 173.3 7.39 135 23.5

CONTROL 80.9 6.65 150 12.2 PHENLZINE - 20 mM 384.4 8.97 111 42.8

Table 12: The effect of phene1zine concentration on the Km value for ethano1, and on the Vmax, in reaction systems using yeast a1coho1 dehydrogenase: Woolf plot.

The values for Km and Vmax-were ca1cu1ated by substituting the values shoWn for

the slopes and constants in equations whichare detailed in the texte

\0 1-' 92

, ,

- "~ dehydrogenase with respect to ethanol. '.':-

Inhibition by Phenelzine with Respect to DPN+:

The data obtained ~or the inhibitory e~~ect o~ phenelzine

with respect to coenzyme (DP~) is summarized in Table 13. The procëdures used have been described in the section on

methods. When concentrations o~, YADH (11.L g.), ethanol (0.6 M) and DPN+ (5 x 10-5 M) were held constant, a 2 x 10-3 M concentration of phenelzine in the medium had no apparent e~~ect on enzyme activity. Ten x 10-3 M and 20 x 10-3 M phenelzine inhibited activity 38% and 45% respectively (Fig. 14).

Increased amounts o~ DPN in the reaction systems reduced in-

hibition in both cases. The values ~or Km and vrnax calculated

by methods o~ Lineweaver - Burk and Wool~ are summarized in Tables 14 and 15 respectively, and the graphie representations o~ these results are shown in Figures 15 and 16. Ten x 10-3 M -3 and 20 x 10 M phenelzine increased the Km ~or ethanol up to

two or three times the control value without signi~icantly

altering the Vmax, indicating competitive inhibition o~ the enzyme with respect to coenzyme. -3 The e~~ect of 2 x 10 M phenelzine on YADH activity

di~~ered from those obtained with the other concentration o~ phenelzine in that an increase in the Vmax was indicated. The nature of the Lineweaver - Burk and Woolf plots suggested that if this concentration of phenelzine had any effect, it

'.' was not inhibitory. • •

DPN+ CONCENTRATION IN THE MEDIUM (10-5 M)

5 10 20 50 100 200

CONTROL 28.8 + 0.7 50.6 + 0.9 88.5 + 2.9 131.9 + 5·.3 169.0 + 4.0 197.0 + 2.0 PHENELZINE - 2mM 32.6 + 3.7 51. 3 + 2.7 88.0 + 2.3 150.4 + 6.1 187.0 + 8.0 239.0 + 8.0 CONTROL 26.1 + 0.3 50.2 + 2.3 81.5 + 3.0 129.8 + 8.5 173.9 + 7.5 215.1 + 15.2 PHENELZINE - 10 mM 21.1 + 1.6 36.8 + 3.6 67.7 + 3.1 123.0 + 6.6 161.9 + 7.6 190.4 + 9.3 CONTROL 26.6 + 1.0 51.9 + 0.9 85.0 + 3.0 139.1 + 2.9 184.9 +5.1 208.7 + 5.7 PHENELZINE - 20 mM 14.8 + 0.9 31.3 + 1.6 56.5 + 2.1 99.3 + 2.8 148.8 + 6.5 188.3 + 7.0

Table 13: The effect of phene1zine concentration on the in vitro activity of yeast a1coho1 dehydrogenase,

with respect to coenzyme {DP~) concentration.

The activity of control and phene1zine inhibited reaction systems is expressed as the ve10city

at which DPNH was formed in llM DPNH/mg. protein/min. Ethanol and phene1zine concentrations

were as shown in the table.

\D VJ 94

50

40

>- ~ ~ t- U < 30 ::I: 0 <>- u.. 0 Z 20 0 co~ ::I: Z PHENELZINE ~ 10 20mM

10 mM

o 5 10 20 50 100' 200 LOG CONCENTRATION OF DPN+

Fig. 14: The per cent inhibition of yeast a1coho1 dehydro­ genase activity, with respect to coenzyme (DPN+) concentration, by10 and 20 mM phene1zine.

l':" • {''i

CONSTANT (a) SLOPE (b) Vmax (pM/mg. /min. ) Km (10-3 M)

CONTROL 4.43 1.51 226 0.34

PRENELZINE - 2 mM 4.01 1.46 249 0.36

CONTROL 3.96 1. 70 253 0.43 PHENELZINE - 10 mM 4.17 2.28 240 0.55

CONTROL 3.64 1.68 275- 0.46

PHENELZINE - 20 mM 2.89 3.22 346 . 1.11 . -

.+ Table 14: The effect of phene1zine concentration on the Km value forDPN ; and on: the Vmax,

in reaction systems using yeasta1cohol dehydrogenase: Lineweaver-Burk plot.

The values for Km- and Vmaxwere ca1cù1ated bysubstituting the values shown

for the constants ands10pes in equations which are deta!led in the teXte

\0 \Jl ' '1 • •

CONSTANT (a) SLOPE (b) Vmax (tiM/mg./min.) Km (10-3 M)

CONTROL 1.54 4.33 230 0.36 PHENELZINE - 2mM 1.62 3.47 287 0.47

CONTROL 1. 76 3.93 254 0.45 PHENELZINE - 10 mM 2.23 4.14 241 0.54

CONTROL 1.60 3.97 251 0.40 PHENELZINE - 20 mM 3.01 3.83 261 0.79 - --- -

Table 15: The effect of phene1zine concentration on the Km value for DPW, and on the Vmax,

in reaction systems using yeast a1coho1 dehydrogenase: Woolf plot.

Those values for Km and Vmaxwere ca1culated by substituting the values

shawn for the slopes and constants in equations which are detai1ed in the texte

\.D 0\ 97

:~ ,

7 20 mM

6

5 10 mM _1 x 10-2 V 4 CONTROL 2 mM 3

2

1

o 2 4 6 810 20

Fig. 15: The competitive inhibition of yeast alcohol dehydrogenase activity, with respect to coenzyme (DPN+) concentration, by 2, 10 and 20 mM phenelzine: Lineweaver-Burk plot. 98 !e 12

20 mM 10mM 10

2 mM 8 [oPN+] V

6

o 0.2 0.4 0.6 0.8 1.0 2.0 [OPN+] x 10-3 M.

,.,:.-,.

Fig. 16: The competitive inhibition of yeast alcohol dehydrogenase activity, with respect to coenzyme

(DPN+) concentration, by 2, 10 and 20 mM phenelzine: Woolf plot. 99

• EFFECT OF PHENELZINE ON LlVER ALCOHOL DEHYDROGENASE (LADH) ACTIVITY: The experimental methods used to determine the inhibitory effect of phenelzine on liver alcohol dehydrogenase activity have been described. The results were analysed by the methods of Lineweaver and Burk (1934), and Woolf (in Dixon and Webb, 1958), as previously outlined.

