INFORMATION TO USERS
This w u produced from a copy of a document sent to us for microfilming. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the material submitted.
The following explanation of techniques is provided to help you understand markings or notations which may appear on this reproduction.
1.The sign or “target” for pages apparently lacking from the document photographed is “Missing Page(s)”. If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent p8ges. This may have necessitated cutting through an image and duplicating adjacent pages to assure you of complete continuity.
2. When an image on the film is obliterated with a round black mark it is an indication that the film inspector noticed either blurred copy because of movement during exposure, or duplicate copy. Unless we meant to delete copyrighted materials that should not have been filmed, you will find a good image of the page in the adjacent frame.
3. When a map, drawing or chart, etc., is part of the material being photo graphed the photographer has followed a definite method in “sectioning” the material. It is customary to begin filming at the upper left hand comer of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning Is continued again-beginning below the first row and continuing on until complete.
4. For any illustrations that cannot be reproduced satisfactorily by xerography, photographic prints can be purchased at additional cost and tipped into your xerographic copy. Requests can be made to our Dissertations Customer Services Department.
5. Some pages in any document may have indistinct print. In all cases we have filmed the best available copy.
University Microfilms International 300 N. ZEEB ROAD, ANN ARBOR. Ml 4B106 18 BEDFORD ROW. LONDON WC1R 4EJ. ENGLAND 8115100
ENEANYA, DENNIS ILOZULIKE
DIETHYLDITHIOCARBAMIC ACID: DOSE DEPENDENT KINETICS, BILIARY SECRETION OF METABOLITES AND CHOLERETIC EFFECTS
The Ohio State University PH.D. 1981
University Microfilms International 300 N. Zeeb Road, Ann Arbor, MI 48106
Copyright 1981 by
ENEANYA, DENNIS ILOZULIKE All Rights Reserved PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V
1. Glossy photographs or pages ______2. Colored illustrations, paper or print _____ 3. Photographs with dark background _____ 4. Illustrations are poor copy ______5. Pages with black marks, not original copy ______6. Print shows through as there is text on both sides of page ______7. Indistinct, broken or small print on several pages ^ 8. Print exceeds margin requirements _____ 9. Tightly bound copy with print lost in spine ______10. Computer printout pages with Indistinct print ______11. Page(s)______lacking when material received, and not available from school or author. 12. Page(s)______iseem to be mtssing in numbering only as text follows. 13. Two pages numbered I______. Text follows. 14. Curling and wrinkled pages 15. Other
University Microfilms International DIETKYLDITHIOCARBAMIC ACID; DOSE DEPENDENT KINETICS,
BILIARY SECRETION OF METABOLITES AND CHOLERETIC EFFECTS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy in The Graduate School
of The Ohio State University.
By
Dennis Ilozulike Eneanya, B.S.
The Ohio State University
1981
Reading Committee: Approved by
Dr. Joseph R. Bianchine
Dr. Brian D. Andresen Adviser Dr. Richard H. Fertel rtment of Pharmacology
Dr. Rao Panganamala ACKNOWLEDGEMENTS
I would like to thank Doctor Joseph R. Bianchine, ay academic adviser with whom I have been priviledged. to work; whose counsel, criticism and support have been invaluable. I will continue to have the highest regards for him.
I would wish to extend my gratitude to Dr. Nicholas Gerber. My experiments have borrowed from his prestigious works and his suggestions have been of immense help to me. I have also been priviledged to work with him.
My sincere thanks to Drs. Brian Andresen, Richard Fertel and Rao
Panganamala. They have taught, challenged and enlightened me.
To my family (especially my wife, Comfort; my daughter, son and mother) I express my deepest appreciation for their closeness, encour agement, understanding and prayers. I also thank the Sterrett's
family whose friendship, kindness and support, I will remain indebted
to.
I would’ like to thank Dr. B.M. Hanumaiah for technical laboratory
assistance, I thank Mrs. Carol Jones for her patience and expertise in
the preparation of this manuscript, and Miss Dorothy Brickman for her educational help. The aupporc o£ the predoctoral scholarship from the Federal Govern** meat of Nigeria is acknowledged.
Finally, to all who helped me during my graduate school career, I gratefully acknowledge. VITA
September 8, 1952 Born- Nri, Nigeria
1976 B.S., Denison University Granville, Ohio
1976-1981 Graduate Research Associate Department of Pharmacology The Ohio State University Columbus, Ohio
PUBLICATIONS AND PRESENTATIONS
Tejwani, G.A., Pertel, R., Hart, R.W*, Eneanya, D., Allison, D.: Asbestos Induced Changes in the Concentrations of Cyclic Nucleotides in Normal Human Fibroblasts. Federation Proceedings 2 J J 1536, 1978.
Eneanya, D.I., Daniel, F.B., Hart, R.W.: Effects of Asbestos (chrysotile intermediate) on the Metabolism of Benzo(a)pyrene to DNA of Syrian Hamster Embryo Cells. Federation Proceedings a3§,: 1653, 1979.
Eneanya, D.I., Daniel, F.B., Hart, R.W.: Asbestos (chrysotile intermediate) Alters the Metabolism of Benso(a)pyrene in Syrian Hamster Embryo Cells. Xlth International Congress of Biochemistry 7932003, Toronto, Canada July 1979
Eneanya, D.I., Duran, D.O., Wu, J., Andresen, B., Bianchine, J.: Characterisation of the Glucuronide of Diethyldithiocarbamic Acid Using Gas Chromatography and Mass Spectrometry. Pharmacologist 22: 458, 1980.
Eneanya, D.I., Bianchine, J.R., Duran, D.O., Andresen, B.: The Actions and Metabolic Fate of Disulfiram. Annual Review of Pharmacology and Toxicology 21: (1981) In Press.
iv Bianchine, J.R., Brys, D.A., Schwarz, R.D., Eneanya, D.I., Duran, D.O., Greenwald, J.E., Andresen, B.D.: Clinical Correlates of Changes in Receptors During Aging, In Neural Regulatory Mechanisms During Aging, Edited by Roberts, J. C19B0T
FIELDS OF STUDY
Major Field: Pharmacology
Drug Metabolism ...... Dr. J.R. Bianchine and Dr. N. Cerber
t Gas Chromatography and Mass Spectrometry...... Dr. B. Andresen
Biochemical Pharmacology. . . Dr. R. Fertel
Pharmacokinetics ...... Dr. N. Gerber
Physiological Chemistry . . . Dr. R. Panganamaia
R a d i o l o g y ...... Dr. R. Hart and Dr. B, Daniel
Tissue Culture...... Dr* R. Hart and Dr. B. Daniel TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS Li
VITA iv
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS ...... xii
INTRODUCTION 1
Literature Review ...... I
Chemistry of D i s u l f i r a a ...... 2
Pharmacokinetics of Disulfiram ...... 4
Administration ...... 4 Absorption 4 Distribution ...... 4 Metabolism ...... 5 Reduction of disulfiram ...... 5 Glucuronidation ...... 9 Non-enzymatic degradation ...... 9 Methylation...... 10 O x i d a t i o n ...... 11
Disulfiram and the Enzymes of the B o d y ...... 12
The Dehydrogenases ...... 12 The Oxidases ...... 13 Dopamine p-Hydroxylase ...... 14 Other Enzymes Inhibited by Disulfiram .... 14 General Mechanism of Enzyme Inhibition .... 14
Pharmacodynamics of Disulfiram...... IS
The Disulfiram - Ethanol Reaction ...... 15 Acetaldehyde and D E R ...... 16
vi Page
Disulfiram and Biogenic Amines ...... 18 Disul£iram~Induced Hepatotoxicity ...... 20 Carbon Disulfide and DS Neuropathies...... 20 Disulfiram Induced Psychosis ...... 22 Disulfiram Induced Acetonemia ...... 23 Inhibition of Oxidative Phosphorylation . . . 23 Other Side Effects of Disulfiram ...... 24
Disulfiram Interaction With Other Drugs ..... 24
Other Drugs with Disulfiram-Like A c t i o n ...... 26
Methods for the Determination of Disulfiram and Its Metabolites 28
Colorimetric methods ...... 28 Other methods ...... 29 Chromatographic assays ...... 29
STATEMENT OF PROBLEM: AN OVERVIEW...... 30
MATERIALS AND METHODS 32
Synthesis of Dithiocarbamic Acids ...... 33
Assay Procedure ...... 33
Extraction 36
Liver Perfusion Experiments ...... 37 Kinetic studies ...... 38 Choleretic effects ...... 40 Excretion of metabolites ...... 41
Intact Animal Experiment ...... 41 Decline of DDC in blood of rat at I.V. dose of 250 mg/kg ...... 42
Effect of Choleresis on Composition of Bile . . . 42
Analytical Methods ...... 42 Unmetabolized DDC ...... 43 DDC-conjugate by alkali hydrolysis ...... 43 Perfusate samples ...... 43 Page
Analysis of DDC-glucuronide ...... 44 Permethylation...... 44 Silylation ...... 45
Decline of DDC in IntactAn i m a l ...... 45
Effect of Choleresis on Bile Consumption . . . . 47
Absorption of Bile ...... 47
RESULTS ...... 48
Synthesis of Internal Standard ...... 48
Decline of DDC in the Perfusate With Doses of 100, 200 and 30 mg/kg N a D D C ...... 48
Choleretic Effect of DDC on Bile F l o w ...... 66
Electrolyte Studies ...... 66
Metabolites in Bile ...... ' ...... 69
Decline of DDC in Intact Rat ...... 70
DISCUSSION ...... 78
Internal Standard ...... * ...... 78
Choleresis ...... 82
Dose-Dependent Kinetics...... 86
DDC-Glucuronide ...... 90
SUMMARY AND C O N C L U S I O N ...... 93
BIBLIOGRAPHY...... 96
v m LIST OF TABLES
Table Page
1. Physical Properties of DDC and D P C ...... 35
2. Amount of Total Metabolites Produced in Bile Following The Addition of NaDDC. Compounds Include Unraetabolized DDC, Methyl* ated DDC and Free DDC fromConjugate .... 63
3. Effect of Choleresis on Major Electrolytes of Bile ...... 65
4. Comparison of DPC and Biphenyl as Internal Standard for D D C ...... 80
5. Chemical Composition of Human B i l e ...... 83
6. Types of Groups in Foreign Compounds to Which Glucuronic Acid Can BeTransferred . . 89
ix LIST OF FIGURES
Figure Page
1. The Structure of Disulfiram (Tetraethylthiuram disulfide), the Precursor of D'iethyldithio- carbamic acid ...... 3
2. Metabolic Pathways of Disulfiram and Metabolites. 6
3. Conjugation of Diethyidithiocarbamic Acid and Glucuronic Acid ...... 8
4. Condensation of Dopamine and Acetaldehyde co Form Salsolinol, a Tetrahydroisoquinoline. . . 19
5. Synthesis of Diethyidithiocarbamic Acid and Dipropyldithiocarbamic Acid . 34
6. Schematic of the Ambec Two/Ten Perfuser...... 39
7. Synthesis of the Sodium Sait of Dimethylsufoxide. 46
8. Typical gas chromatogram of MeDDC and the internal standard MeDPC in rats' blood, obtained isother- mally (140*C) on a 32 OV-17 glass column . . . 49
9. Mass Spectrum of MeDDC with Molecular Ion M*"163 and Fragment Ions at m/e 116, 88 and 60 . 50
10. Mass Spectrum for MeDPC with Molecular Ion Mf^iSl and Fragment Ions at m/e 176, 144, 102 and 9 1 ...... 51
11. Mass Spectrum for EtDDC with Molecular Ion, M*m177 and Fragment Ions at m/e 148, 116 and 88 . 52
12. Maas Spectrum for DDPC With Molecular Ion, M+^194 and Fragments m/e 148, 102 and 94 . . . 53
13. Nuclear Magnetic Resonance Spectrum Confirming the Structure of DDC ...... 54
x Figure Page
14. Nuclear Magnetic Resonance Spectrum Confirming the Structure of D P C ...... 55
15. Nuclear Magnetic Resonance Spectrum Confirming the Structure of E t D D C ...... 56
16. Standard Curve of DDC with DPC as InternalStandard. Each point is a Mean of 5 values from different Experiments...... 57
17. Dose-Dependent Metabolism of DDC in Perfusate of Isolated Perfused Rat Liver ...... 59
18. Choleretic Effect of DDC with Doses of 100 and 300 M g / K C ...... 61
19. Metabolites in Volume of Bile During Choleresis . 64
20. Absorption of Bile at Choleresis Following the Administration of 80 and 100 mg of DDC to an Intact Rat ...... 67
21. Effect of Choleresis on Absorption of Bile . . . 68
22. Gas Chromatogram of Control Perfusion Bile and DDC Perfusion Bile. The bile samples were permethylated...... 71
23. Gas Chromatogram of Control Bile and DDC Perfusion Bile. The bile samples were treated with BSTFA and IX TMCS ...... 72
24. Total Ion Trace of Bile Obtained from Isolated Perfused Rat Liver ...... 73
25. Mass Spectrum of TMS Derivatized DDC Glucuronide in B i l e ...... 74
26. Decline of DDC in Intact Rat . . .' * ...... 76
27. Structures of DPC and Biphenyl Compared to DDC for Use as Internal Standard...... 79
28. Michaelis-Menten Kinetics ...... 87
xi LIST OF ABBREVIATIONS
DDC Diethyidithiocarbamic Acid
DPC Dipropyldichiocarbanic Acid
DS Disulfiram
BSTFA N,0-bis-(TrinieChyl»ilyl)Trifluoroacecamide
TMCS Trimethylchlorosilane
W/V Weight by volume
-SH Sulfhydryl
DBH Dopamine beta-Hydroxylase
DER Disulfiram-Ethanol Reaction
NADP Nicotamide Adenine Dinucleotide Phosphate
ALDH Aldehyde Dehydrogenase
GC/MS Gas Chromatography and Masa Spectrometry
ID Inner Diameter
OD Outer Diameter
NaCI Sodium Chloride
KC1 Potassium Chloride
CaCl2 Calcium Chloride
MgCl2 Magnesium Chloride
Na2S04 Sodium Sulfate
NaH2P04 Dihydrogen Sodium Phosphate
NaHC03 Sodium Bicarbonate
xii co2 Carbon Dioxide 02 Oxygen
HaOH Sodium Hydroxide
DMSO Dimethyl Sulfoxide
NaDDC Sodium Diethyidithiocarbamic Acid
MeDDC Methyl Eater of Diethyidithiocarbamic Acid
MEDPC Methyl Eater of Dipropyldithiocarbamic Acid
M/e Maaa to Charge Ratio
TMS Trimethylailylation
EtDDC Ethyl Eater of Diethyidithiocarbamic Acid
UDPGA Uridine Diphoaphate Glucuronic Acid , , INTRODUCTION
Literature Review
Before the 1940'a, it was known that exposure to certain organic
sulfur compounds, including carbon disulfide, tetramethylthiuram
disulfide and tetraethylthiuram monosulfide, produced unpleasant re
actions upon coincident ingestion of ethanol (Jacobsen and Larsen,
1949). Tetraethyl disulfide (Disulfiram) (DS), first produced in
1881, was used to accelerate the vulcanization of rubber (Twiss
ejt 1922). In the 1930's, it was introduced into medicine as a
scabiescide (Hammerskjold, 1943). Disulfiram was found to be toxic
to lower forms of life which used copper containing respiratory
enzymes. Its toxicity appeared to be directly related to its ability
to chelate copper. Intestinal worms were especially sensitive and
hence DS was introduced as a vermicide. By chance, Held and Jacobsen
(1948b) consumed ei:hyl alcohol shortly after DS and promptly noticed
a peculiar and highly disagreeable reaction. Based upon this ethyl
alcohol-DS interaction, DS was proposed for the prophylaxis of
chronic alcoholism (Jacobsen, 1945). A new concept was then proposed
that the patient taking DS chronically, would not avoid alcohol for
fear of this disagreeable reaction*
Most studies of disulfiram, especially those reported two and a
half decades after DS was introduced for treatment of alcoholism,
1 were poorly designed and commonly lacked adequace control populations.
Statistical analysis of the data derived from the clinical studies as well as accurate and sensitive methods for determining DS and its metab olites in body fluids were not ideal in chose early years of investi gation. The rate of improvement in alcoholics using DS varied between 19-83% in different studies (Gerard and Saenger, 1966; Gairman,
— Jlk** 1951; Hoff, 1955; Hoff and McKeown, 1953; Wallerstein, 1958).
A definitive criterion for measurement of of improvement of alcoholism
is still required to obtain a clearer picture. Nevertheless, most clinicians experienced in the treatment of alcoholism are convinced
that DS is a useful drug in treatment of selected patients.
Chemistry of Disulfiram
Disulfiram (Fig. 1) is an off-white or light gray, odorless,
almost tasteless crystalline powder that is practically insoluble in water (0.7% W/V) but has varying solubilities in organic solvents
such as ethanol (3.82% W/V), anci ether (7.14% W/V). Its melting
point is 70°C-72°C and it has a density of 1.30. It has a molecular
weight of 296.54 and is a potent chelator of iron, copper and zinc.
Disulfiram and its major metabolite, diethyidithiocarbamic acid (DDC)
are both stable in basic medium (pH 7.0-9.0) but unstable in acid
acid medium up to pH 7.0. 3
C2 H3^ M 5 /C2 H5 N- C— S — S — C — c2 h / c2 h 5
FIGURE I
The Structure of Diaulfirem (Tetraethylthiuram disulfide), the Precursor of Diethyidithiocarbamic Acid.
< Pharmacokinetics of Disulfiram
Administration
In the 1950's, a daily oral dose of 1.5 g of DS was administered for prophylaxis to alcoholic patients.. As a result of the toxicity commonly manifested at such a high dose, it was reduced to an initial dose of 500 mg followed by a daily maintenance dose of 250 mg. The implantation of DS under the skin in a depot reservoir'was initiated in 1955 (Wilson, 1975). Subcutaneous implantation was attractive because it provided long-term treatment for up to six months for the noncomplying patient. However, inadequate knowledge of the physico chemical characteristics of such an implants as well as the variable bioavailability of DS have made this approach to treatment of alcoholism questionable.
Absorption
Disulfiram is rapidly but incompletely absorbed from the human gastro-intestinal tract. Hald and Jacobsen (1948a) found 20% of administered DS unchanged in feces. DeSaint-Blanquet et al. (1976) reported that 70-90% of the orally administered DS was absorbed in rats. In these studies the presence of DS and DDC were qualitatively demonstrated in blood, liver, kidney and muscle two hours after dosing.
Distribution
Because of its high lipid solubility, DS accumulated in the various fat depots. The brain consistently revealed the lowest con- Generations of DS and its metabolites have been found in the thyroid, thyroid, adrenals, pancreas, stomach, small and large intestines, muscle, liver, testes, kidney, lung, spleen and heart. These findings by Faiman et al. (1978a) and Stromme (1966) are largely based upom metabolism studies using radioactive DS. Following intraperitoneal administration of ^S-diethyldithiocarbamic acid (DDC), the thiol was found in highest concentration in the plasma, liver and kidney, with the lowest concentration detected in the brain.
Metabolism
Reduction of Disulfiram
The metabolism of DS [l] outlined in Figure 2 proceeds first by a reduction of the disulfide linkage to its corresponding thiol,
DDC [II] metabolite. The kinetics of this reaction may possibly be first order with respect to the disulfide (Fava et si., 1957) and may
Lead to formation of mixed disulfides with protein sulfhydryl groups.
The glutathione reductase system of the erythrocytes has been shown to reduce DS (Keleti, 1964; Stromme, 1965). It has been estimated that up to 50 g of disulfiram can be reduced by the adult human erythrocytes within 24 hours (Stromme, 1963b). This brisk reduction reaction causes the rapid disappearance of the parent compound from the blood stream
(Cobby et al., 1977a; Domar et al., 1947; Eldjarn, 1950; Linderholm and Berg, 1951; Prickett and Johnston, 1953). In in vitro studies,
Cobby et al., (I960) found that upon addition to whole blood, disulfiram was promptly reduced to the thiol within 4 min. Disulfiram may also FICURE 2
Metabolic Pathways Of Disulfiram and Metabolites
I Tetraethylthiuram disulEide (disulfiram) (a) Glutathione reductase
11 Diethyidithiocarbamic acid (b) Conjugation
III Glucuronide of Diethyldithiocarbamate (c) Non-enzyaatic degradation
IV Diethylamine •(d) Oxidative desulfuration (C-P450)
V Carbon disulfide (e) Oxidation
VI Sul fur (£) Sulfoxidase
VII Carbonyl sulfide (g) Esterases
VIII Carbon dioxide (h) S-aethylation: S-adenosyl methionine transmethylaae IX Sulfate
X Methyl ester of diethyldithiocarbamate
XI Methyl mercaptan
XII Formaldehyde
XIII Tbiocarboxylic acid CZH 7 FIGURE 3
Conjugation of DDC with Glucuronic Acid
CD 9
be pharmacologically inactivated by an interaction with protein by a mechanism similar to the drugs reaction with cystamine derivatives
(Eldjam and Phil, 1956). The DDC produced is further metabolized
via four different pathways. These are glucuronidation, non-enzymatic
degradation, methylation and oxidation.
Glucuronidation
Conjugation of DDC with glucuronic acid (Figure 3) is a major
detoxification mechanism of DS in man and ocher animals (Kasalder, 1963).
Metabolism studies of radioactive DS in the rat revealed that about 502
of a dose was excreted in urine as the glucuronide metabolite [III]
(Stromme, 1965a). However, it is possible that a small portion of the
glucuronide conjugate may be hydrolysed by the esterases in the intes
tine to yield DDC during enterohepatic recirculation.
Non-enzymatic degradation
Aspila ad. have shown that the rate of decomposition of
DDC is pH dependent. In acid, DDC decomposes rapidly to diethylamine
[IV] and carbon disulfide [V] (Stromme, 1966). Diethylamine may be
excreted unchanged by man (Williams, 1959) but further degradation to
ammonia and acetaldehyde may occur. An enzymatic degradation follows.
In the presence of NADPH and cytochrome P-450 (a mixed function oxidase),
carbon disulfide is oxidatively desulfurated to carbonyl sulfide [VII]
and elemental sulfur (Matteis, 1974). The carbonyl sulfide can be
be further oxidized to carbon dioxide and sulfur. The sulfur, which
can be covalently bonded to carbon and other compounds, can be metabo- 10
lized to sulfate by the sulfoxidases (Rainey, 1978). Carbon disulfide • * the sulfoxidases (Rainey, 1978). Carbon disulfide also reacts with
certain amino acids to yield dlthiocarbamates (Brand and Fleet,
1970). In addition, dithiocarbamate can be cleaved back to carbon
disulfide and amino acids or isothiocynates and hydrogen sulfide (H2S).
Both of these pathways may subsequently yield sulfate ions (Davidson and
Feinleib, 1972). These observations account for the large amount
.of sulfate ions in the urine of man and animals during intoxicated with
carbon disulfide poisoning or DS.
Methylation
In radiochemical experiments (Cobby et al., 1977b) DS was
administered intraperitoneally and 2 hours later the methyl ester of
DDC (MeDDC) was calculated to represent 0,05% of the given dose.
In another experiment, DDC was administered to dogs intravenously
and S-methylation accounted for approximately 27% of metabolism of
the administered dose (Gessner and Jakubowski, 1972). A highly
active S-adenosyl methionine transmethylase catalyzes the methylation
of DDC. This enzyme, found both in kidney and liver is associated
with the 100,000 x g microsomal fraction. Biotransformations from
mercaptans to methylthioesters has also been reported for some ali
phatic thiol compounds (Snow, 1957; Stripp il., 1969). Similarly,
S-methylation is involved in the metabolism of thiopurines, thiopyri-
midines (Reray, 1963; Sarcione and Sokal, 1958; Sarcione and Stulzman,
1960) and thiopentals (McBain and Menn, 1969). The methyl ester of 11 the DDC derivative [X] may be accacked by esterases, generating a methyl mercaptan which may in turn be oxidized co sulfate and formaldehyde* In support of this conclusion, the observation is that the sulfate is produced rapidly from other thioester compounds. For example, 6-methyl-raercaptopurine generates sulfates more rapidly
Chan 6-mercaptopurine when administered co rats (Canellakas and
Tarver, 1952). This may be due to cleavage of methyl mercaptan from the S-methyl compound and the letter's oxidation to produce inorganic sulfate (Cobby et al., 1977; Maw, 1954). S-methylthio- glycollic acid is also readily oxidized to sulfate while the corresponding S-phenyl-thioglycollic acid yields no significant amount of sulfate.
A similar oxidative pathway may act as an important source of sulfate in the metabolism of disulfiram. Approximately 62-74% of sulfate formed from the metabolism of DS involves oxidation of raethyl-mercaptan (Cessner and Jakubowski, 1972). The methyl ester of DDC is chemically very stable in blood (Cobby et al., 1977).
While slowly cleared from the body, it does not go through the covalent disulfide interchange reaction by which DS inhibits aldehyde dehydro genase (Kitson, 1976). A thioalcohol [XIII] formed from this methyl- » ’ ester, can be glucuronidated and excreted in urine.
Oxidation
Stromne (1965b) demonstrated the reoxidation of DDC back to DS, However, this reoxidation involved only about 4% of DDC formed. It is known chat in the presence of atmospheric oxygen, DDC 12 can be reoxidized co DS. Such an oxidation can be effected by the oxidases in the body.
Diflulfiram and the Enzymes of the Body
The Dehydrogenases
Shortly after it's introduction in the management of alcoholism, the effect of DDS on important enzymes system was investigated. In in vitro experiments, DS produced a long lasting decrease in liver alde hyde dehydrogenase activity (Deitrich and Erwin, 1971). This suggested that recovery of activity might depend upon synthesis of new enzymes.
Certain workers claimed that DDC was the active inhibiting com pound in vivo (Li and Vallee, 1969), while others found DDC co be inef fective, at inhibiting aldehyde dehydrogenase (Deitrich and Erwin, 1971).
Stromme (1965b) found that both ^g-DDC and 35g_ug actively bind to
liver and plasma proteins. The interaction of either DS or DDC with protein -SH groups can yield mixed disulfides. Alternately, it is also possible that the sulfhydryl groups of aldehyde dehydrogenase
(Deitrich and Hellerman, 1963; Deitrich, 1967) might participate in
this reaction in vivo. Additionally, one of the mixed disulfides could interact with and inhibit aldehyde dehydrogenase. Inhibition of aldehyde dehydrogenase (ALDH) can be prevented but not reversed by sulfhydryl containing compounds (Li and Vallee, 1969) such as dithiothretol.
Fructose-1,6-diphosphate dehydrogenase and succinic dehydrogenase
are also inhibited by DS (Chefurka, 1952; Keilin and Rartree, 1940).
Therefore, glycolysis, the tricarboxylic acid cycle, and pentose phos 13 phate shunt are affected by DS. Clyceraldehyde -3-phosphate dehydro genase is also inhibited by DS (Nygaard and Sumner^ 1952).
The Oxidases
Rjeldgaard (1949) found that DS at a concentration of 1.2 x 10"7 mole per ml in rabbit liver homogenate caused marked inhibition of aldehyde oxidase, a flavoprotein. He also demonstrated that the reduced disulfiram (i.e. DDC) was not as effective an inhibitor of aldehyde oxidase as was DS. Further, following inhibition by DS, the enzyme could not be reactivated with glutathione. He concluded that the S-S linkage of DS may be important for the inhibition of aldehyde oxidase. Another flavoprotein enzyme, xanthine oxidase, not
-only oxidizes certain purines but also utilizes acetaldehyde as a substrate, converting it to acetic acid by direct utilization of oxygen. Disulfiram inhibits xanthine oxidase activity in rat liver, lung and spleen (Richert et al., 1950) but not in milk. Studies of this enzyme (Ball, 1939; Corran et al., 1939) suggest that it is composed of two different prosthetic groups; one an oxidase system which is responsible for reoxidation of the reduced enzyme by atmos pheric oxygen, and the other, a dehydrogenase action capable of transferring hydrogen from xanthine to methylene blue.
In a study of 32 male alcoholics, 7 developed psychosis. Inter estingly, the platelet monoamine oxidase and plasma amine oxidase
activity were significantly lower and the red cell catechol-O-methyl
transferase was higher in the patients who developed psychosis than
those that did not develop psychosis (Major et al., 1979). It is 14
possible thee this decrease in amine oxidizing activity may result in a persistence of catecholamine action and hence the psychotic response. The disulfide grouping of DS is important for the irrever sible inhibition (Niems et al., 1966) on D-amino oxidase by DS.