Inhibition by Phenelzine with Respect to Ethanol: Procedure 1: The inhibitory effect of phenelzine added to the reaction mix9~re before the enzyme, was measured for the time interval between six and ten minutes after initiation of

the reaction. The changes in optical density at 340 ~ for control and phenelzine inhibited reaction systems were converted to reaction velocities and tabulated in Table 16. These results are expressed graphically by the Lineweaver-Burk and Woolf methods in Figures 17A and 17B. The rate of ethanol oxidation by LADH was much slower, in terms of J.LM DPNH formed/mg. protein/

minute, than the rate observed with yeast alcohol dehdrogenase j possibly because the velocities for the LADH reaction systems were calculated in terms of total protein in the reaction mixture; liver alcohol dehydrogenase presumably comprised only a small portion of the protein in the enzyme preparation used. An intrinsic difference in the ability of YADH and LADH to oxidize ethanol to acetaldehyde might also be an explanation (Singer ~ and Kearney, 1954). • Il

ETHANOL CONCENTRATION IN THE MEDIUM (10-3 M)

0.5 1.0 2.5 5.0 CONTROL 5.45 + 0.19 7.71 + 0.62 11.55 + 0.28 13.28 + 0.74 PHENELZINE - 0.1 mM 3.12 + 0.20 1.92 + 0.47 6.52 + 0.06 9.73 + 0.48

Table 16: The effect of phene1zine on the in vitro activity of mouse 1iver a1coho1 dehydrogenase, with

respect to substr,ate (ethano1) concentration: Procedure 1. '

The activity of control and pllene1zine inhibited reaction systems is expressed as the ve10city

at which DPNFl was fopned, ,in llM DPNH/mg. protein/min. x 10-3~ Ethanol and phe~e1zine

concentrations were as shawn in the table.

,1 1-'o o .' 101 ·If'.

50

40 -.L xlO 0.1 mM V 30

20 CONTROL

o 100 200 1 M-1 [ëiHANoi] x

50

40 A CONTROL. [ETHANOL) x 104 V 30

o 234 5 [ETHANOL] x IO'M

B

Fig. 17: The erfect of phenelzine on the in vitro activity of liver alcohol dehydrogenase, with respect to substrate (ethanol) concentration. These results were obtained using procedure I, as described under methods and results. A. Lineweaver-Burk plot B. Woolf plot .~~ ~y' 102

The decrease in LADH activity produced by 0.1 x 10-3 M phenelzine was antagonized by increasing the concentration of ethanol in the medium. The interaction between lower con- centrations of phenelzine and ethanol was not tested in this procedure. The inhibition by 0.1 x 10-3 M of phenelzine was essentially competitive, although the formof the Lineweaver­ Burk plot suggested .the possibility that a non-competitive . element May also be involved. The changes observed in the Km for"ethanol, and in the Vmax, in the Lineweaver-Burk analysis (Table 17) supported this possibility. The Km for ethanol increased 22% from 0.9 x 10-3 M to 1.1 x 10-3 M and the Vmax decreased 40% from 15.0 x 10~3 ~/mg./min. to 9.2 ~/mg./min. AlI parameters derived by the Woolf method emphasized the competitive nature of the inhibition. Procedure 2: The inhibitory effect of phenelzine added to a liver alcbhol dehydrogenase system eight minutes after initiation of the reaction, was measured for the time interval between eight and ten minutes. The control was the activity of the same reaction mixture measured for the two minute interval immediately preceding the time of addition of inhibitor. The calculated velocities are tabulated in Table 18. and expressed graphically by the Lineweaver-Burk method in Fig. 18 and by the Woolf method in Fig. 19. Control activities of the enzyme preparation were not constant from series to series, necessitating the use of a different graph for each • concentration of inhibitor. Inhibition of LADH activity by - '", • •. .

CONSTANT SLOPE Vmax (mg. min. / lJM) (lJM/mg./min. x 10-3) (10~ M)

CONTROL 66.6 60.5 15.0 0.91 A PHENELZINE - 0.1 mM 108.8 120.5 9.2 1.10

CONTROL 6.5 6.4 15.7 1.02 B PHENELZINE - 0.1 mM 17.5 7.3 13.7 2.39

Table 17: The effect of phenelzine on the Km value for ethanol, and on the·Vmax, in reaction systems using

mouse liver alcohol dehydrogenase: Procedure 1 •.

The values for Km and Vmaxwere calculated by'substituting the values shown for the constants

and slopes inequations which are detailed in the text.

A. Lineweaver-Burk plot·

B. Woolf plot

of-I lJJ '- •

ETHANOL CONCENTRATION IN THE MEDIUM (10-3 M)

0.25 0.5 1.0 2~5

CONTROL 2.22 + 0.11 2.80 + 0.12 4.18 + 0.19 5.37 + 0.16 PHENELZlNE - 0.05 mM 1.81 + 0.08 2.23 + 0.12 2.90 + 0.08 3.91 + 0.16 CONTROL 4.67 + 0.24 5.62 + 0.17 . 7.13 + 0.33 9.12 + 0.38 PHENELZINE - 0.1 mM 2.70 + 0.12 3.30 + 0.13 4.17 + 0.56 5.48 + 0.25

Table 18: The effect of phene1zine concentration on the in vitro activity of mouse 1iver a1coho1

dehydrogenase, with respect to substrate (ethano1) concentration: Procedure 2.

The activity of control and phene1zineinhibited reaction systems is expressed as the

ve10city at which DPNH was formed, in ~M DPNH/mg. protein/min. x 10-3• Ethanol and phene1zine concentrations were as shown in the table.

f-I o +:- 105 "

60 0.05 mM

, lXl0 CONTROL , V

10

0, 2 3 4 _1_ X 10 3 M-f [ETHANOL]

50 _1 xl0 V 40 A 0.1 mM

CONTROL

i o 100 200 300 400 1 1 _, [ETHANOL] xM

. ~ ,

B

Fig. 18: The effect of phenelzine concentration on the in vitro activity of liver alcohol dehydrogenase, with respect to substrate (ethanol) concentration: Lineweaver-Burk plot. These results were obtained using procedure 2, as described under methods and results. A. 0.05 mM phenelzine B. 0.10 mM phenelzine 106·

70 0.05 mM 60 [ETHANOL] " ID Y 50 CONTROL

40

30

20

o 0.5 1 1.5 2 2.5 . [ETHANOL] " ICi" M

60

50 0.1 mM [ETHANOL] " 103 .. A -y-. ·40

1 30 CONTROL!