Dopamine-g-Hydroxylase
Another important amine degradative enzyme inhibited by disulfiram is dopamine-6-hydroxylase (DBH), an enzyme that catalyses the conversion of dopamine to norepinephrine (Goldstein Other Enzymes Inhibited by Disulfiram The aldolases are inhibited by DS and Stromme (1963a) has shown that DS (but not DDC) is a potent inhibitor of hexokinase. General Mechanism of Enzyme Inhibition The reduction of the disulfide bond of DS by glutathione SH-con- taining compounds leads to the formation of mixed disulfides which in turn, may inhibit selected enzymes. It is possible that disulfiram 15 inhibits more or less, all -SH enzymes and cofaccors with -SH groups e.g., CoA and thioctic acid. DS and DDC are potent chelators of copper and other metals. Chel ation of the metal portion of an enzyme by DS or DDC might lead to the inactivation of that enzyme. Metal containing enzymes inhibited by either DS or DDC include zinc containing enzymes such as aldehyde dehydrogenase and glyceraldehyde phosphate dehydrogenase (Keleti, 1964; Stoppani jet , 1966) as well as the copper dependent enzymes, aldehyde oxidase and dopamine - B“hydroxylase (Kaufman and Friedman, 1965). Pharmacodynamics of Disulfiram The Disulfiram-Ethanol Reaction (PER) Administration of DS to a subject 12 hours prior to about 15 ml of alcohol produces a series of unpleasant effects within 15 minutes. These reactions begin with a The hypotension may produce pallor, weakness, vertigo, nausea and vomiting. Confusion, drowsiness and sleep usually follow with eventual complete recovery within two to four hours (Lundwall and Baekeland, 1971; Raby, 1956; Weisman, 1968) providing, of course, that the amount of alcohol ingested is modest. Frequently, there are transient changes in the electrocardiographs such as flattening of the T-waves, depression 16 of the S-T segment end Q-T prolongation in a pattern suggestive of right ventrieulat "strain" (HaCabe and Wilson/ 1954; Markham and Hoff, 1953). These reactions are collectively called the disulfiram -ethanol reaction (DER). As might be expected, absorption of very small amounts of alcohol by a patient on DS treatment may not precipitate the DER. For example, a small amount of wine ingested during religious communion, the absor ption of the dilute alcohol medium in a bronchial nebulizer spray, or ear drops containing ethyl alcohol did not result in DER in some patients (Rothstein, 1970). Acetaldehyde and DER In the beginning, the reactions that developed following the administration of alcohol and DS were attributed to increase in acetal dehyde formation in the body. Disulfiram was thought to inhibit the dehydrogenases, aldolases and oxidases that utilize acetaldehyde as a substrate. Hald and Jacobson (1948c) documented an eight-fold increase in acetaldehyde concentration in the blood when 40 ml of alcohol was consumed after 1.5 g of DS was taken the previous day. Using gas chromatographic methods, Truitt (1962) showed that the blood concentration of acetaldehyde in a nonalcoholic subject who had consumed a moderate amount of alcohol was less than 1.0 mg/ml. However, the level of acetaldehyde produced by a standard dose of alcohol in an alcoholic subject appears to be highly variable. Majchrowicz et al. (1968) found no difference in the rate of alcohol or acetaldehyde 17 metabolism in rats after a two month period of forced consumption of 202 alcohol or acetaldehyde. This finding is in agreement with other results published (Forney and Hager, 1969; Himwich, 1956; Kalanc, 1962; Mardones, 1963). On the other hand, there is some evidence that chronic administration of alcohol may increase formation of acetaldehyde by inducing the synthesis of alcohol dehydrogenase or NADP dependent hepatic microsomal enzymes (Lieber and DeCarli, 1968). The highest level of acetaldehyde found in the central nervous system appears in the cerebellum which controls movement, position and equilibrium. Mitochondria have the greatest sensitivity to acetaldehyde in vitro (Keissling, 1962a; 1962b). Alcohol dehydrogenase found in the soluble fraction of brain homogenates (Raskin and Sokoloff, 1968) may be responsible for the higher leveL of acetaldehyde found in brain following alcohol consumption. Twice as much acetaldehyde has been reported (Keissling, 1962a; Ridge, 1963) in the brain following alcohol treatment (Duritz and Truitt, 1966). The amount of acetaldehyde in rabbit blood increases with increasing dose of DS with the alcohol dose as a constant (Hald and Jacobsen, 1948c). Again, when the animals were adequately dosed with DS, the amount of acetaldehyde increased with the increase in alcohol (Hald et al., 1949), Following perfusion • • of liver from normal and DS treated animals with blood to which ethyl alcohol had been added, there was a marked increase of acetaldehyde in the liver treated with DS as compared to the untreated liver. The tentative inference from this study is that the liver may be the major organ involved in the acetaldehyde production which in turn is important to the DER phenomena (Jacobsen and Larsen, 1949). While ocher tissues 18 may be Involved in this acetaldehyde production, they play only a minor role when compared to the amount of acetaldehyde produced in the liver. Recently, it has been made clear that acetaldehyde alone does not duplicate all of the reactions noted during DER. In dogs, anesthetized with chloralose, intravenous administration of acetaldehyde (8-9 mg/kg body weight) caused an increase in arterial blood pressure, because of peripheral vasoconstriction (Handovsky, 1934; 1936). However, peripheral vasoconstriction does not explain cutaneous flushing, vasodilation and hypotension that may attend the DER. Interestingly, acetaldehyde inhibits dopamine-8-hydroxylase and thus the conversion of dopamine co norepinephrine. The resultant depletion of norepinephrine stores in the heart and blood vessels then allows acetaldehyde to act directly on these tissues to yield hypotension and vasodilation (Truitt and Walsh, 1971). This might explain this seeming paradox. At concentra tions of 0.2 to 0.7 mg X in blood or plasma, acetaldehyde infused intravenously into the body of normal humans produced marked increase in heart rate and ventilation similar to the hyperventilation observed during DER (Asmussen , 1948; Gyorgy, 1932; MacLeod, 1950). Diaulfiram and biogenic amines Through a condensation reaction, acetaldehyde can react with dopamine in vitro to form salsolinol, a tetrahydroisoquinoline (Cohen and Collins, 1970; Collins aU, 1979). The cetrahydroisoquinolines are suspected of being involved in the addiction to alcoholism. It is possible that DS treatment causes the production of salso linol via two different mechanisms. First, DS greatly increases the N i N l f l M f k l H ADN ALBM [01 Schtff?a I w FIGURE 4 Condensation of Dopaalne and Acetaldehyde to Fora Salsollnol, A Tecrahydrolsoqulnollne. 20 accumulation of acetaldehyde by blocking aldehyde dehydrogenase. Secondly, DS inhibits dopamine-g-hydroxylase and thus allows dopamine to accumulate. As a result, the in vivo condensation of these two substances to form salsolinol is enhanced (see Figure 4). A possible role of salsolinol in DER has not been established. Disulfiram induced hepatotoxicity Some of the adverse effects and toxicity of DS may be disguised or overlooked as ethanol induced reactions. Disulfiram has caused hepatotoxicity in patients (Eisen and Ginsberg, 1975; Keefe and Smith 1974; Knutsen, 1949; Ranek and Andreasen, 1977). Although Goyer and Major (1979) failed to find dose related hepatotoxicity in 35 alcoholics receiving the drug over a three week period, nine of the patients did show subclinical hepatotoxicity unrelated to the dose. These investigators attributed these findings to an "idiosyncratic1' response in the patients. Other investigators demonstrated that chronic DS and ethanol treatment in animals significantly enhanced hepatic lipid peroxidation. Further, incubation of liver homogenates with acetaldehyde damaged membrane lipids of the hepatocyte (Stege et al., 1977). Carbon disulfide and DS neuropathies Peripheral nervous ayatem effects of disulfiram Disulfiram has been implicated in the development of sen sorimotor peripheral neuropathy (Gardner-Thorpe and Benjamin, 1971; Graveleau, 1972; LeQuesne, 1975; Moddei et , 1978). In ail cases, this neuropathy improved upon discontinuation of DS treatment and 21 reappeared on resumption of DS therapy (Dyck, 1975). The denervation potentials were detected in all cases in distal muscles and the con** duction velocities ranged from normal to slightly reduced. These electrophysiological findings suggest that DS induces degeneration of the axon, similar to that observed in experimental isoniazid neuropathy (Schaepfer, 1964a; 1964b) or during the early stage of Wallerian degeneration after crushing injury to nerve (Vial, 1958). With no observed myelin breakdown in the absence of axonal degeneration, the stages of the degeneration resemble axonal lesion (Dyck, 1975). Disulfiram has been shown to cause optic atrophy and encephalopathy (Hotson and Langston, 1976). These neuropathies occur in the absence of the DER suggesting that more than inhibition of acetaldehyde dehydro genase or DBH may be involved in these neuropathies. Carbon disulfide, a metabolite of disulfiram, has been shown to cause axonals degeneration and neuropathy in animals (Linnoilal et al., 1976; Zendzikovski 1974) and in man (Alpers and Levy, 1940; Hopkins, 1975). Carbon disulfide distributes in all tissues in the body, (Brieger, 1971) with the brain having the slowest uptake and longest retention time. Generally, 76Z to 95Z of the dose is deposited and metabolized by body tissues. Five to 301 is exhaled and less than one percent is excreted in the urine. Lewey et al. (1941a; 1941b) has suggested possible explanations of the mechanism underlying carbon disulfide induced generalized neuropathy. They proposed that DS may induce a pyridoxine deficiency similar to that described with carbon disulfide (LeQuesne, 1975; Vasak and Kopecky, 1967b). The carbon disulfide reacts with pyridoxamine causing pyridoxine deficiency 22 (Dassault and LePage, 1976). In general, carbon disulfide interferes with primary amino acid groups. In intact animal models carbon disulfide leads to decreased excretion of pyridoxic acid, enhanced excretion of xanthurenthic acid (which are indicators of pyridoxine deficiency) and decreased plasma level of pyridoxal phosphate (Djuric et al., 1973. Vasak and Kopecky, 1967a). The thiamine deficiency that results from chronic liver injury, commonly present in alcoholic patients receiving DS treatment, makes them especially’vulnerable to the neuropathy associated with DS. Further, such alterations may be of major importance in perpetuating liver injury since interference with dependent biochem- ical reactions may contribute to liver injury and interfere with its repair (Alpers and Levy, 1940). Disulfiram induced psychosis Since its introduction into clinical medicine, DS has provoked psychological and neurologic abnormalities (Liddon and Satran, 1967). Major symptomB include, a peculiar disorientation in which certain patients are conscious but disinterested in their environ** ment, gradual amnesia, anxiety, insomnia, and violent restlessness* Often, these symptoms necessitate reduction of DS dose by the consulting physician (Martensen-Lersen, 1951). These psychoses might relate to the personality difficulties of the patient that originally led to alcoholism and then become manifest after pharmacologic prohibition of alcohol. The psychological difficulties may be handled by regressing to a psychotic reaction. 23 Disulfiram induced acetonemia DeMaster and Nagasawa (1977) have shown chat chronic DS administration increases the concentration of serum acetone in both man and the rat. For example) administration of an oral dose of disulfiram (0.5 g/kg) to rats for three consecutive days increased the fasting blood acetone twenty-five fold. Similarlyt in humans treated with 250 mg of DS daily for a minimum of one month, the increase in acetone in expired air was fifteen-fold greater than in matched controls. Acute administration of DS 0.5 g/kg to rats orally raised the serum acetone level five-fold. This acetonemia may not be related to ALDH inhibition by DS because other ALDH inhibitors such as cyanamide and pargyline did not produce increase in blood acetone. The exact mechanism leading to the increased production of acetone in man by DS remains unclear. Inhibition of oxidative phosphorylation Disulfiram enhances the toxicity of normobaric oxygen to rats in a manner similar to paraquat (Deneke et al., 1978; Fisher et al., 1973). An interaction of DS with the electron transport system may increase the production of superoxide ions (Hassan and Friudorich, 1977) which in turn may cause edematous lung lesions. DS inhibits NAD*-dependent mitochondrial oxygen consumption (Edwards, 1949; Rassinen, 1967) and oxidative phosphorylation and ion transport in rat liver mitochondria. 24 Other side effects of disulfiram Disulfiram has led to convulsions in alcoholics receiving therapy (Price and Silberfarb, 1976). While the mechanism of this disulfiram-induced seizure in alcoholics remains unknown, it must be understood that the chronic alcoholic is particularly prone to seizure disorders for a variety of reasons. Discontinuation of DS therapy has been suggested in these cases. An unusual sign of increased difficulty with colostomy due to DS has been reported. This difficulty was relieved on discontinuation of DS (Miller, 1977). Disulfiram should be avoided in pregnancy because of suspected teratogenicity (Nora et al., 1977). Disulfiram Interaction With Other Drugs The duration and intensity Of drug action often are modified by the activities of drug metabolizing enzymes primarily located in the hepato- cytes. These enzymes catalyze the metabolism of drugs by many pathways such as hydroxylation, dealkylation, deamination, sulfoxidation and glucuronide formation. One drug can inhibit the metabolism of a second drug by inhibiting the enzyme necessary for the detoxification of the second drug. As a result, the plasma and tissue half-life is prolonged and both the therapeutic and the toxic effects of the drug are potentiated. In contrast, the administration of one drug may reduce the pharmacologic activity of a second drug by stimulating the hepatic drug metabolizing enzymes and thus increasing the inactivation of the second drug. Fewer drugs are known to inhibit drug metabolizing enzymes than are known to induce the activity of the same enzymes* Some of the drugs known 25 co inhibit drug metabolizing enzymes include nortriptyline (Vessel et al., 1970), methandrostenolone (Weiner et al., 1965) oxyphenbutazone, allo- purinol, methylphenidate (Garrettson ^ al., 1969) phenyramidol (Solomon And Schrogie, 1966) and DS (Rothstein, 1968; Vessel et al,, 1971). Drugs that that stimulate drug metabolism include the barbiturates, antihistamines, hypoglycemics and uricosuric agents. Disulfiram increases the half-life of antipyrine (Vessel et al., 1971) by inhibition of the hepatic microsomal mixed function oxidases. Rothstein (1968) reported the enhanced effect of warfarin following DS administration. O'Reilly (1973) studied the pharmacologic mechanism for Che interaction of DS and warfarin and suggested that DS inhibited the enzymes responsible for the hydroxylation of warfarin. This impairs Che metabolic clearance of warfarin, prolongs the serum half-life for the unchanged warfarin which in turn inhibits the synthesis of vitamin K dependent clotting factors resulting in an exaggerated hypoprothrom- binemic effect. Disulfiram inhibits the metabolism of phenytoin (Olesen, 1966; 1967). Svendsen et al (1976) confirmed the latter study on phenytoin but showed chat DS did not inhibit tolbutamide metabolism. The metabolism of phenytoin, antipyrine and coumarins all involve hydroxylation by the hepatic microsomal enzyme system of man and animals. However, Svendsen et al., (1976) found that although tolbutamide is also oxidized by hepatic microsomal enzymes, its metabolism was not inhibited by DS. He suggested that DS may be a more specific inhibitor in the microsomes enzyme system. Other drugs whose biotransformations are inhibited by DS include chlordiazepoxide, hexobarbital and thiopental (Giarman 26 ec al., 1951). The coxicity of certain centrally acting drugs have been increased by DS in rats. These drugs include morphine, meperidine, amphetamine and barbital (Sharkawri, 1978). Disulfiram increases the carcinogenicity due to ethylene bromide (Yodaiken, 1978). While the combined use o£ DS and metronidazole are not synergistic in the manage** ment of alcoholics, their combined use may increase the incidence of confusional psychosis (Rothstein, 1970b). Lang et al., (1976) demonstrated that DS and DDC diminished the t inductive effect of phenobarbital on cytochrome P-450 content and p-nitroaniaol O-demethylation, while both compounds showed additive effects in inducing NADPH-dependent cytochrome reductase. Contrary to other investigations (Bahr and Bartilason, 1971; Remmer et al., 1976), Maraelos et al., (1976) found that glucuronidation and hydroxylation enzymes are not in close functional relationship as might be suggested by their similar response to induction or inhibition by foreign compounds. It is possible that glucuronidation could be stimulated while there is coincident inhibition of the hydroxylation enzymes. Other Drugs With Disulfiram-Like Actions Tetramethylthiuram disulfide, tetramethylmonosulfide, tetraethyl thiuram monoBulfide, all affect the metabolism of ethanol in a fashion similar to disulfiram. Interestingly, in rabbits, the thiuram mono sulfides have greater capacity to induce acetaldehydemia than do the corresponding disulfides (Barnes and Fox, 1955). Citrated calcium cyanamide was introduced as a less powerful alternative to DS (Collins and Brown, 1960; Ferguson, 1954). This compound inhibits rabbit liver aldehyde dehydrogenase in vivo. However, the severity of the ethanol withdrawal reaction in mice (Deitrich and Erwin, 1975) is less than chat demonstrated with DS. Cyanamide also potentiates the ethanol induced shift in the metabolism of norepinephrine. The same unpleasant symptoms of DER are experienced on drinking alcohol after eating the common mushroom, Coprinus Atramentarlus. The active ingredient chat elicits this reaction is under investigation (Hatfield et al.., 1974; 1975). Some hypoglycemic sulfonylurea compounds such as carbutamide, chlorpropamide and tolbutamide exhibit a disulfiram-like action (Assaad and Clarke, 1976; Rothstein, 1970b; Royer e t ^ U , 1962; Svendsen et al., 1976; Truitt et al., 1962). Fodgainy and Brassier found (1968) that these sulfonylureas noncompetitively inhibit an aldehyde dehydrogenase and stated that this observation accounts for the DER-like syndrome either by an accumulation of acetaldehyde, or by an alteration in the metabolism of serotonin. However, Assaad and Clarke (1976) found no specific interaction in vitro between the sulfonylureas and aldehyde dehydrogenase. Pyrogallol also increases acetaldehyde concentrations during ethanol metabolism (Collins e££l*« 1974) by inhibiting the aldehyde oxidation pathways. Other compounds with DS-like action include hydrogen sulfide, tetraethyl lead, animal charcoal and aminophenasone with phenylbutazone (PullarStrecker, 1955). Metronida zole has been reported to reduce the craving for alcohol (Mottin, 28 Methods For The Determination of Disulfiram and ita Metabolites A severe handicap to the study o£ the metabolism of DS has been lack of very sensitive assay methods for determining the drugs and its metabolites. Analytical methods which have been used include (a) colori metric assays (Divatia et a U , 1972; Domar et al., 1947; Farago, 1967; Fried, 1976; Linderholm and Berg, 1951; Sauter et al., 1976; Tompsett, 1964), b) polarographic methods (Brand and Fleet, 1970; Brown et al., 1974; Gregg and Tyler, 1950; Prue a^., 1972; Taylor, 1964), c) pro- magnetic resonance (Sheinin, 1978), d) radiometric assay (Eldjam, 1950; Faiman et al., 1978a; Iber et al^., 1977; Stromme, 1965b) and e) chromatographic assays. Colorimetric Methods The basis for colorimetric assays is the ability of a compound to form a complex that has a color. Disulfiram forms a chemical compound with copper ion, which is yellow in color and stable in carbon tetra chloride. Cupric diethyldithiocarbamate is determined photometri cally at 430 nm Domar _et art., 1947; Linderholm and Berg, 1951; Tompsett, 1964). Cul + (C2H5)2 NCSSSCSN (C2H5)2 (C2H5)2 NCSSCu SCSN (C2H5>2 + l/2I2 Diethyldithiocarbamic acid also reacts with copper to yield a yellow color which absorbs at 430 nm (Domar et al., 1947; Linderholm and Berg, 1951; Tompsett, 1964). 2 ( c 2h 5 )2 ncss + cu2+ (c 2h 5)2 ncsscuScsn (c 2h 5 )2 Disulfiram, cyanide and ethanol react to give a color maximum between 520 and 580 nm (Faiman et al., 1978b) with an absorption that can be spectrophotometrically determined. The pitfalls of these methods include lack of sensitivity and specificity, and often proteins interfere with the spectrophotometric determinations. * * Other Methods Polarographic and proton magnetic resonance methods are not suitable for biological fluids and tissues. The radiometric assay is of limited use, a consequence of high protein-binding characteristics of disulfiram* Chromatographic Assays The assays developed so far involve the methylation of DDC which is then chromatographed either by HPLC or gas chromatography (Cobby et al., 1977a; Faiman et al., 1978b; Sauter and Wartburg, 1977). In the HPLC assay, DDC is methylated using dimethyl sulfate and chromato graphed on Porasil column. In gas chromatography, methyl iodide is used in the methylation process. We have confirmed the gas chromato graphic assay and also made some modifications. We have successfully ethylated DDC and methylated dipropyldithiocarbamic acid for use as internal standards. We believe that the methyl ester of dipropyldith iocarbamic acid has more of the extraction characteristics of MeDDC and would serve as a better internal standard than biphenyl as pre viously reported internal standard. STATEMENT OF PROBLEM: AN OVERVIEW r * The isolaced perfused rat liver has been used as a convenient technique for the investigation of the biotransformation of drugs and other chemicals. Generally, the metabolites secreted in bile are similar to those observed in the intact animal, especially when the drug is metabolized mainly by the liver (Thompson et al., 1973; Gerber et al., 1971; Gerber 1977; Lynn et al., 1977). The rat liver has been shown to have a high capacity for conjugation of drugs with glucuronic acid and the conjugates are then excreted in bile (Abou-El-Makarem £t a U , 1966). Some drugs, e.g. valproic acid, naltrexone, and diethylmaleate have been shown to increase the rate of bile flow. The choleretic effect has been attributed to several factors among them, an osmotic effect resulting from the excretion of metabolites, especially ester glucuronides in bile (Dickinson e£ al., 1979). Diethyldithiocarbamic acid has produced such choleretic effect in our experiments. Disulfiram is metabolized, first, by a rapid reduction of the % * disulphide to its corresponding thiol, diethyldithiocarbamic acid (DDC). Cobby et al., (1977a) have found in in vitro studies that the levels of Disulfiram are not detectable 4 min after its addition to whole blood. In view of this brisk reduction, we decided to look at the reduced product DDC and study its kinetics. In modification of the method of 30 31 Cobby et al., (1977a) we sought: co synthesize an analog o£ DDC for use as internal standard in gas chromatographic assay. After developing this assay for DDC| it would be used to determine the half-life of DDC at different doses. Further identification and quantification of ocher metabolites of Disulfiram excreted in bile was undertaken. Experiments were undertaken to a) study the disposition of DDC in the perfusate and bile, b) identify the biotransformation products of DDC in bile, c) examine the effect of DDC or metabolites on the rate of bile flow and d) study the enterohepacic recirculation of DDC in intact rat. MATERIALS AND METHODS Reagents Disulfiram was purchased from Ayerst Laboratories. Sodium diethyl- dithioearbamate was supplied by Sigma Chemical Company; methyl iodide was purchased from J.T. Baker Chemical Company, carbon disulfide, hexane and chloroform were purchased from Mallinckrodt Inc.; ethyl iodide was purchased from Fisher Scientific Company and dipropylamine and lodomethane~d3 were purchased from Aldrich Chemical Company Inc. Apparatus Gas chromatography was performed with a Varian instrument, Model 3700, equipped with a flame ionization detector. A coiled glass column (1.82 m x 2 mm I.D.) was packed with 32 OV-17 on gas chrom-Q, 100- 200 mesh. Dry helium (30 ml/min) was used as the carrier gas. The oven injector port and detector block temperatures were maintained at 130, 250 and 320*0, respectively. The detector response was monitored by a Varian strip chart recorder Model 9176, and the peak heights were measured directly. Electron impact mass spectra were obtained at 70 eV on a Hewlett Packard Model computer guided 5985 gas chromatograph/mass spectrometer system. Electrothermal melting point apparatus was used to determine the melting points of DDC and DPC. 32 Synthesis of dithiocarbamic acids Dipropylamine (0.05 mole) was placed in a 50 ml tube containing 10 ml of hexane. The tube was placed in a beaker of water and 0.05 mole of carbon disulfide was added dropwiae. On cooling, dipropyldithio- carbamic acid (DPC) crystallized out and was washed repeatedly with hexane. Diethyldithiocarbamic acid (DDC) was synthesized using the above procedure but substituting diethylamine for dipropylamine (Fig. 5). The yield as calculated by using the following formula: actual yield (grams) X Yield ■ theoretical yield (grams) x 100 Theoretical Yield ■ Number of moles of limiting reagent X molecular weight of product Assay Procedure Methylation of DDC or DPC A 1 mg sample of DDC or DPC was dissolved in 1 ml of water. In a 15 ml screw-capped (Teflon-lined) culture tube, 0.A ml of methyl iodide was added. The tube was immediately mixed for 40 seconds on a Vortex shaker. This procedure constituted the methylation step. Ethylation of DDC is also achieved using the above procedure but by substituting ethyl iodide for methyl iodide and mixing for 60 sec. Assays in whole blood and urine were accomplished by substituting the biologic fluid for water. \ N-C-SH+CH3I v 'N-C-SCH3-*-HI / s /: R R Methyl Ester of the Dithiocarbamate C 3 H 7 \ C 3 H 7 \ N H + C S , ------» N — C — S H C H / r L I / ■ !i 3 7 3 7 S CARBON DIPROPYLDITHIOCAR8AMIC DIPROPYLAMINE DISULPHIDE ACID (DPC) C2 Hg \ C22 Hg S\ 'NH + CS2 ► Nj-C C 2 H 5 c2 H g s CARBON DIETHYLDITHIOCARBAMIC DIETHYLAMINE DISULPHIDE ACID (DDC) FIGURE 5 * Synthesis of Dlechyldlthiocarbaaic Acid and Dlpropyldithlocarbamlc Acid. 35 TABLE I Physical Properties of DDC and DPC Synthesis of DDC Synthesis of DPC Color of crystals ■ white Color of crystals ■ white Melting point ■ 80-8l*C Melting point ■ 112-113*C Yield - 70* Yield - 71* 36 Hxtrmotion A 5 ml amount of chloroform was added and the tube inverted gently every second for 10 mint The tube was then centrifuged for 10 min to separate the phases. The organic phase was transfered to a 5 ml Reactivial (Pierce Chemical Co., Rockford, IL) and evaporated to dryness under a 4 stream of dry nitrogen and reconstituted in 100 pi of chloroform. One pi was injected into the gas chromatograph or gas chromatograph/mass spectrometer. 