20 1

1 t t

~ __~ __~ ______~ ____~ 1i 1 2 3 i [ETHANOL] " lo"M 1

B

Fig. 19: The effect of phenelzine concentration on the in vitro activity of liver alcohol dehydrogenase, with respect to substrate (ethanol) concentration: Woolf plot. These results were obtained using procedure 2, as described under methods and results. A. 0.05 mM phenelzine B. 0.10 mM phenelzine 107

,.' phenelzine doubled approximately when the concentration of • inhibitor in the medium was increased from 0.05 x 10-3 M to 0.10 x 10-3 M. Inhibition occurred immediately after the

addition of phenelzine to the reaction mixture~ and the

reaction velocity~ though diminished~ remained linear. Figures 18 and 19 demonstrate that the inhibitory effects obtained with both phenelzine concentrations were antagonized non-competitively by increasing the ethanol concentration in

the medium. Calculated values for the ethanol Km and the Vmax are tabulated in Table 19. They reflect a non-compet- itive type of inhibition in that phenelzine markedly decreased the maximum velocities (by as much as 50% with the highest concentration of inhibitor) but had little or no effect on the Km for ethanol. The results obtained by the Lineweaver- Burk and Woolf methods were in very close agreement for this particular series.

Inhibition by Phenelzine with Respect to DPN+:

Procedure 2~as described under methods~ was used to determine the inhibition of LADH with respect to coenzyme concentration. The reaction velocities derived from these experiments for control and phenelzine inhibited reaction mixtures are tabulated in Table 20 and expressed graphically by the Lineweaver-Burk method in Fig. 20 and by the Woolf method in Fig. 21. At a co~centration of 0.1 x 10-3 M~ ~ phenelzine had little effect on LADH activity with respect • "

CONSTANT SLOPE , Vmax Km (mg. min. /llM) (llM/mg./min. x 10-3) (10-3 M)

CONTROL 173.1 74.9 5.7 0.43

PHENELZINE - 0.05 mM 234.0 85.9 4.3 0.37 A 1 CONTROL 109.4 28.5 9.1 0.26

PHENELZINE - 0.10 mM 182.3 50.6 5.5 0.28

CONTROL 89.2 152.1 6.5 0.58

PHENELZINE - 0.05 mM 108.6 212.7 4.7 0.50 B CONTROL 378.4 966.8 1.0 0.39 -.- PHENELZINE - 0.10 mM 675.6 1592 0.6 0.42

Table 19: The effect of phene1zine concentration on the Km value for ethano1, and on the Vmax, in reaction

systems using mouse 1iver a1coho1 dehydrogenase: Procedure 2.

The values for Km and Vmax were ca1cu1atéd by substituting the values shown for the constants

and slopes in equations which are detai1ed in the texte

A.. Lineweaver-Burk plot f-Jo B. Woolf plot co .- 1

DPN+ CONCENTRATION IN THE MEDIUM (10-3 M)

0.05 0.1 0.25 0.5

CONTROL 3.05 + 0.18 4.65 + 0.11 7.87 + 0.09 11.08 + 0.35

PHENELZINE - 0.1 mM 2.50 + 0.06 3.98 + 0.11 6.35 + 0.23 8.80 + 0.27

CONTROL 2.02 + 0.22 3.02 + 0.35 4.55 + 0.34 8.58 + 0.44

PHENELZINE - 0.2 mM .... _~ 2.43 + 0.30 3.75 + 0.26 6.76 + 0.48

~----- ~ ------

Table 20: The effect of phene1zine concentration on the in vitro activity of-mouse 1iver a1coho1

dehydrogenase, witll respect to coenzyme (DPN+) concentration.

The activity of control and phene1zine inhibited reaction systems is expressed as the

ve10city at which DPNH was formed, in J..lM DPNH/mg. protein/min. x 10-3 .:n;éN+;. and

phene1zine concentrations were as shown in the table.

f-Io \0 110 !,

5

4 0.1 mM 1 x 10 2 V CONTROL 3

2

0 2 4 6 8 10 20 _'_1_ X 103 ~ [DPN+] 80 0.2 mM

70

60

CONTROL A 50 L xlO V 40

30

20

o 5 10 15 20 1 103M-1 [OPN+t

B

Fig. 20: The effect of phene1zine concentration on the in vitro activity of 1iver a1coho1 dehydrogenase, with respect to coenzyme (DPN+) concentration: Lineweaver-Burk plot.

A. 0.10 mM phene1zine B. 0.20 mM phene1zine . III "

6 0.1 mM

CONTROL

o 1 2 3 4 5 [OPN +] x 10· 4M

Fig. 21: The effect of 0.1 x 10-3 M phenelzine on the in vitro activity of liver alcohol dehydrogenase, with respect to coenzyme (DPN+) concentration: Woolf plot. • .''..~-;- ."

CONSTANT SLOPE Vmax 3 Kln (mg. min ./lJM) (lJM/mg./min. x 10- ) (10-3 M)

CONTROL 68.9 13.9 14.5 0:20

PHENELZINE - 0.1 mM . 90.3 15.7 11.1 0.17 A CONTROL 115.9 21.5 8.6 0.19

PHENELZlNE - 0.2 mM 105.8 34.3 9.5 0.32 - CONTROL 14.8 .62.4 16.;1 0.24 B PHENEL~INE - 0.1 mM 17.1 81.8 12.2 0.21

Table 21: The effect of phene1zine concentration on the Klnva1ue for DPN+, and' on the Vmax, in reaction

systems using mouse 1iver a1coho1 dehydrogenase.

The values for Km and Vmax were calculated by substituting the values shown for the constants

and slopes in equations which are detailed in the texte

A. Lineweaver-Burk plot

B. Woolf plot ~ ~ N 113

to DPN+ concentration. Although the graphie plot suggested a slight inhibitory effect at this concentration of phenelzine, no significant change from the control value was noted for the Km. of DPN+ or for the maximum velocity. A modest inhibition of LADH activity, reversible by larger concentrations of DPN+, was ob"tained with 0.2 x 10-3 M phenelzine (Fig. 20 B). The Km for DPN+ was increased 69% over "the control value, from 0.19 x 10-3 M to 0.32 x 10-3 M. The results for 0.2 x 10-3 M phenelzine were studied with the Lineweaver-Burk method only. With the data available, the Woolf method was unsatisfactory. 114

DISCUSSION

EFFECT OF PHENELZINE ON ETHANOL SLEEPING TIME: Pretreatment of mice for one half hour with 40 mg.!kg. of phenelzine prolonged ethanol induced sleep from a mean time of 27 ± 2 minutes to 98 + 7 minutes. A consideration of what is known of the pharmacology and metabolism of ethanol suggests that at least four mechanisms might have been involved in this alteration. They are: 1) increased ethanol concentration 2) increased acetaldehyde concentration 3) potentiation of the depressant action of ethanol on the central nervous system 4) potentiation of the depressant action of acetaldehyde on the central nervous system. The relevance of these mechanisms will be discussed in relation to the results reported in this thesis and the information available in the literature. The discussion is equally relevant to an interpretation of the altered lethality of ethanol.

Increased Ethanol Concentration: The duration of ethanol induced sleep in mice can be prolonged by administering larger doses of ethanol or by maintaining hypnotic concentrations of ethanol for longer periods of time. That one of the hydrazipe monoamine oxidase inhibitors could prolong the actions of ethanol by maintaining blood ethanol concentrations above control levels was reported by 115

Smith et al (1961). Dogs pretreated with pheniprazine • (3 mg./kg.) _~or two days prior to the administration of a subhypnotic dose of ethanol, exhibited depression) drowsiness and impairment of behavior greater than that occurring in control animaIs. These symptoms were correlated with a substantial decrease in the rate of disappe.arance of ethanol from the blood. Pheniprazine given alone produced no sedation or depression and the interaction with ethanol was dose dependent since no alteration in the effects of ethanol were

observed at pheniprazine doses of 0~5 and 1.0 mg./kg. An analogy can be drawn between the interactions of the hydrazine monoamine oxidase inhibitors with ethanol and those observed with the barbiturates, another group of general central nervous system depressants. The prolongation of barbiturate induced sleep in mice pretreated with iproniazid (Fouts and Brodie, 1956) was shown by Laroche and Brodie (1960) to be mediated by an inhibition of barbiturate metabolism. In the experiments reported here, it was shown that pre­ treatment of mice with phenelzine for one half hour reduced the rate at which ethanol disappeared. Concentrations of ethanol in control and experimental mice were similar immediately after injection of ethanol, but were 22% higher in phenelzine pretreated animaIs two hours later (Table 8). A comparison of the regressions for the elimination of ethanol by both groups (Fig. 8) showed that phenelzine significantly reduced the rate of disappearance

of ethanol from 0.55 g./kg./hr. to 0.38 g~/kg./hr. 116

~ The presence of higher concentrations of ethanol in phenelzine pretreated mice for longer periods of time might explatn the observed prolongation of sleeping time.

Increased Acetaldehyde Concentration: The increased sleeping time observed after phenelzine pretreatment may not be due entirely to the depressant action of ethanol on the central nervous system, but might also involve an hypnotic action by acetaldehyde, a metabolite of ethanol. Support for this hypothesis is derived from studies reported by Larsen (1948) on the inhibitory effect of disulfiram (Antabuse) on acetaldehyde metabolism. Of fifteen rabbits treated with disulfiram and given ethanol (0.2 ml. of 20% solution per g.) a day later, twelve (80%) went to sleep as compared to two of ten (20%) control animaIs. Larsen showed that the oxidation of ethanol to acetaldehyde was unimpaired in the disulfiram treated animaIs, but acetaldehyde con­ centrations were increased to 1 to 2 mg.%, values which were five times higher than those observed in control animaIs. In the same paper, it was reported that disulfiram pretreatment prolonged ethanol induced sleep in mice by 50%,although no

values for acetaldehyde concentrat~ons were given. The depressant properties of acetaldehyde were also pointed out by Skrog (1950) who reported a sudden onset and rapid dis- appearance of anesthesia in rats administered acetaldehyde (~ subcutaneously. It is doubtful, however, that the increased 117

~ sleeping time observed in mice pretreated with phenelzine ,~ could be attributed to a depressant action by acetaldehyde.

The studies in this thesis have shown that the maximum con- centrations of acetaldehyde occurring after the administration of ethanol to phenelzine pretreated mice were reduced by 20%, rather than elevated, as compared to acetaldehyde levels in

control animals(Ta~le 9). Although not consistent with the hypothesis of an action . synergistic with that of ethanol, the reduction in acetaldehyde

concentrations might ~e an explanation for increased sleeping times if interpreted according to the findings of Larsen et al (1952). These authors investigated the interaction between the pharmacological effects of ethanol and acetaldehyde in rabbits and they found a pronounced antagonism between the depression of respiration produced by ethanol and the stimulation of respiration produced by acetaldehyde. This result has not been reported for mice, but on the assumption that it is valid for this species, a reduction in acetaldehyde concentration might enhance the central nervous system depression produced by ethanol, and consequently prolong sleeping times.

Potentiation of the Depressant Action of Ethanol on the Central Nervous System! The phenomenom of potentiation was considered by Fouts and Brodie (1956) in an effort to explain the prolongation of Î~ hexobarbital sleeping time in mice pretreated with iproniazid. 118

(~. They defined a potentiating agent, as opposed te a pro- longing agent, as a compound able to reinduce sleep in animaIs

awakening from hexobarbital induced hypnosis~ and able to induce sleep when combined with a sub-hypnotic dose of this barbiturate. The results of experimental work associated with this thesis indicated that phenelzine potentiated the depressant action of ethanol on the central nervous syst;em in addition to prolonging it by inhibiting the metabolism of éthanol. In work not reported here, it was shown, by duplicating the criteria determined by Fbuts and Brodie, that phenelzine injected i.p. jnto mice awakening from ethanol hypnosis reinduced sleep in some of these animaIs. Success in this respect seemed to depend, however, on how long the mice slept initially. Phenelzine did not reinduce sleep in mice having an initial sleeping time longer than about twenty minutes. Further reason to classify phenelzine as a potentiatGr of ethanol sleeping time was the finding that phenelzine pretreated mice remained asleep at body concentrations of ethanol which were sub-hypnotic in control animaIs, and awoke with concentrations as much as 35% less than those found in control mice (Table 6). It is of interest to note that the hydrazine monoamine oxidase inhibitors phenelzine, pheniprazine and iproniazid, and the tri-cyclic antidepressant, imipramine, have been reported to potentiate the depressant effects of ethanol and the barbiturates by what may be a similar mechanism involving the (~ central nervous system. Smith et al (1961) noted that iproniazid, 119