37 Liver Perfusion Experiments Materials Male Sprague-Dawley rats (400 g) were obtained £rom Laboratory Supplies, Indianapolis, IN and housed in stainless-steel cages in a well ventilated room at 21*C and maintained on Lab Chow and water ad libitum. Diethyldithiocarbamic acid was purchased from Sigma Chemical Company; chloroform was purchased from Mallinckrodt Inc. and ether and methyl iodide from the J.T. Baker Chemical Company. Procedure Perfusion Medium. The perfusion medium contained the following NaCl 109 mM; KC1 4.0 mM; CaCl2 1.6 mM; MgCl2 0.5 mM; Na2S(>4 0.5 mM; NaH2P(>4 1.0 mM; Sucrose 12.0 mM; Glucose 10 mM; NaHC03 25 mM. The medium was saturated with C02/02 (5:95 v/v) prior to entry into the portal vein. Surgical Technique. Light ether anesthesia was used during cannu- lation of the bile duct and portal vein. A midline incision 4-6 cm in length was made along the abdomen wall. The intestinal tract was gently removed to the gauze-protected outside of the body wall and wrapped in saline-saturated gauze to prevent dehydration. The duodenal loop was exposed and the bile duct ligated approximately 2.5 cm from the liver. The duct becomes filled with bile a few seconds after ligation. A second ligature is placed loosely around the duct between the first ligature and the liver, taking care to avoid damage to any blood vessels. Using a 25 gauge needle, and pulling the loose ends of the first ligature to preserve tension, the duct is punctured on one side approximately 2 cm from the liver. Immediately, a length of polyethylene cannula (0.011" tnm ID and 0.024" OD) having a slightly tapered end, is introduced 0.5 cm into the duct and secured by tightening of the second ligature. The bile duct is cannulated proximal to the liver to ensure that only bile and not pancreatic juice is collected. In order to give extra anchorage, the loose ends of the first ligature are also tied around the cannula. Bile now flowed freely from the cannula into a collecting tube. One hundred units of heparin was injected into the abdominal artery from which 10 ml of blood was drawn and added to the perfusate. The hepatic portal vein was quickly cannulated using a metal cannula attached to a saline drip. The posterior vena cava was quickly severed to effect unobstructed flow of perfusate through the liver. At this point the liver should be completely blanched. Gentle massage may help in relieving unblanched portions. The liver was separated from the rest of the body with the cannulation to the bile duct and portal vein intact. Perfusion The liver was moved to an Ambec extracorporeal perfusion unit Model two/ten and the metal cannula connected to the tubing in which the perfusate circulated (Fig. 6). The flow rate for the perfusate was set at 20 ml/min and maintained at a temperature of 37*C. Control bile was collected for 30 min. Drug Administration and Kinetic Studies In different kinetic studies, 100, 200 and 300 mg/kg of the sodium salt of diethyldithiocarbamic acid, dissolved in I mi of saline, was ORGAN Ur iirlttry I n at Wi-lli HI liiilriiMiil/liilu«|oo ports V///I ______Venous o r out lot for s IimjIm p a ss p e rtu s Io n rm llnaleil I Itl F3 Oxyyen in In le t vent Oullel venl & l<» (In water bath) / sorter 1.11 pressor «■ H m i lo r ir/y/l llp.it ««i:lom|nr (In »ilnr loth) FIGURE 6 Ul Schematic of the Ambec two/ten Perfuser. * ^ added to the perfusate. Portions .of the perfusate, about 0.5 ml, were sequentially removed at 2,3,4,5,6,10,15,20,30,40,60,80,100,110 and 120 minutes. One |ig of dipropyldithiocarbamic acid contained in 100 pi of a 100 pg/ml stock solution was added to each sample as an internal standard. The amount of 0.4 ml of methyl iodide was added to the sample and the tube vortexed immediately for 40 sec. Methyl DDC, and the internal stardard were extracted with 5 ml of chloroform. The chloroform layer was evaporated to dryness under a gentle stream of nitrogen and reconstituted in 100 pi of chloroform. One pi was injected into the gas chromatograph using a glass column (1.82 m x 2 mm ID) packed with 32 OV-17 on gas chrom-Q 100-120 mesh (Applied Science Laboratories, Inc). Dry helium (30 ml/min) was the carrier gas. The oven temperature was isothermal at 140*C, injector port temperature was 250*C and detector was 320*C. Standard curves were prepared by the addition of known amounts of DDC to the blank perfusate. The ratio of the peak heights of DDC and the internal standard, DPC was measured and the concentration determined by comparison with the standard curve. The standard curves were linear between 0.5 and 5 pg/ml with correlation coefficients routinely exceeding 0.99. Choleretic Effect of DDC on Bile Flow The tubing to the bile duct was attached to the tip of a uniformed diameter pipet marked in 20 pi divisions with a total volume of 200 pi. The time was taken at which the menicus of the bile progressed to each 20 yI mark. The rate of bile flow was obtained by dividing the time into the volume of bile and these values were plotted in a 41 progressive manner from the control and after the addition of 100f 200 and 300 mg/kg of NaDDC, respectively. The bile flow was mdnitored in at least two rats at each dose. Excretion of Metabolites Control bile was collected for 30 min before the addition of 80 mg NaDDC to the perfusate. The perfusion was continued for 3 hr until a approximately 1.0 ml had been collected. Intact Animal Experiments Decline of DDC in the blood of rats given I.V. dose of NaDDC (250 mg/kg) Catheters were especially constructed by joining a short (ca 3.0 cm) piece of polyethylene tubing (0.023" ID, 0.038" OD) to a longer and less flexible polyethylene segment (0.034" ID, 0.050" OD). This was subsequently heat-molded to the required shape. Rats were anesthesized with ether. The left jugular vein was exposed and ligated* The short portion of the catheter was inserted into the superior vena cava through a small incision in the jugular vein. The less flexible polyethylene portion of the catheter was passed subcutaneously to the back of the animal and exited between the scapulae. The exited tubing was protected by a flexible coiled metal spring attached to the skin. Rats were allowed to recover overnight before the experiments and were maintained unrestrained in a metabolic cage with free access to food and water. Patency of the catheter was maintained by injection of heparinized saline (sodium heparin, Riker Laboratoriees, Inc., Northridge, CA). One hundred units of heparin were injected into the rat to prevent coagulation of the blood. 42 Ac least two rats were studied at the 500 mg/kg dose; 100 mg NaDDC dissolved in 0.5 ml saline and administered intravenously through the catheter over a period of 30 sec. The endpoint of the injection was designated as zero time. Samples of blood (0.3 ml) were drawn at 2,4,6, 8,10,15,20,30,40,45,50,55,60,70,80,90,100,110 and 120 min. Sample venous blood was first drawn in excess of the volume of the dead space of the catheter. Following the collection of each sample, the equivalent volume was replenished by infusion of heparinized saline. Samples of blood were assayed as described below. Effect of Choleresis on Bile Composition Following the catheterization of the jugular vein, the bile duct was also cateterized as described above. Control bile was collected for one hour. 80 mg of NaDDC was administered to the rat through the jugular vein. Bile was collected for 30 minutes. 120 mg of NaDDC was administered to the rat and bile collected for 30 min. More bile was collected for the subsequent 30 minutes. Analytical Methods Metabolized and unmetabolized DDC was identified by gas chromatography using DPC as the internal standard. Three ml of chloroform were added % to tubes containing Che bile samples collected. This was vortexed and centrifuged. The chloroform portion was cranafered to a 3 mi reacti-vial (Pierce Chemical Co., Rockford, IL). The mixture was equilibrated, cen trifuged and evaporated to dryness under a gentle stream of dry nitrogen. This was reconstituted in 10 til of chloroform and 2-5 yl of the chloroform 43 were injected for analysis in the GC. Gas chromatography was performed isothermally. Gas chromatographic conditions are as described above. Unmetabolized DDC Methyl iodide (0.3 ml) was added to methylate the unmetabolized DDC. Five ml of chloroform was added to each tube to extract the methylated DDC for analysis as described above. Analysis of DDC conjugates by alkali hydrolysis The aqueous portion from the remaining unmetabolized DDC analyses was treated with 1 ml of 3M NaOH and the mixture agitated and placed in an oven at 80-85*C for 30 min. After cooling, 0.7 ml of 6 M HC1 was added to the mixture and the tubes were vortexed. Free DDC from the alkali hydrolysis was analyzed as described above for unmetabolized DDC. When quantitating for the concentration of drug and the metabo lites in bile, separate analyses for unmetabolized and methyl. DDC were omitted. The bile samples were hydrolysed as described above and quantitated using DPC as internal standard. Perfusate samples Internal standard^DPC was added to each perfusate sample, methy lated as described above and analyzed with the GC. In each experiment, a standard curve was prepared by the addition of NaDDC to the blank perfusate to cover the range of the desired concentration. The ratio of the height of the DDC peak to that of the DPC was used to calculate the concentration of DDC by comparison with the standard curve. A graph of the decline of DDC in the perfusate was plotted against time. 44 Analysis of DDC-glucuronide Permethylation One hundred pi of bile was evaporated to dryness under a gentle stream of nitrogen. The vial was flushed thoroughly with dry nitrogen and then capped with a rubber septum to exclude moisture. The reagents were added to the vial by piercing the septum. The residue remaining after the evaporation was dissolved in 100 ul of anhydrous DMSO. Fifty ml of DMSO" sodium salt were added, followed after 15 min by 20 pi of methyl iodide. The reaction was stopped after 90 min by the addition of 1 ml of water and equilibrated with 1 ml chloroform. The organic phase was washed two times with 1 ml of water* The chloroform extract was transfered to a clean vial and evaporated to dryness. The residue was redissolved in 10 pi of chloro form and analyzed with the gas chromatograph and mass spectrometer. Gas chromatographic analysis was performed using a Varian Model 3700 equipped with flame ionization. The glass column (1.82 x 2 mm ID) was packed with 3% OV-17 on gas chrom-Q, mesh 100-200 from Applied Science Labs., Inc. The column temperature was run isoehermally at 100*C for 5 min and programmed at 330*0 at 5*C per min. The injector and detector temperatures were 250*G and 340*C respect ively. Hydrogen, air and helium flows were 30, 300 and 30 ml/min respectively. Mass spectrometric analysis was performed on a Hewlett Packard Model computer-guided 5985 gas chromatograph/mass spectrometer system. A 0.9 on x 2 mm ID glass column was packed as described for gas chromatography. The oven temperature was programmed as described above. 45 Synthesis of sodium salt of DMSO Sodium hydride (Ventron Corp.) as a dispersion of gray powder in an industrial white oil was washed with toluene. Dry DMSO was prepared by distillation under vacuum. Sodium hydride was added to excess dimethyl sulfoxide with stirring under nitrogen at 65*70*0 until hydrogen was no longer evolved. This was stored in aliquots in I ml vials and sealed to prevent moisture (Fig. 7). IMS derivatization One hundred pi of bile were evaporated to dryness in a 3 ml Reacci-viai under a gentle stream of nitrogen gas. Dry pyridine (50 ml) were added, followed by 50 pi of N,0-bis(Trimethyl silyl tri- fluroacetamide + 1% trimethylchosilane (Pierce Chemical Co). The mixture was incubated at 70*C for 5*hrs; 4 pi of the mixture were analyzed using the GC and the combined GC/MS. The temperature of the ion source was maintained at 220*C. The ion source was at 70 eV. Electron impact spectra was obtained and interpreted. Analysis of DDC in intact animal Ac each time indicated in the sampling schedule noted above, 0.2 ml of ratio blood was collected placed in a 15 ml tube with its inside cap lined with Teflon material. Next, 0.5 pg of internal standard was introduced to each tube* Following the addition of 0.4 mi of methyl iodide to the mixture was immediately vortexed for 30 sec. Five mi of chloroform was added to the tube and inverted every second for 10 min, centrifuged and the organic layer removed and evaporated to dryness and reconstituted in 10 pI of chloroform. 0 0 ~ 0 CH 3 SCH 3 + NaH— ►[CH3 S «CH^*CH3S -CH 2 ]n -h2 FIGURE 7 Synthesis of the Sodium Salt of Dlmethylsulfoxide 47 One co two microliters were used for gas chrotnaeographic analyses. The ratio of che DDC peaks co DPC were compared co the standard curve carried out in Che same biological fluid. Efface of Choleresis on Bile Composition One-half ml of bile for each of che following time - a) control, b) 30 minuces following 80 mg NaDDC, c) 30 minutes following 120 mg NaDDC and 30 minuces following(c) were analysed for sodium, potassium, chloride and bicarbonate ions at the Clinical Chemistry Laboratories of The Ohio State University Clinics. Absorbance of Bi*le A one to ten dilution of aliquots of che bile samples were scanned on the Beckman Model 26 spectrophotometer between wavelengths of 600 to 330 nm to determine how the different fractions collected were absorbed in the yellow range of the light spectrum. RESULTS Synthesis of Internal Standard A chromatogram of DDC and DPC extract of water, urine and blood showed two sharp peaks. In Fig. 8, the chromatogram trace in blood shows the two peaks with retention times of 2.94 and 5.23 oin. There were no significant interfering peaks from the urine or blood. A solution of disulfiram in chloroform yielded no peaks with or without the addition of 0.3 ul methyl iodide. Spectral data from GC/MS were used to identify the components of the peaks. The mass spectral data are shown in Figs. 9 and 10. Nuclear magnetic resonance was used to further confirm the structure of the compounds (Figs. 13, 14, and 15). A standard curve of DDC in water, urine and blood shows a decline in sensitivity in the following order: water>urine>blood. This decline may be due to protein binding by DDC. Endogenous substances in the biological fluids may also interfere with the assay. A Standard curve of DDC in water obtained in 5 experiments is shown in Fig. 16. Decline of DDC in the perfusate with in vitro liver perfusion study with doses of 100, 200 and 300 mg/kg NaDDC The concentrations of DDC measured against time following the addition of the NaDDC to the perfusate are shown in Fig. 17. When 100 mg/kg of NaDDC were added, the half-lives obtained in two different 48 49 Solvent Front 2.94 min (MeDDC) 5.24 ^ (MeDPC) VJ TIME FIGURE R Retention Times (Minutes). Typical Gas Chromatograph of MeDDC andQthe Internal Standard MeDPC In rats' blood, obtained lsothermally (140 C) on a 31 QV-17 glass column. Retention time Is noted above each GC peak. « FW 0326 5PEC 61 REF. TIME 3 . 9 101) C II -M -C-S H -OS 'S CoatrlbutiM 20 90 *30 I K tCQ ficure 9 Ibu Specliw of llvUllC wllh Ho I ecu] nr Inn, H*“141 and frogoewl luio M 6, OS a n l 60, Ul . o FRII 0131 DTEC 374 RET. Tine S. 1 1(1(1 c-sai, C.lt.-H - O S n 5 s n V i - \ t II - o s Vi. . H - t.— . n , c3», V i I 3 S'V s ^H-C-S S"; I S Contribution y 10 20 40 IQ SO 70 BO 90 100 110 120 ISO 140 ISO ISO 170 ISO I DO 200 210 220 230 240 2SO riGURE 10 Hass Spectra* for meOK WIUi Molecular Ion, H -191 and fratpcnls at 116, 144, 102 and 91. FRM 0130 EPEC 367 BET. TlttE 0. 0 100 S Contribution 20 90 <0 60 70 00 I DO 110 120 190 140 160 ISO 170 160 I DO 200 210 290 210 290 FIGURE 11 Hass Spec Iron for EtOOC tilth Molecular Ion. H'*177 and Frajpaents a t IU , 116 and 88. I n to FRtl 0132 6PEC 360 RET. TME 0 . B 100 ^B-C-Scn, -SCO C3 V c* ! *v*rT j»^S Coatrlbat loa ■»T-" r T JjfhiJyii ifcf .<*n nywN^wrWi^ r rnir "'r r 0 10 20 90 40 SO 00 70 BO SO 100 110 190 190 140 ISO ISO 170 I BO ISO 900 910 *90 290 940 210 FIGURE 12 Hist Spectrin for DDPC If lit* Molecular Ion, H4»194 and Fragnent Iona 128, 102 and 94. toI n !*»■?■* • * I **)' t f T f f lt * | » * • • | » *»T f H-C-S-U ^=4 l l t . gal « r i n r “ * *v MWB r-'V'"1* ad /• »* ta riOME . 13 Nuclear Magnetic Icsuiw cc Spectrin* Confirming (In Structure of Pletliylilltlilocarbaalc Acid, a - C llj- i>- -ai2_ c - -Sll J - CUClj •C-(JI «-| • * i** I • v* f r* »■* f-rr* * | t r* » | * ■“* ► | CU, — --_-- « tt-n. e w * **• w* zzz3 n J* —. w m* J* *-* rictmi' n Nuclmr Magnetic Keummce Spcctrua Confining the S tru c tu re of PPC. a - CU,*1 d - -Sll b- -aij- c- atci. c - -ai2- m til h *• •• »* n u n m M W X t « i f r rt*! • *-*-» y »-»*r |‘* f- * t | • *■*-** »*ir | • r#*» | * r« » ( t | i • • • | • t « » | t r r-t | • -* r T * | T- T-Tf |»i I f f »«>*<.