• in a dose of' 10 mg./kg. f'or two da ys and once again one half' hour bef'ore ethanol, produced higher stages of' drunkeness in dogs at a given dose of' ethanol, in the absence of' any change in the rate at which ethanol was eliminated. Pheniprazine pretreatment produced the same ef'f'ect, in addition to prolonging the action of' ethanol by inhibiting its metaboliam. Imipramine was shown by Kato et al (1963) to alter pentobarbital sleeping time in a manner similar to that occurring in the prolongation of' ethanol sleeping time by phenelzine. An injection of' imipramine reinduced sleep in animaIs recovering f'rom pentobarbital hypnosis and induced sleep when combined with sub-hypnotic doses of' pentobarbital; it was not stated whether imipramine administered alone in the doses used could produce hypnosis or sedation. Imipramine, like phenelzine and pheniprazine, also exhibited a prolonging action by inhibiting the enzymic degradation of' pentobarbital. The results reported by Kato é,t al, and those in this' thesis, suggest that monoamine oxidase inhibitor and tri-cyclic antidepressant drugs might potentiate central nervous system depression produced' by ethanol and the barbiturates, by a common pharmacological action. Potentiation of' the ef'f'ects of' ethanol is not conf'ined to antidepressant drugs. Chlorpromazine, an antip$ychotic agent with sedative properties,has been shown by Smith et al (1961) to increase ethanol induced sleeping time by a mechanism similar r. to the one reported as associated with the actions of' iproniazid 120

'. and phenipraz ine • The ,monoamine inhibitors, tri-cyclic antidepressants and chlorpromazine are thought to affect the function of the central nervous system by altering the concentration of endogenous amines. This suggests that the potentiation of ethanol and barbiturate sleeping time by these compounds could derive from changes in brain amine levels, particularly of 5-hydroxytryptamine. Parenterally administered 5-hydroxytryptamine

has been shown to increase the duration of hypnotics in mice by a central mechanism (Shore et al,1955) and its depressant effect on the central nervous system has been demonstrated by Feldberg and Sherwood (1954) and Curtis and Davis (1962).

Pbtentiation of the Depressant Action of Acetaldehyde on the Central Nervous System:

No evidence was found during experiments with a~etaldehyde,

or in the literature, to suggest that the hydrazine monoamine oxidase inhibitors might potentiate the depressant action on the central nervous system that acetaldehyde is known to possess (Skrog, 1950). This mechanism will not, therefore, be further discussed although the possibility that such a mechanism is involved in the production of the results ob'served cannot be discounted.

EFFECT OF PHENELZINE ON THE LD 50 OF ETHANOL:

. . . . '. Kalant (1961) postulated that the activity of the central nervous system reticular format:ion, and particularly of the reticular activating system, was selectively depressed by low doses of ethanol, resulting in a decreased state of wakefulness. The lethal effect of increasing concentrations of ethanol was due to inhibition of respira tory and vaso-motor centers in the medulla. Potentiation of the central nervous system depressan~ action of ethanol might further inhibit both reticular and medullary centers at any given dose of ethanol. In view of the probable participation of a potentiating effect by phenelzine

in increasing ethanol sleeping time, it was anticipate~ that the hydrazine compound would potentiate the lethal effect of ethanol. Unexpectedly, phenelzine pretreatment mitigated the toxicity of ethanol in mice, significantly increasing the LD 50 for ethanol from 4.14 g./kg. to 5.16 g./kg. (Fig. 6). An explanation for this dichotomy of ," action by phenelzine was not pursued in this laboratory, nor was one found in the lite rature • The finding was an interesting one, however, and invited speculation concerning a possible mechanism. The analeptic properties of the hydrazinemonoamine oxidase inhibitors might be evoked to explain the phenomenom of increased LD 50's in phenelzine pretreated mice, but this hypothesis is not compatible with the prolongation of ethanol sleeping time in the same animaIs. It is also unlikely that phenelzine per se possesses analepticactivity (Biel et al, 1959). An explanation involving two distinct mechanisms for the hypnotic and lethal effects of ethanol was considered, but abandoned for 122

'. lack of evidence. The protection afforded mice by phenelzine against the lethal action of ethanol was aIl the more interesting becaUse ethanol concentrations were elevated concurrently. As was discussed for sleeping time, it is not known for certain whether ethanol toxicity is due to the alcohol itself, or to acetaldehyde. Acetaldehyde is capable of inducing respiratory embarrassment andcirculatory collapse in animaIs (stotz et al, 1944; McLeod, 1950) and is tolerated only in very small quantities (Westerfeld, 1955). However, the significance of reduced acetaldehyde concentrations in phenelzine pretreated mice with respect to the antidotal.effect of phenelzine is uncertain. The importance of acetaldehyde as the toxic agent was diminished by the finding that a five fold increase in acetaldehyde con­ centrations in disulfiram·treated rabbits was not accompanied byan increase in the mortality rates (Larsen, 1948). In addition, dogs administered ethanol were shown to have in­ sufficient quantities of acetaldehyde in their blood for the aldehyde to be responsible for the state of intoxication observed (McLeod, 1950). An interesting and plausible mechanism is suggested for the protective effect of phenelzine by the findings of Greenberg (1967). Certain primary, secondaryand tertiary amines', with and without analeptic activity, prevented the Mean energy changes in the EEG pattern of rabbit cortex usually associated with ethanol. d-Amphetamine was 97% effective in this regard, 123

lysergic acid diethylamide 98% effective, dimethylaminoethanol '.' 80% effective, and desmethylcarnitine 87% effective. The se drugs did not significantly alter the mean energy content of the EEGwhen given alone, nor were any changes noted in the blood alcohol disappearance curves. The relevance of these findings to the phenomenom of phenelzine protection against the effects of ethanol is emphasized by the fact that although , the EEG reflected marked prevention of the cortical depressant effects of ethanol, no such prevention occurred in behavior. Righting reflex and respiration remained depressed, indicating that the depression of medullary centers by ethanol was not relieved. Kalant's (1961) hypothesis of progressive depression by increasing doses of ethanol may be .viewed in terms of the affinity of various brain structures for ethanol. Those whose activity is depressed at low ethanol concentrations (e.g. the reticular activating system) might have a high affinity for the ethanol molecule, whereas those depressed only at higher concentrations (e.g. the medullary centers for respiration and vaso-motor tone) might have correspondingly lower affinities. Greenberg (1967) implied that the amines mentioned previously might have prevented cortical depression by interacting with ethanol receptor sites in the brain. If phenelzine, shown in this thesis to compete with ethanol for an enzymic site, can also compete for an ethanol receptor in the central nervous ~ system, it would probably be more effective at sites having a 124

. ... , '.~ low affinity for the a1coho1. By this mechanism~ phenelzine cou1d antagonize the depression of regu1atory centers in the

medul1a caused by ethano1~ thereby reducing the 1etha1"factor~ without affecting the depression of systems invo1ved in

maintaining wakefulness. Alternately ~ phenelzine could in- crease the concentration of endogenous brain amines at these

sites by inhibiting mono~ine oxidase~ and the amines themselves might compete with ethanol for the appropriate receptor sites.