• >»► .w — “ ^ > 1 01,-0^ ~ , 3? -1 1 * MM*A ■ IIM 1 >• 1"-±ir'»r f J Yfi I I, v.^“ i i.',.?.“^‘r.’ 1 i . v\*r>’. \\“ r* M M fW|l M n If H h o m e is: Hue]car Magnetic B eminence Spcctrw Cuoftrelng tie Structure - of EtUOC. e- CJ13_ c- UjO I** -CII2 - tl> CUC1. 0 1 0 1 57 FIGURE 16 Standard Curve of DDC in water. Each point is a mean of 5 values from 5 experiments. Linear regression on the values gave a correlation coefficient of .995. The standard error of the mean for each concentration is shown below. Standard Error Concentration of che Mean 0.5 ug .045 1.0 ug .024 2 . 0 ug ,017 3.0 ug .031 4.0 ug .037 5.0 ug .158 58 STANDARD CURVE OF DDC IN WATER £ 5 . 0 - c* d» X 1 4 . 0 - Q. O CL O ^ 3 . 0 - -C o> 0> X *o 2.0- « CL O Q Q 1.0- 1.0 2.0 3 . 0 4 . 0 5 . 0 Concentrations of DDC in (iq 59 FIGURE 17 Dose-dependent metabolism of DDC in perfusate of isolated perfused rat liver. One-half ml of perfusate was collected at times noted in text. Internal standard was added to each sample and the samples were methylated and gas chromatographed. The peak height ratio obtained for each sample and internal standard was compared to the standard curve and che concentrations of DDC were determined. 0 — 0 300 mg/kg • 200 mg/kg 0— 0 100 mg/kg jug/ml DOC IN PERFUSATE 2000 1000 o H soo 100H 0 3 20 0 4 TIME (mini 0 6 0 6 0 120 100 60 61 FIGURE 18 Choleretic effect of DDC with doaee of 100 end 300 mg/kg. ‘t At this time, DDC was added to the perfusate. The volume of bile collected was divided by the time to obtain the rate of flow and the race plotted against time. • • 300 mg/kg O — O 100 mg/kg O— □ Control RATE OF BILE FLOW (/J.l/m ln) 0 2 16 12 0 4 8 50 TIME ( min) ( TIME 100 150 63 TABLE 2 Amount of Total Metabolites Produced in Bile Following the Addition of NaDDC. Compounds Include Unmetabolized DDC, Methylated DDC and Free DDC From Conjugate — ------J - 100 mg/kg 300 mg/kg . r 0-25 mins 0-25 mins Vol. of bile - 400 I Vol. of bile ■ 400 1 Z of given dose ■ 25 X of given dose ■ 18 26-68 mins 26-54 mins Vol. of bile - 600 I Vol. of bile ■ 400 I X of given dose ■ 8 X of given dose - 16 55-106 mins. Vol. of bile - 600 I X of given dose ■ 5 - -- -- * — PER CEN.T OF TOTAL METABOLITES IN BILE 1 0 8 - 0 5 - 0 5 5 2 Metabolites in and Voluae of Bile During Choleresis Bile During of in andVoluae Metabolites Metabolites Volume ofBile 0 5 TIME IN MINUTES IUE 19 FIGURE 5 7 S) 100 800 r rBOO 0 0 4 - 0 0 4 - VOLUME OF BILE -O o* 65 TABLE 3 Concentrations of Some Electrolytes in Bile Before end During Choleresi9 in Intact Rat with Different Doses of NaDDC Electrolytes Before Drug . 80 mg/NaDDC 120 NaDDC Experiment I Na+ 177 160 156 K+ 4.1 4.5 5.0 Cl" 80 80 86 HCO3" % 30 29 29 Experiment II Na+ 161 152 L48 K* 4.4 4.6 5.3 Cl" 86 90 97 h c o 3“ 27 29 28 Experiment III Na+ 164 - 159 155 K* 4.5 4.7 5.2 Cl" 84 87 93 HCO3' 28 30 29 N * , K+ and Cl" are expressed in milliequivalents per liter* HCO3- is expressed as CO2 in millimoles per liter. 66 liver perfusions were 6*1 end 6,3 (Mean 6.2 rain.) Ac 200 mg/kg, che half-lives were 7.85, 7.87, 7.A (Mean 7.7 rain). Ac 300 mg/kg, Che half- lives were 10.0 and 10.2 (Mean 10.I rain) In all doses, chere was inicial non-linear decline of DDC in che perfusaCe followed by an apparenc linear decline. There was concinued plaCeauing of che curve shorcly after che inicial decline before Che linear decline and this effect increased with che rising dose. Cholerecic effect of DDC on bile flow In Fig. 18, che stimulating effect of DDC on che race of bile flow following che addition of NaDDC co the perfusate is shown. With a dose of 100 mg/kg Che race of bile low rose sharply from 7.6 l/min co 17.0 l/rain. A dose of 300 rag/kg DDC increased che race of bile flow from 3.8 l/min to 16.0 l/rain. A control race of bile flow is also shown in che figure for comparison. A comparison of Figs. 18, 19 and Table 2 shows chat inicial surge and subsequent decline in appearance of Che metabolices of DDC coincided well wich Che increasing and decreas ing race of bile flow. Again, Fig. 18 shows Chat che cwo doses used stimulated che race of bile flow but chat che duration of stimulation was longer wich che larger dose. Figures 18, 19 and Table 2 are representative of Che kind of daca obtained in 4 experiments using 100 mg/kg and 300 mg/kg doses and 3 controls. Electrolyte Analysis Table 3 shows results obtained from che elecCrolyte studies of bile in intact rac. There was a regressive decline in che concentration of sodium wich increasing choleresis. Potassium ions barely increased. 67 300 350 400 450 500 550 600 —*■ WAVELENGTH (nm) FIGURE 20 Absorption of whole bile at choleresis following che administration of 80 and 100 mg of DDC to an intact rat. Wavelength for maximum absorption was between 410-415 nm. a " control bile; b ■ bile collected 30 min following che administration of 80 mg; c* bile collected 30 min following the administration of 120 mg; d * bile collected for period following "c". % ABSORPTION AT WAVELENGTH OF 4IOnm 100 50- 0 Of Bile at Wavelength of 410 - 410 - 415 Bile at ofOf Wavelength nm Effect of Choleresis on Absorption Effect ofon Absorption Choleresis g OFDDC mg FIGURE FIGURE -21 80 120 69 che chloride ions increased and che bicarbonace ions slightly decreased. Sodium is che major cacion in bile. SpeccrophoComecric decerminacions show a dilution in Che color of bile which can be read as a reduction in che absorbance of bile ac che wavelength of yellow lighc (Fig. 20), following choleresis. This dilution and consequent reduction in absorbance appeared dose-dependent when plotted as reduction in absorbance vs. dose (Fig. 21). Metabolites in bile Careful analyses of bile samples revealed the presence of unmetabo- lized DDC, MeDDC and DDC-glucuronide* DDC and MeDDC were characterized by GC/MS having che following mass speccral data. Molecular ion at 1634* and fragment ions at 148, from the loss of che CH^4 group, 116 from che loss of a sulfur and methyl, 88 from the loss of an ethyl group, sulfur and methyl groups. The TMS derivative of che glucuronide was characterized by che following: molecular ion M+-15, ac 598 and fragment ions at m/e 148 (aglycone); m/e 116 (loss of a sulfur from aglycone); m/e 88 (loss of a sulfur and an echyl group from the aglycone) and prominent ions at m/e 375, 333, 257 and 204 generated from Che fragmen tation of the TMS-glucuronic acid moeity. Permethylation procedure did not yield mass spectral evidence for the glucuronide. It is possible chat a quarterenary ion may form during the permethylation procedure yielding che highly polar structure noted in Fig. 22 and hence, is not evident in a GC tracing. Silylated bile is shown in Fig. 23 and the total ion trace is shown in Fig, 24. 70 Decline of DDC in the Intact Rat The blood concentrations of DDC measured at different times after the I.V. administration of 500 mg/kg of DDC to the rat are shown in Fig. 26. There was a definable distribution or alpha phase which gradually entered an ill-defined elimination or beta-phase. During this beta-phase, there was a secondary rise in the concentration of DDC. The Mean half-life of DDC in these experiments was 12 minutes. PEHMETHTLATED CONTROL BILE PEHMETHXLATED SAMPLE BILE FIGURE 22 Gas Chromatogram of Control Perfusion BLle and DDC Perfusion Bile. The bile samples were permethylated. 72 TMS CONTROL BILE u . JUL • TMS SAMPLE BILE FIGURE 23 Gas Chromatogram of control bile and DDC perfusion bile. The bile samples were treated with BSTFA and 1% TMCS , Peak A is suspected DDC-G-TMS peak. 116. 175. Cl TI I t » FIGURE 24 Total Ion Trace of Bile Obtained from Isolated Perfused Rat Liver. An 80 mg of DDC was added to the perfusate. The derlvatlaed sample was Injected onto a 1.5 ft column paced with 3Z OV-17. Fragment Ions 375 for the glycone, 116 for the aglycone -g and the molecular Ion 613 were monitored. FIGURE 25 Mabs Spectrum of TMS Derivatized DDC-Glucuronide with M -15, 598 and fragment ions, 375, 333, 257 204 for the glycone and fragment 116, 88 for the aglycone. I KM UUU1 poc-oLUcuiotiiDC ins oniiviiiivc. i.s r i. 32 ov-n uuics c o l u m ioo- o El\ • *«C*S E l' II lie ai» TMS-O^a f|Vct5 in 'a,s 00 '] l«9 2i r 204 I ■ ftijH 4-y n ., r i ,1.1 I ^ I. , ■1f,n.y4tUTiir^4T.r in.yK*^fw^.r., [f , JL ■■ph .y.-i.yt|^y 1 0 0 - TMS ilMS IMS ISO I M I M I N 910 no 390 910 3S0 940 970 410 no 190 410 ISO 4*0 470 4*0 4BO 100 1119 — s IMS 7 M3 I M3 SIM SIO SID 590 510 550 SMI 570 SIM CDO MO 110 MO 090 BIO S50 UK *70 70U 710 710 790 7(0 Itt vl in FIGURE 26 Decline of DDC in intact rat. Dose administered was 500 mg/kg. DDC was administered intravenously through the left jugular vein and blood samples were collected through the same route. Internal standard was added to each sample, methylated and gas chromato graphed* Each point is a Mean of values from two experiments. CONC l)DC x 100 (//g/ml) 10 - TIME(mins) 8060 100 120 77 DISCUSSION Internal Standard Gas chromatography is a recognized powerful separative technique which combines sensitivity and specificity in analyses. Also, its use calls for very small sample sizes. A mass spectrometer furnishes a spectrum of an unknown compound which can give considerable information about the compound structure which can be established on comparison with an authentic sample. When a gas chromatograph is coupled to a mass spectrometer, an integrated instrumental system is developed that allows for utilization of both instruments to maximal advantage. The information by a mass spectrometer shows a highly characteristic pattern of fragments into which a molecule splits when it is ionized in the vapor phase. Both masses of the major fragments and their relative abundances are characteristic of a given molecule under a constant set of conditions. These data constitute the mass spectrum. The development of a good assay is of paramount importance in pharmacokinetic studies especially when using the gas chromatographic technique. The internal standard serves an important role in precise methods of quantification. Comparison of component peak areas or peak heights with that of the internal standard, compensates for major sources of potential error. Some of these errors include variation in sample size and inconsistencies during sample preparation procedures. 78 DIPROPYLDITHIOCARBAMIC ACID Hi N — C — SH > ll H, S DIETHYLDITHIOCARBA.MIC ACID FIGURE 27 Structures of DPC and Biphenyl in Comparison co DDC as Internal Standard. 8 0 TABLE 4 Comparison of DPC and Biphenyl as Internal Standard for DDC DDC DPC Biphenyl Is a carbamic acid Is a carbamic acid Not a carbamic acid Is a thio compound Is a thio compound Not a thio compound Has close retention Has a close retention time to DDC time to DDC Binds to protein Binds to protein Has not been shown to bind to protein Is methylated and Is only extracted extracted with DDC with DDC 81 che use of Che internal scandard becomes even more imporcanc when che assays are carried ouc in a biological macrix e.g. blood, bile and urine. Ic is-necessary Co add che incernal sCandard before che clean up of che sample. Ideally, an incernal scandard should exhibic chemical characceriscics chac are nearly indiscinguishable co che compound co be analyzed. Ic muse be a pure, well defined compound chac resolves in che chromacogram wich a retention time near chac of che compound co be analyzed under che given chromacographic condicions. Gas chromatographic technique had been used in analyzing methylated DOC wich biphenyl as Che internal scandard. Boch compounds , DDC and biphenyl, resolve within che same range of retention times. However, beyond this similarity in chromacographic characteristics, chey have liccle chemical similarity. These two compounds are contrasted in Table 4. Two analogs of MeDDC synthesized in this laboratory for use as internal standard are EtDDC and MeDPC. Mass spectra and nuclear magnetic resonance analyses show these compounds co be pure and well defined. In Table 4, a comparison of DPC and biphenyl as incernal standard compounds for DDC is made. These structures are shown in Fig. 26. The advantage of DPC as internal scandard over EtDDC is because DPC is added to Che sample and meChylated wich DDC. EcDDC is already alkylaced and is involved only with extraction, whereas, DPC is involved wich che entire sample preparation procedure. 82 For incernal scandard calibration, a calibracion mixture con taining known amouncs of che drug and che incernal scandard are prepared and che racio of che drug co incernal scandard peak heights is estimated. In Che experiments carried out, DPC solution of known concentrations were added co che samples before mechylation was carried out. The correlation coefficient was routinely 0.99 or betcer and was consistent in biological samples. Anocher modification of Che method of Cobby et al., (1977a) involved che use of chloroform instead of carbon tetrachloride as the extraction solvent. This modification has the decided advantage of lessening the risk of liver and kidney damage chat might result in the from exposure to carbon tetrachloride fumes. Choleresis The process involved in the formation of bile are not very well known. However, it is known that bile is formed by the parenchymal hepatocytes. These hepatocytes excrete the bile into the bile canaliculi which drain into larger ductules. They in turn join to form bile ducts (Popper and Schaffner, 1957; Steiner and Carruthers, 1961). The epithelial cells of the ducts have prominent secretory golgi apparatus and microvilli projections. The composition of bile varies from species to species. The average water content in liver bile is 97-98% (Dittmer, 1961; Table 5 ). Half of the 2-3% of solids is made up of bile salts which include salts of glycine and taurine conjugates of cholic and chenodeoxycholic acid. Inorganic electro lytes, phospholipids and bile pigments make up the rest of the bile. TABLE 5 Chemical Composition of Human Bile* Hepatic bile Gallbladder bile Gentral compoiition (s/100 ml) Dry nutter 2 .3 -3 .3 18 Inorganic matter 0.2-0.9 0J-1.1 BDe tilts 0.6-1.4 11.5 Total tolidt 1.0-4.0 4.7—16 J Water 97 .S 85.9 Eltctrolyttt {mMIequl valent!//) Bicarbonate 40 8 - 1 2 Chloride 75-110 1 5 -3 0 Calcium 2 .0 -4 .5 5 - 7 Potassium 2.6-12 - Sodium 1 3 1 -1 6 4 — Magnesium 1.5 — Iron (as Fe **) 2.4 0 .0 1 -0 6 Organic zubtiancti (mg %) Protein (total 273 3 1 5 -5 3 9 Bilirubin 2 0 -2 0 0 1000 Urea . 23.6 2 0 -4 5 Nitrogen Total N 6 7 -9 2 490 A m ino ad d N 5.4 ' 6 .0 -2 1 .6 Cholesterol 120 630 Fatty ad d s 110 970 Lecithin 100-575 3500 (From Dittmer, 1961) 84 - Generally, che concentrations of electrolytes in the bile approximate those in che plasma. Sodium, potassium and calcium are the major inorganic cations while bicarbonate and chloride ions are che major anions. There is a difference in concentration of substances that make up hepatic bile and gall-bladder bile. The major difference in composition is due co the concentrating activity of the gall-bladder. The rat, which served as the animal model in these experiments, lacks a gall-bladder. Water transport is believed to be initiated by osmotic filtration. Non-reabsorbable solutes induce choleresis in a manner analogous to that whereby osmotic diuretics may induce choleresis by establishing a local osmotic gradient. Bile secretion, a complex phenomena, involves several different mechanisms (Forker, 1977; Erlinger and Dhumeaux, 1974). One of the major mechanisms is, the secretion of bile acid. Bile acids have been shown to stimulate bile production in many species including dog, rabbit, rat and the isolated perfused rat liver, the Rhesus monkey and man. This increase in bile flow is canalicular in origin as it is accompanied by enhanced erythritol clearance. A linear relationship has also been found between bile acid excretion and erythritol clearance, a good estimate of canalicular flow. Bile acid may possibly create an osmotic gradient for passive filtration of water and electrolytes. This process is espoused as the bile acid-dependent mechanism. Another mechanism is one in which sodium ions are thought to be actively transported and in this process to generate osmotic gradients. 85 Upon addition of DDC, the rate of bile flow was increaaed by * about 2.5 to 3.0 times. The extent of the choleretic effect differed slightly with each liver but the overall effect was unequivocal. In view of the high concentrations of DOC we used, we believe that in each case the liver might have attained its maximum rate of bile production. However, the duration of time the rate‘remained at the maximum increased with concentration. Analyses of bile samples showed that the increase in bile flow was coincident with the exit of metabolites. Simildr cases of increased bile flow have been attributed to the osmotic effect of diethyl maleate conjugate metabolites (Barnhart and Combes, 1978), naltrexone metabolites (Rodgers et al.), and suspected to be caused by exit of valproic acid conjugates (Dickinson et al., 1973). Conjugation is a major metabolic pathway of DDC and the DDC-glucuronide conjugate is secreted in bile. The reduction in absorbance of the different fractions of bile collected was similar to an effect obtained by using water to dilute the bile. The yellow color became less intense. The reduction in the concentration of sodium also indicated a dilution effect. The mechanism underlying DDC-induced choleresis remains unclear. Perhaps the drug may change the permeability of the parenchymal cells of the liver and with the exit of the metabolites, molecules of water are passively filtered through to dilute the bile. Some exogenous substances that have also been shown to stimulate bile flow include ethacrynic acid (Chendrovitch et al., 1975), ouabain (Graf and Feterlik, 1976), and probenecid (Erttmann and Darnm, 1976). 86 The significance of the choieresis caused by DDC is not: known but in che light of che induced hepacocoxicity caused by Disulfiram, Che precursor of DDC, in patients (Gisen and Ginsberg, 1975; Keefe and Smith, 1974; Knutsen, 1949; Ranek and Andreasen, 1977) and in laboratory animals (Stege £t a K , 1977) it is necessary to evaluate che relation** ship of this choieresis to the induced hepatotoxicity of the drug. Dose-dependent kinetics The pharmacokinetic analysis of the perfusate concentration of DDC did not give a simple first-order kinetics. The half-lives increased from a Mean of 6.2 min at a concentration of 100 mg/kg to 7.6 min at 200 mg/kg and 10.0 min at 300 mg/kg. With increasing dose, there was a developing plateau, following a short distribution phase, alpha, suggesting saturation of one or more of the biotrans formation pathways. Another possible explanation may be the inhibition of metabolism by substrate or product. Similar observations have been shown with valproic acid in rats (Dickinson ££ a U , 1979) with phenytoin in mice, rats and humans (Gerber and Wagner, 1972); Gerber £t a U , 1971; Gerber and Arnold, 1969) and with gamma-hydroxybutyric acid in rats (Lettieri and Fung, 1979). These observations can be explained in terms of the Michaelis-Menten kinetics. There is a limitation in the capacity to the enzymatic and active transport processes that occur, in the liver. There is only so much enzyme present in the liver, and therefore, there is a maximum rate at which metabolism can proceed. Also, this limitation can be imposed by the availability of the cosubstrates and cofactors required in the Vm Km + C o /Km Plasma Drug Coneanlrallon 4 Vm Km + C Vm t - 2 Km Plasma Drug Concentration, FIGURE M a* Rate of metabolism Increases toward maximum Value Vm with Increasing drug concentration. b. Metabolic clearance falls with Increasing drug concentration. 88 enzymatic reaction. In a fashion similar to active biliary excretion, there is a maximum rate at which a drug can be excreted into the bile* All drugs therefore, theoretically are capable of exhibiting saturation kinetics if the dose is sufficiently great. Drugs that are toxic to the liver may not be in high enough concentration to attain this saturation effect. The knowledge of enzyme kinetics is derived from in vitro studies in which substrate, enzyme and cofactor concentrations are controlled. Many factors that are involved in vivo cannot be isolated. Nevertheless, the basic principles of enzyme kinetics have application in pharmacokinetics. The behavior displayed by che curve immediately following Che distributive phase displays Michaelis-Menten kinetics i.e., the metabolism by the enzyme approaches the maximum race with the following relationship, Vm C Rate of Metabolism * km+ C where Km is a constant, the Michaelis-Menten constant. "C" is the con centration of the substrate. In enzyme kinetics the value of Vm is directly proportional to the concentration of enzyme, and Km is an inverse function of the affinity between drug and enzyme. An estimate of Km requires measurement of drug concentration at the metabolizing enzyme site. It is not possible to do this in vivo, hence, the plasma concentration is conventionally used. At concentrations above Km, the rate approaches the value of Vm. Metabolic clearance is defined as the rate of metabolism relative to the plasma concentration, it it therefore follows that its value Vm Metabolic Clearance * Km-*- C TABLE 6 Types of Groups in Foreign Compounds to Which Clucuronic Acid Can be*Transferred Type of group Structure* Example of compound forming glucuronidc Hydroxyl Phenolic AiOM Morphine Enolic —CH=COM • 4*hydroxycoumarin Alcoholic (primary) —CM, OH Chloramphenicol Alcoholic (secondary) ^CMOM - Alcoholic (tertiary) frrf-bulanol llydroxylaminic -HOH MhydroxyW-2-fluorenylaceianude Carboxyl Aromatic A t C O O H Salicylic acid . Aliphatic -CM, COOH Indomelhsdn Amino and Aromatic A t N H , 4,4’-diaminodiphenylsulphonc imino Carbamate -OCONH, Meprobamate Sulphonimide -SO,NT/- Sulphadimelhoxine Heterocyclic' > Sulphisoxazole Sulphydryl Thiol -S H 2*mercaplobcnzol hiazole Carbodilhioic -CSSH Diethyldithiocarbamic acid * The hydrogen italicized is replaced by the glucuronyl residue CaNtO* to form the conjugated glucuronic add. Based .on Smith and Williams (1966). 90 decreases at drug concentrations approaching and exceeding the value of Km* Following this Michaelis-Menten kinetics the curve follows a first-order kinetics. Glucuronide of DDC The glucuronic acid conjugation is one of the most important metabolic conjugation reactions from the point of view of biliary excretion. Many drug and their metabolites appear in bile largely as glucuronic acid conjugates. For conjugation to occur, the compound should either possess (or acquire during its metabolism) a group at which a conjugation reaction can occur. The groups are a) a hydroxyl group which may be alcoholic, hydroxylaminic, phenolic or enolic, b) an amine group which is usually aromatic and in some cases a substituted sulphonamide group, c) a carboxyl group which may be aromatic or aliphatic and, d) a sulphydryl group (Smith and Williams, 1966). The structures of these groups which can act as receptors for glucuronic acid in transfer reactions are shown in (Table 6). Glucuronide formation is the most versatile of the conjugation mechanisms from the point of view of the many chemical groups to which the conjugating agent can be transferred. In conjugation, a non-polar compound is made much more polar and secondly, its molecular weight is increased by 176 units. These two factors, polarity and molecular weight, are important for biliary excretion. The synthesis of the glucuronide proceeds by way of transfer of the glucuronic acid from a nucleotide, uridine diphosphate glucuronic * 91 acid (UDPGA) Co an acceptor molecule, the reaction being catalyzed by the enzyme glucuronyl transferase. The UDPGA is formed initially from uridine diphosphate glucose which is oxidized by a soluble enzyme, UDP glucose dehydrogenase. The cat is the only species that appears to have a defect in the glucuronide conjugation mechanism. Although formation of glucuronide . occurs mainly in the liver, it can also take place extrahepatically in che gastrointestinal tract, kidneys, lungs and spleen. Drugs that have conjugation as a major pathway of biotransformation may be subsequently hydrolyzed by che esterases in che intestinal lumen leading to increase in concentration in the central compartment. This results in secondary peaks in serum and this is commonly attributed to an enterohepatic recirculation. These secondary serum peaks do not appear in kinetic studies of rats with exteriorized bile or in studies of the isolated perfused liver. Hence, we did not observe these secondary peaks in either the isolated perfused rat liver study or in the intact rat. Secondary increases in concentration in drugs that are conjugated have been shown with morphine (Dahlstrom and Paalzow, 1978), phenolpthalein (Colburn et_ A U , 1979) and valproic acid (Dickinson et al., 1979). Earlier works have suggested glucuronic acid conjugation as a major pathway of DDC elimination. Identification of this possibility has been shown by hydrolysis of bile to yield DDC. In our experiments, we tried to identify the DDC-glucuronide by GC and GC/mass spectrometer techniques following derivatization with either permethylation or 92 silylation techniques. Permethylation yielded no derivatized conjugate and we suggest that this nay be due to the methyl group attacking the nitrogen of the amine to yield a quarternary C2H5 N+-C-S-Glucuronide-CH3 I II C2H5 CH3 S ion which will be very polar and not show up in the gas chromatography However, the silylated sample gave a peak analyzed by mass spectrometer that is characteristic of a DDC-glucuronide conjugate. This is the first time this major metabolite of Disulfiram has been distinctly characterized. Unchanged DDC and methylated DDC were also clearly and unambiguously identified in the bile studied. SUMMARY AND CONCLUSION Dipropyldithiocarbamic acid, an analog of DDC, was synthesized for use as internal an standard in the quantification of DDC. The synthesis involved a reaction between dipropylamine and carbon disulfide in cold hexane. The white DPC crystals were methylated with methyl iodide and analyzed using the gas chromatography and mass spectrometry. Further analysis was done using nuclear magnetic resonance techniques. Both analytical techniques confirmed the structure of DPC. The retention times for the methyl esters of DDC and DPC were 2.94 and 5.24 minutes, respectively, in rats blood and remained within the same range in water, bile, urine and plasma. Assays on DDC performed with DPC as the internal standard were consistent and reliable* The kinetics of DDC was studied in the isolated perfused rat liver and in the intact rat. In the in vitro experiments, the liver was and separated from the rat and perfused on an extracorporeal perfuser. The liver was perfused with Kreb's-bicarbonate buffer and the rat's blood to a total volume of 110 ml of perfusate. Prior to entry into the liver, the perfusate was saturated with oxygen. Doses of 40, 80, 120 mg of DDC or 100, 200, 300 mg/kg of DDC relative of entire rat were studied. The decline of DDC in the perfusate at the doses described did not follow a simple first-order kinetics. A short 93 94 distribution phase was followed by a kinetics that indicated satur ation of the enzymes, Michaelis-Menten kinetics. The high concentration of the drug saturated the enzyme system leading to decreased metabolic clearance. Following the saturation kinetics the decline of DDC followed a partial first-order kinetics. The half-lives were determined at this part of the curve and were 6.2, 7.7 and 10.I minutes for 100, 200 and 300 mg/kg, respectively. The halflife of the drug increased with increas ing dosage. In the intact rat, the left jugular veing was cannulated and 500 mg/kg of DDC was administered intravenously through this route. Blood samples were withdrawn from the same route and the decline of DDC was determined. There was a short distribution phase followed by a saturation kinetics phase and a partial first-order elimination phase. A half-life of 12 minutes was described for DDC at a dose of 250 mg/kg. The calculated volume of distribution indicated that most of the DDC remained in the central compartment. No extra peaks were observed for secondary concentrations of DDC in the blood. The rate of bile flow was monitored in the isolated perfused rat liver. Administration of DDC caused an increase in the rate of bile flow. This effect was dose-dependent. Increase in the concentration of the drug produced a longer increase in rate of bile flow. In the intact rat, bile collected before and after doses of 200 and 300 mg/kg of DDC were injected intravenously showed a reduction in the concentration of sodium, the most prominent ion in bile. Again, the yellow color of the bile became less intense as determined by spectro- photometric analysis. Both reduction in the sodium concentration and lightening of the yellow color were dose-dependent. 