There is presently~ however~ no experimental evidence other than that reported by Greenberg (1967). to substantiate this speculation.

EFFECT OF PHENELZINE ON ETHANOL METABOLISM: The observations by Masoro et al (1953) and Hughes and Forney (1961) that the degradation of ethanol was rectilinear with time were confirmed by the in vivo results reported in this the"sis for the elimination of ethanol by control and phenelzine,

pretreated mice (Table 8~ Fig. 8)~ Some deviation from the

rectilinear pattern~ suggestive of the exponential form of

degradation reported by Marshall and Fritz (1953)~ was detected at ethanol concentrations of less than 0.20 g./kg. These low , values, determined at twelve hours after the injection of ethanol,

could have been artifacts~ however~ resulting from the .inaccuracy of the quantitative assay at low concentrations of ethanol. Ethanol can be eliminated from the body by exhalation from .. ~ the lungs.andby rena1 excrètion~ but these routes account for 125

less than 15% of the total eliminated (Lundquist and Wolthers, 1958) and were not considered primary routes of elimination

" during these studies. Ethanol disappears from the body mainly by chemical transformation to acetaldehyde in the liver (Westerfeld, 1961) and inve·stigations into the effect of phenelzine on the rate at which ethanol disappeared were pursued at this level only. The two enzymes involved in the hepatic degradation of ethanol are catalase, located in the microsomal fraction, and alcohol dehydrogenase, a soluble protein found in the cytoplasmic fraction. Keilin and Hartree (1945) showed originally that catalase,in the presence of peroxide, would oxidize ethanol to acetaldehyde, but subsequent studies suggested that catalase was not an important factor in ethanol metabolism (Kinard et al, 1956). It MaY, however, be involved sufficiently to account for the occasional deviation from the rectilinear pattern of disappearance (Jacobsen, 1952). Alcohol dehydrogenase is now accepted as the primary mechanism for the hepatic oxidation of ethanol to acetaldehyde (Westerfeld, 1961). Inhibition of yeast (YADH) and liver (LADH) alcohol dehydrogenase by pheniprazine, iproniazid and isocarboxazid was reported by Redetzki and O'Bourke (1961). The inhibition by pheniprazine was reversed by the addition of DPN+ to the medium (Sankar et al, 1961), suggesting that inhibition was mediated through this coenzyme. However, the significance • of a DPN+ -hydrazine interaction in the inhibition of LADH was 126

questioned by Smith et al (1961) who f'ound that additional • DPN+ d~d not reverse ,the i~hibition of' rat LADH produced by pheniprazine. The results reported in this thesis show that increased concentrations of' DPN+ in the reaction media competitively antagonized the inhibition of' both YADH and LADH by phenelzine, thus conf'irming the inhibitory ef'f'ect of' the hydrazine monoamine oxidase inhibitors on alcohol dehydrogenase, and substantiating the hypothesis that inhibition of' this enzyme 'by hydrazine derivatives is mediated in part through an interaction with DPN+ at some site on the enzyme. Zatman et al (1954) proposed that certain DPN+- nucleosidases caused an exchange of' iproniazid f'or the nicotinamide portion of' DPN+ to f'orm an inactive analogue'of' the coenzyme. The absence of' pyridine in the structure of' phenelzine, and the rapidity with which phenelzine inhibited the alcohol dehydrogenases, preclude a similar mechanism being invoked to explain its inhibitory ef'f'ect in these studies. A mechanism more compatible with the rapid inhibitory action observed would be a' direct interaction of' phenelzine with a DPN+ receptor site, possibly mediated by the hydrazine moiety. The importance of' the hydrazine group in the inhibitory mechanism is emphasized by the f'inding that the hydrazines iproniazid, pheniprazine and isocarboxazid were 90% ef'f'ective in blocking alcohol dehydrogenase activity, whereas tranylcypromine; a primary amine, inhibite.q the enzyme by only 23% (Redètzki .~~ and O'Bourke, 1961). The f'ormation of' a reversible complex 127

,~ between phenelzine and the zinc moiety of the DPN+ site was suggested by the similarity of inhibition of liver alcohol dehydrogenase by phenelzine to that by,a zinc chelating agent" 1,10-phenanthroline. Inhibition by both compounds was instantaneous and competitîve with DPN+, but non-competitive with ethanol. The non-competitiveness of inhibition by these compounds with respect to ethanol was indicative of a binding, or interaction of ethanol, at a location on the enzyme ~part from the DPN+ receptor site (Vallee and Hoch, 1957).

. , This was in conttast to the competitive nature of the interaction between phenelzine and ethanol in the inhibition of yeast alcohol dehydro'genase, which might indicate some difference' in the ethanol receptor sites of the two enzymes. Another plausible site of interaction betweenphenelzine and DPN+ are the sulfhydryl groups which are thought to play a role in the binding of DPN+ to the enzyme (Hoch and Vallee, 1959; Snodgrass et al, 1960). Sank&r et al (1961) showed that inhibition of .YADH and LADH by pheniprazine could be reversed. by the additton of the' sulfhydryl containing amine . ac id, cys te ine, to the reac t ion me d iwn·. Inhibition mediated through the blockade of sulfhydryl groups has not been confirmed or disproved by other investigators and remains a viable explanation for the action of pheniprazine and phenelzine, the compound studied in our experiments. There is strong evidence to support the hypothesis of an acyl or alkyl radical derived from hydrazine compounds as 128

~ the active inhibitor substance for the enzYme monoamine oxidase (Schwartz, 1961; Kory and Mingioli, 1964) and some - _. evidence for the formation of a hydrazone complex capable of inhibiting the enzyme L-glutamic acid decarboxylase (Bain and Williams, 1960). In certain instances, the formation of a similar hydrazine derivative might be necessaryfor the inhibition of alcohol dehydrogenase. Freshly prepared solutions of pheniprazine were shown to have no inhibitory effect on YADR, but the solutions became markedly inhibitory if incubated at room temperature for four hours (Sankar et al, 1961), presumably through the formation of an active oxidation product, In contrast, however, was the strong and immediate inhibition of liver alcohol dehydrogenase on addition to the medium of freshly made solutions of pheniprazine. The structural change required to develop this inhibitory power was not determined by Sankar et al, but it rnay have centered about the a-methyl group. The a-demethylated derivative of pheniprazine, phenelzine, was shown in this thesis to instantaneously inhibit both YADH and LADR. The rapidity

of action sugge~ted that chemical transformation to an active derivative was not necessary for inhibition of alcohol dehydrogenase by phenelzine. Further support for this hypothesis was derived from the finding that .there was no increase in the ability of phenelzine to inhibit LADH after incubation with the enzyme at 370 C for one half hou~-. Studies to test the inhibitory effect of phenelzine left at room 129

tempe rature ·:for a numbero:f hours were not done, and in view 1 o:f the color changes .in phenelzine solutions noted a:fter one day, the :formation o:f an inhibitory oxidative derivative cannot be discounted. The in vitro studies reported in this thesis have demonstrated that inhibition o:f yeast alcohol dehydrogenase was not due entirely to the compet.itive interaction between phenelzine and DPN+, as seemed the case :for inhibition o:f LADH. Phenelzine inhibition o:f YADH was also competitively antagonized by the addition o:f substrate (ethanol) to the medium. The Km :for ethanol, alréady ten times greater than the Km. :for DPN+, was increased ten :fold by phenelzine (20 mM), as compared to a three :fold increase in the Km of DPN+. This might indicate that phenelzine has an even greater a:f:finity :for the ethanol receptor site on YADH than :for the' DENt s~te. There remains one observation ror which no de:finite explanation is available, namely the delay required :for the .. LADH reaction system to reach maximum velocity. Whatever the reason, the velocity became ma·rimal within :five minutes and remained so fOT. at least twenty minutes.

EFFECT OF PHENELZINE ON THE METABOLISM OF ACETALDEHYDE: .. The effect o:f phenelzine pretreatment on acetaldehyde metabolism in mice given ethanol appears to be two :fold. The maximum concentration observed in experimental animaIs about twenty minutes after the administration o:f ethanol was decreased 130

'. 20% relative to the control value (Table 9) and was succeeded by a rapid, but short-lived decline, similar to that occurring in the initial portion of the control 'elimination curve. This

~slight decrease was followed by a, stabilization of acetaldehyde concentrations, with a slight tendency at the two hour interval towards the re-establishment of the original peak level. The form of the elimination curve beyond two hours was not determined experimentally in control or phenelzine pretreated

an~ls. Acetaldehyde was shown to be metabolized rapidly and

~xponentially by cats in vivo (Lubin and Westerfeld, 1945) and by rat and mouse brain and muscle tissue in vitro (WesterfeŒd et al, 1949). In contrast to these findings, a multiphasic pattern of elimination was reported to occur in rat brain in vivo by Ridge (1963), the rate of acetaldehyde decay fluctuating. sharply a number of times before becoming constant after one hour. The control degradation,curves for acetaldehyde illustrated in Fig. 10 would probably assume an exponential character at concentrations lower than those determined within the two hour interval. The second change in direction of the eliminationcurve for acetaldehyde in phenelzine pretreated mic'e (Fig. 10) was unusual and was attrlbuted to enzymic inhibition by phenelzine. The initial decrease in maximum concentration would be expected if the oxidation of ethanol'were inhibited. The period of stabilization following this may reflect a reduction in the rate of acetaldehydel:lmetabolism 131

~t because of a blockade of aldehyde dehydrogenase," a DPN+ dependent enzyme which oxidizes acetaldehyde to acetic acid (Westerfeld, 1961). Phenelzine was shown in this thesis to interfere with DPN+ at "a site on alcohol dehydrogenase, and it is not unlikely that a similar inhibitory mechanism could operate with respect to the DPN+ -aldehyde dehydrogenase interaction. A second mechanism for the stabilization of acetaldehyde concentrations noted in phenelzine pretreated mice may also be postulated. The hydrazine monoamine oxidase inhibitors have been shown to form hydrazone complexes in vivo with carbonyl compounds, such as pyridoxal phosphate (Williams and Abdulian, 1956; Dubnick et al, 1960), and iproniazid has been reported to interfere with yeast glycolysis by forming the hydrazone of acetaldehyde (Neuberg and Forrest, 1953). In view of these findings, a chemical reaction between phenelzine and acetaldehyde is a distinct possibility. The resultant hydrazone might not be subject to degradation by aldehyde dehydrogenase, or perhaps would be metabolized at a slower rate than free acetaldehyde, without altering the ability of the assay method to detect the acetaldehyde component. 132 '. SUMMARY AND CONCLUSIONS The antidepressant monoamine oxidase inhibitors have been contraindicated with ethanol because of reports that these compounds prolong and intensify the central nervous system depressant effects of ethanol. Studies have been conducted to determine if phenelzine, a hydrazine monoamine oxidase inhibitor in therapeutic use, could alter the pharmacological properties of ethanol in vivo, and if an interaction did occur, to determine whether it could be explained on the basis of an inhibition of ethanol Metabolisme The following results were . obtained: 1) Ethanol sleeping time in mice pretreated for one half hour with phenelzine, 40 mg./kg., was increased from a control value of 27 + 2 minutes to 98 + 7 minutes. 2) Body concentrations of ethanol determined in phenelzine pretreated mice on awakening were as much as 35% lower than ethanol concentrations in control mice on awakening. 3) Pretreatment with phenelzine mitigated the lethality of ethanol. The LD 50 of ethanol increased in experimental animaIs to 5.16 + 0.25 g./kg. from a control value of 4.14 + 0.43 g./kg. 4) Phenelzine significantly reduced the rate of the in vivo disappearance of injected ethanol from 0.55 g./kg./hr. to 0.38 g./kg./hr. The linear pattern of disappearance was not altered. 5) Maximum acetaldehyde concentrations occurring in 133

'e:"", phenelzine pretreated mice administered ethanol were decreased by 20% to 0.20 rng.%, but this concentration showedlittle tendency to decrease over the two hour interval during which the study was conducted, as compared to the rapid decline in the acetaldehyde content of control mice during the same periode 6) Phenelzine inhibited the oxidative activity of yeast alcohol dehydrogenase (YADH) in vitro by cornpetitively inter­

fering with both the coenzyme ~-diphosphopyridine nucleotide

(DP~) and the substrate, ethanol.

7) The Km values for ethanol and DP~ in the reaction mix­ tures inhibited by phenelzine suggested thatphenelzine had a greater affinity for the ethanol receptor site on YADH than for the DPN+ site. 8) Phenelzine inhibited the oxidative activity of liver alcoho1 dehydrogenase (LADH) in vitro through a non-competitive interference with ethanol and by cornpetitively interacting with DPN+. 9) Incubation of phenelzine in a concentration of 0.1 x 10-3 M with LADH for six minutes at room temperature produced competitive inhibition of the enzyme with respect to ethanol concentration. 10) Phenelzine in concentrations of 0.05 x 10-3 M and 0.1 x 10-3 M inhibited LADH non-competitively with respect to substrate concentration in the absence of an incubation periode • The inhibition was greatest at the higher concentration of 134

' :• ~ inhibitor and was reversed by the addition of larger amounts of ethanol to the medium. Il) Phenelzine in a concentration of 0.1 x 10-3 M had little effect on LADH activity with respect to coenzyme concentration, but a modest competitive inhibition was observed when the inhibitor concentration was doubled.

The principal conclusions were: 1) A pharrnacological interaction between ethanol and phenelzine occurred in mice. The interaction was rnanifested as a prolongation of ethanol induced sleep and a decrease in ethanol toxicity. 2) The prolongation of ethanol sleeping tirne in phenelzine pretreated rnice was due,in part, to an inhibition of ethanol rnetabolism. 3) Phenelzine inhibited yeast alcohol dehydrogenase by a competitive interaction with both substrate and coenzyme. 4) The inhibition of liver alcohol dehydrogenase by phenelzine was mediated primarily through a competitive interference at the coenzyme level. Many new problerns have been suggested by the results of

the studies reported in this t~esis. It would be of immediate interest to determine the rnechanisrns underlying: 1) the decrease in the toxicity of ethanol in phenelzine pretreated mice 2) the action of phenelzine on the central nervous system • responsible for potentiation of ethanol induced depression of 135

" . . . , , • the central nervous system. 3) the interaction of phenelzine with ethanol and DPN+ at receptor sites on the alcohol dehydrogenase enzymes.

~' ~ 136

APPENDIX l

DRUGS: . Phenelzine Sulphate: Phenelzine sulphate was kindly supplled by Warner-Lambert of Canada Ltd. Properties: White crystalline salt. Molecular weight (salt): 232 % basebyweight: 58 Highly soluble in distilled water. Phenelzlne was stable in the crystalline form but unstable ln aqueous solution. Color changes due to oxidation occurred within twenty-four hours at room temperature. Preparation and Administration: For in vivo studies phenelzine was made fresh daily in 0.9% sodium chloride solution at a concentration of 0.4%, W/V. The volumes Injected were based on a ratio of 0.01 ml./g. mouse. For in vitro studies phenelzine was freshly prepared ln

distilled water Immediately pr~or to use each time. Concentration was expressed in terms of molarity. The dilution of concentration followed the formula: (vol. of stock soIn.) x (molarity of stock soln.) = (vol. of diluted soln.) x desired molarity. 137

A constant volume of 0.1 ml. of phenelzine solution was added to aIl incubation media requiring inhibitor. The pH of the solution was not adjusted before ad­ dition.

Ethanol: Ethanol was obtained as a 95% V/V solution from Commercial

Alcohols~ Ltd.~ Gatineau~ P.Q., Canada. Prope rtie s : Molarity: 16.25 Density: 0.810 at 250 C %ethanol by weight: 92.3 Molecular weight of ethanol: 46 Boiling point of ethanol: 690 C

~he stock 95% solution was stable for months when kept refrigerated at 4°c in a glass stoppered bottle. Preparation and Administration: AlI solutions used for in vivo and',. in vitro studies were dilutions of the 95% stock solution made in distilled water. For !p vivo studies, 'the preparation of a solution of predetermined percentage followed the formula: (vol. of 95% soIn.) x 95% = (vol. of diluted soln.) x desired % AlI injections were given as a 40% solution containing 315 mg. of ethanol per ml. The volumes of injection 138 '.' were calculated as follows: Weight of mouse in g. x 3.5 mg./g. 315 mg./ml. For in vitro studies, the concentrations of ethanol

solutions were expressed in ter~s of molarity. Preparation of solutions of a predetermined molarity followed the formula; (vol. of 16.25 M soIn.) x 16.25 M = (vol. of diluted soIn.) x desired molarity. 139 ,., . • 1 •, , . APPENDIX II REAGENTS: Unless indicated otherwise, chemicals used were purchased from the Fisher Scientific Co., MOntreal, P.Q., Canada. Ethanol Assay In the Total Mouse: Potassium dichromate reagent: 2.13 g. of reagent grade potassium dichromate were dissolved in approximately 200 ml. of distilled water and diluted to 1 liter with concentrated nitric acid. This solution was cooled to room tempe rature before being made up to final volume. Sulphate-tungstate reagent: 100 g. of sodium sulphate and 100 g. of sodium tungstate were dissolved in 600 ml. of distilled water and diluted to a final volume of 1 liter with distilled water. Sulphate-sulphuric acid reagent: 200 g. of sodium sulphate were dissolved in 500 ml. of water. 30 ml. of concentrated sulphuric acid were added and the v.olume made up to 1 liter with distilled water. Chromic nitrate reagent (for standard curve only): 0.58 g. of chromic nitrate were dissolved in approx­ imately 20 ml. of distilled water and diluted to 100 ml. with concentrated nitric acid. 140

'.'".. ' 1 Acetaldehyde Assay In the Total Mouse:

10~ sodium tungstate reagent: made in volumes or 2 liters and stored at 4°C. o 0.66 N sulphuric acid reagent: stored at 4c. 2% sodium bisulrite: rreshly prepared berore each series or distillations. 5% copper sulphate reagent: Sulphuric acid reagent: S.G. 1.84, reagent grade. This reagent must be pretected rrom contamination. It was dispensed rrom a glass burette having a terlon stopcock. p-hydroxybiphenyl reagent: (K & K Laboratories, Inc., Plainview New York, N.Y., U.S.A.) l.g. or the crystal11ne chemical was dissolved in 25 ml. or hot 2N sodium hydr oxi de, and 75 ml. or water was added berore cooling. This reagent was stable ror months when stored in a brown bottle (stotz, 1943). Paraldehyde, B.P.: (Glaxo-Allenburyl's (Canada) Ltd., Toronto" Ontario, Canada): Boiling point: 1200 è. 5 ml. portions were puriried by redistillation in a micro-distillation apparatus using a gentle rlame rrom a Bunsen burner. Unused portions or the distillate were discarded. A stock solution baving a concentration or l mg. paraldehyde per ml. was prepared by dissolving l ml. ~. W' or redistilled paraldehyde in 800 ml. or distilled 141

water and dllutlng to 1 llter. The reagent was stable ror at least two rnonthswhen rerrlgerated. A worklng standard was prepared as requlred by dilutlng 1 ml. or stock to 500 ml. wlth dlstllled

water, to glve a rlnal concentration or 2 ~g./rn1.

Acetaldehyde: (J.T. Baker Chernical Co., Phi111psburgj N.J.,

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