95 Conjugation is a major pathway for the metabolism of diethyldi- thiocarbamic acid. Attempts to analyze the rat bile for the glucuronide conjugate by permethylation was unsuccessful. However, derivatization of the bile with BSFTA + 1% TMCS yielded a peak of the sample bile in the gas chromatogram that was absent in a similarly treated control bile. The extra sample peak was identified and characterized as che glucuronide of DDC by a Hewlett Packard computer- guided gas chromatography and mass spectrometry unit. This is the first time this glucuronide has been discretely identified and characterized by this technique. Although disulfiram has been used since 1945 for adjunct treatment of alcoholism, the mechanism of action of the disulfiram-ethanol reaction is still not well-known. With pharmacokinetic and pharmaco dynamic studies such as I have carried out, more necessary information will become available for Che greater understanding of the pharmacology of disulfiram. BIBLIOGRAPHY Abou-El-Makarem, M.M., Millburn, P., Smith* R.L., Williams, R.T, 1966, The biliary excretion of foreign compounds in different species, Biochem, J. 99: P3 1979, Alpers, B.J., Levy, F.H. 1940. Changes in nervous system following carbon disulfide poisoning in animal and in man. Am. Med. Assoc. Arch. Neurol. Psychiatr. 44: 725-739. Asaad, M., Clarke, D. 1976. Studies on biochemical aspects of the disulfiram-like reaction induced by oral hypoglycemics. Eur. J. Pahrmacol. 35: 301-307. Asmussen, E., Hald, J,, Larsen, V. 1948. The pharmacological action of acetaldehyde on the human organism. Acta Pharmacol. 4: 311-320. Aspila, K., Sastri, V.S., Chakrabarti, C.L. 1969. Studies on the stability of dithiocarbamic acids. Talanta 16: 1099-1102. Bahr, C., Bartilsson. 1971. Hydroxylation and subsequent glucuronide conjugation of desmethylimipramine in rat liver microsomes. Xenobiotica I: 205. Bali, E. 1939. Xanthine Oxidase: purification and properties. J. Biol. Chem. 128:51-67. Barnes, B.A., Fox, L. 1955. Screening some thiuram disulfides and related compounds for acute toxicity and Antabuse-like activity. J. Am. Pharmaceut. Assoc. Sci. Ed. 44: 756-759. Barnhart, J.L., Combes, B., 1978. Choieresis associated with metabolism and biliary excretion of diethyl maleate in the rat and dog. J. Pharmacol. Exp. Ther. 206: 614-623. Brand, M.J.D., Fleet, B. 1970. The application ofpolarography and related electro-analytical techniques to the determination of tetramethylthiuram disulphide. Analyst 95: 1023-1026. Brieger, H. 1961. Chronic carbon disulfide poisoning. J. Occup. Med. 3: 302-308. Brown, M.W., Porter, G.S., Williams, A.E. 1974. The determination of disulfiram in blood, and of exhaled carbon disulphide usingcathode ray polarography. J. Pharm. Pharmacol. Suppl. 26: 95 97 Cannelakis, E., Traver, H. 1952* The metabolism of methyl mercaptan in the intact animal. Arch. Biochem. Biophys. 42: 446-455. Chefurka, W. 1956. Oxidative metabolism of carbohydrate in insect. II. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in che housefly. Enzymologica 18: 209-226. Chenderovitch, J., Raizman, A., Infante, R., 1975. Mechanism of ethacrynic acid induced choieresis in the rat. Am J. Physiol. 229: 1180-1187. Cobby, J., Mayersohn, M., Selliah, S. 1977. The rapid reduction of disulfiram in blood and plasma. J. Pharmacol. Expt. Therap. 202: 724-731. Cobby, J., Mayersohn, M., Selliah. S. 1977. Methyl diechyldithiocarbamace. A metabolite of disulfiram in man. Life Sci. 21: 937-942. Cohen, G., Collins, M. 1970. Alkaloids from catecholamine in adrenal tissue: Possible role in alcoholism. Sciene 167: 1749-1751. Colburn, W.A., Hiron, P.C., Parker, R.J., Milburn, P. 1979. A pharmacokinetic model for enterohepatic recirculation in che rat: Phenonphthalein, a model drug. Drug Metab. Dispos. 7: 100-102. Collins, J.M., Brown, L.M. i960. Calcium carbimide, a new protective drug in alcoholism. Med. J. Austr. 47: 835-838. Collins, M., Nijra, W., Borge, G., Teas, G., Goldfarb, C. 1979. Dopamine related tetrahydroisoquinolines: Significant urinary excretion in alcoholics after alcohol consumption. Science 206: 1184-1186, Collins, M., Rubenstein, J.A., Bigdeli, M.A., Gordon, R., Custod, J.T. 1974. Pyrogalloi: a potent elevator of acetaldehyde during ethanol metabolism. In, Alcohol and Aldehyde Metabolizing Systems, ed. R.G. Thurman, J.R. Williamson, T. Yonecari and B. Chane, pp. 523-528. Academic Press, New York. Corran, H., Dewan, J., Gordon, A., Green, D. 1939. Xanthine oxidase and milk fiavoprotein. Biochem. J. 33: 1694-1706. Dahlstrom, B.E., Paalzow, L.K. 1978. Pharmacokinetic interpretation of the enterohepatic recirculation*and first-pass elimination of morphine in the rat. J. Pharmacokin. Biopharm. 6 : 505-519. Davidson, M*, Feinleib, M. 1972. Carbon disulfide poisoning: A review. Am. Heart J. 83: 100-114. Deitrich, R.A., HeHerman, L. 1963. Diphosphopyridine nucleotide- linked aldehyde dehydrogenase. II. Inhibitors. J. Biol. Chem. 238: L683-1689. 98 Deitrich, R.A., Erwin, V. 1975. Involvement of biogenic amine metabolism in ethanol addicition. Fed. Proc. 34: 1962*1968. DeMaster, E., Nagasawah, I. 1977• Disulfiram-induced acetanemia in rat and man. Res. Common. Chem. Path. Pharmacol. 18: 361*364. Deneke, S., Bernstein, S., Fanburg, B. 1978. Enhancement by disulfiram (Antabuse) of toxic effects of 95 to 97% Oj on the rat lung. J. Pharmacol. Expt. Therap. 208: 377-380. DeSaint-Blanquet, G . , Vidaillac, G . , Lindenbaum, A. et al., 1976. Absorption digestive, fixastion tissulaire et excretion du disulfirame admiistre oralement chez le rat. Arch Int. Pharmacodyn. 223:339*350. Dickinson, R.G., Harland, R.C., Rodgers, R.M., Gordon, W.P., Lynn, R.K., Gerber, N. 1978. Dose-dependent metabolism and enterohepatic recirculation of valproic acid in the rat. Proc. West Pharmacol. Soc. 21: 217-220 Dickinson, R.G., Kaufman, S.N., Lynn, R.K., Cook, M.J., NoVy, M.J., Gerber, N. 1979. Materno-fetal pharmacokinetics and fetal distribution of valproic acid in the monkey. Proc. West. Pharmacol* Soc. 22: 57-61. Dickinson, R.G.,Harland, R.C., Ilias, A.M., Rodgers, R.M., Kaufman, S.N., Lynn, R.K., Geber, N. 1979. Disposition of valproic acid in the rat: Dose-dependent metabolism, distribution, enterohepatic recirculation and choleretic effect. J. Pharmacol. Exp. Ther. 211: 583-595. Dittmer, D.S. 1961. Ed., Blood and Other Body Fluids. Federation of American Societies for Experimental Biology. Washington, D.C* Divatia, K.J., Hine, C.H., Burridge, T.N. 1972. A simple method for the determination of tetraethylthiuramdisulfide (Antabuse) and blood levels obtained experimentally in animals and clinically in man. J. Lab. Clin. Med. 39: 974. Djuric, D. et al., 1973. Disulfiram as an indicator of human susceptibility to carbon disulfide. Excretion of diethyldithiocarbamate sodium in the urine of workers exposed to CS2 after oral administration of disulfiram. Arch. Environ. Health 26: 287-289. Domar, G., Fredga, A., Linderholm, H. 1947. A method for quantitative determination of tetraethylthiuram disulphide (Antabuse, Abstinyl) and its reduced form, diethyldithiocarbamic acid as found in excreta . Acta Chemica Scand. 3: 1441-1442. Duritz, G., Truitt, E. 1966. Importance of acetaldehyde in the aciton of ethanol on brtain norepinephrine and 5-hydroxytryptamine. Biochem. Pharmacol. 15: 711-721. Dussault, P., LePage, M. 1976. Effects of pyridoxine deficiency on the composition of plasma and liver fatty acid in rats fed low and high fat diets. J. Nutr. 105: 1371-1376. 99 Dyck, P.J. 1975. Pathological alterations of the peripheral nervous system in man. In, Peripheral Neuropathy, ed. P.J. Dyck, P.K. Thomas, E.H. Lambert, pp. 29$-55o. W.B. Saunders Co, Philadelphia. Edwards, T.P. 1949. The effect of tetraethylthiuram disulfide (Antabuse) on cellular respiration. Tex. Rep; Biol, and Med. 7: 684. Eisen,. H., Ginsberg, A.L. 1975. Disulfiram hepatotoxicity. Ann. Int. Med. 83: 673-675. Eldjarn, L. 1950. The metabolism of tetra-methylthiuram disulphide (Antabuse, Aversan) in the rat, investigated by means of radioactive sulfur. Scand. J. Clin. Lab. Invest. 2: 198-201. Eldjarn, L. Phil, a. 1956. On the mode of action of X-ray protective agents. I. The fixation in vivo of cystamine and cysteamine to proteins. J. Biol. Chem. 223: 341-3T2, Erlinger, S., Dhumeaux, D. 1974. Mechanisms and control of secretion of bile water and electrolytes. Gastroenterology 66: 281-304. Erttmann, R.R., Damm, K.H. 1976. Probenecid-induced effects on bile flow and biliary excretion of ^H-ouabain. Arch. Int. Pharmacodyn. Ther. 223: 96-106. Fava, A., lliceto, A., Camera, E. 1957. Kinetics of che thiol disulfide exchange. J. Am. Chem. Soc. 79: 833-838. Faiman, M., Dodd, D., Hanzlik, R. 1978. Distribution of disulfiram and metabolites in mice and metabolites of S^5 disulfiram in the dog. Res. Commun. Chem. Path. Pharmacol. 21: 543-567. ( Faiman, M., Dodd., D,, Minor, S., Hanzlik, R. 1978. Radioactive and non radioactive methods for the in vivo determination of disulfiram, diethyldithiocarbamate and dlethyldithiocarbamate-methyl ester. Alcsm: Clin. Expt, Res. 2: 366-369. Farago, A. 1967. Arch. Toxicol. 22: 396. Ferguson, J.K. 1954. A new drug for alcoholism treatment. Canad. Med. Assoc. J. 74: 793-795. Fiala, E., Bobota, G,, Kulakis, C., Wattenberg, L., Weisenberger, J. 1973. Inhibition of 1,2-dimethyl hydrazine metabolism by disulfiram. Xenobiotica 7: 5-9. Fisher, K.H., Clements, J.A., White, R.P., 1973. Enhancement of oxygen toxicity by the herbicide paraquat. Am. Rev. Resp. Dis. 107: 246-252, 100 Forker, E.L. 1977. Mechanisms of hepatic bile formation. Annu. Rev. Physiol. 39: 323-347. Forney, R.B., Hager, R.N. 1969. Toxicology of ethanol. Ann. Rev. Pharmacol. 9: 379-392. Fried, R. 1976. En2ymatic oxidation of diethyldithiocarbamate by xanthine oxidase and its colorimetric assay. Ann. N.Y. Acad. Sci. 273: 212-218. Gardner-Thorpe, C., Benjamin, S. 1971. Peripheral neuropathy after disulfiram administration. J. Neurol. Neurosurg. Psychiatry 34: 253-259. Garrettson, L.K., Perel, J.M., Dayton, P.G. 1969. Methylphenidate interaction with both anticonvulsants and ethyl biacoumacetate. A new action of methylphenidate. J.A.M.A. 207: 2053-2056. Gerard, D.L., Saenger, G. 1966. Outpatient treatment of alcoholism. Toronto: University of Toronto Press. Gerber, N., Arnold, K. 1969. Studies on the metabolism of diphenyl- hydantoin in mice. J. Pharmacol. Exp. Ther. 167: 77-90. Gerber, N., Weller, W.L., Lynn, R., Rangno, R.E., Sweetman, B.J., Bush, M.T. 1971. Study of dose-dependent metabolism of 5,5-diphenyl- hydantoin in the rat using new methodology for isolation and quantitation of metabolites in vivo and in vitro. J. Pharmacol. Exp. Ther. 178: 567-579. Gerber, N., Wagner, J.G. 1972. Explanation of dose-dependent decline of diphenylhydantoin plasms levels by fitting to the integrated form of the Michaelis-Menton equation. Res. Commun. Chem. Pathol. Pharmacol. 3: 455-466. Gessner, T., Jakubowski, M. 1972. Diethyldithiocarbamic acid methyl ester. Biochem. Pharmacol. 21: 219-230. Giarman, N.J., Flick, F.H., White, J.M. 1951. Prolongation of thiopental anesthesia in the mouse by premedication with tetraethylthiuram disulfide (Antabuse). Science 11: 35. Goldstein, M., Musacchio, J., Kopin, I., Axelrod, J. 1964. Inhibition of dopamine- -oxidase by disulfiram. Life Sci. 3: 763-767. Gayer, P., Major, L. 1979. Hepatotoxicity in disulfiram treated patients. Wuat. J. Stud. Ale. 40: 133-137. Graf, J., Peterlik, M. 1976. Ouabain-mediated sodium uptake and bile frmation by isolated perfused rat liver. Am. J. Physiol. 230: 876-885. Graveleau, J. 1972. Les neuropathies peripheriques du disulfirame (Antabuse). Revue Neurologique 126: 149-153. 101 Gregg, E.C., Tyler, W.P, 1950. Polerography of Che bis (diethyldithiocarbamyl) disulfide-diethyldithiocarbamate ion oxidation- reduction system. J. Am. Chem. Soc. 72: 4561-4565. Gyorgy, P. 1932. Acetaldehyd-Ein Kreislaufmitted Klin Wechnschr 11: 277. Hald, J., Jacobsen, E. 1948. The sensitizing effect of tetraethylthiuram disulphide (Antabuse) to ethyl alcohol. Acta Pharmacol* 4: 285-296. Hald, J., Jacobsen, E. 1948. A drug sensitizing the organism to ethyl alcohol. Lancet 2: 1001-1004. Hald, J., Jacobsen, E. 1948. The formation of acetaldehyde in the organism after ingestion of Antabuse (Tetraethylthiuram disulphide) and alcohol. Acta Pharmacol. 4: 305-310. Hald, J., Jacobsen, E., Larsen, V. 1949. Formation of acetaldehyde in the organism in relation to dosage of Antabuse (Tetraethylthiuram disulphide) and to alcohol concentration in blood. Acta Pharmacol. 5: 179-188. Hammerskjold, S. 1943. Tenurid-Ett nytt skabbmedel. Svenska Lakartidinngen 40: 2813-2814. Handovsky, H. 1934. C.V. Au sujet de proprietes biologiques et pharmacodynamiques de 1'acetaldehyde. Compt. Rend. Soc. Biol. (paris) 117: 238-241. Handovsky, H. 1936. Au sujet de l'effect de 1*acetaldehyde sur la rythmicite et le tonus des muscles non-volontaires. Compt. Rend. Soc. Biol. (Paris) 123: 1242-1244. Hassan, H.M., Fridorich, 1. 1977. Regulation of the synthesis of superoxide dismutase in Escherichia coli in induction by methyl viologen. J. Biol. Chem. 252: 7662-7672. Hassinen, I. 1967. Effect of disulfiram (Tetraethylthiuram disulphide) on mitochondrial oxidation. Biochem. Pharmacol. 15: 1147. Hatfield, 6.M., Schaumberg, J.P., Brummel, R.G. 1974. Isolation of the active constituent of coprinus Atramentarium. Lloydia 37: 643. Hatfield, G.M., Schumberg, J.P. 1975. Isolation and structural studies of coprine, the disulfiram-like constituent of coprinus Atramentarium. Lloydia 38: 489-496. Hionrich, H.E. 1956. Alcoholism, Alcohol and Brain Psychology, eds, G.N. Thompson, chapter 5. C.C. Thomas Co., Springfield, IL. 102 Hoff, E.C. 1955* Use of disulfiram in comprehensive therapy of a group of 1020 alcoholics. Conn. Med. 19: 793-798. Hoff, E.C., McKeown, C.E. 1953. An elevation of the use of tetraethylthi uram disulfide in the treatment of 560 cases of alcohol addiciton. Am. J. Psychiatr. 109: 670-673. . Hopkins, A. 1975. Toxic neuropathy due to industrial agents. In, Peripheral Neuropathy. eds, P.J. Dyck, P.K. Thomas, E.H. Lambert, pp 1207- 1226. W.B. Saunders, Philadelphia. Hotson, J.R., Langston, J.W., 1976. Disulfiram induced encephalopathy. Archl Neurol. 33: 141-142. Iber, P., Dutta, S., Sehaszad, M., Krause, S. 1977. Excretion of radioactivity following the administration of 35 sulfur-labeled disulfiram in man. Alcoholism 1: 359-364. Jacobsen, E. 1945* Medical treatment of alcoholism. Austr. Quart. J. Stud. Ale. 10:145. Jacobsen, E., Larsen, V* 1949. Site of the formation of acetaldehyde after ingestion of Antabuse (Tetraethylthiuram disulphide) and alcohol. Acta Pharmacol. 5: 285-291. Kalant, H* 1962. Some recent physiological and biochemical investigation on alcohol and alcoholism. Quart. J. Stud. Ale. 23: 52-93. Kasalder, J. 1963. Formation of S-glucuronide from tetramethylthiuram disulfide (Antabuse) in man. Biochem Biophys Acta 71: 730-732. Kaufroann, S., Friedman, S. 1965. Dopamine- -hydroxylase. Pharmacol. Rev. 17: 71. Keefe, E.B., Smith, F. 1974. Disulfiram Hypersensitivity hepatitis. J.A.M.A. 230-435. Keilin, D., Hartree, E. 1940. Coupled oxidation of alcohol Proc. Royal Soc. Lond. Biol. Sci. 129: 277. Keleti, T. 1964. Studies on D-glyceraldehyde-3-phosphate dehydrogenases. Biochem Biophys Acta 89: 422-430. Kiessling, K.H. 1962. Effect of acetaldehyde on rat brain mitochondria and its occurrence in brain after alcohol ingestion. Ext. Cell Res. 26: 432-434. Kiessling, K.H. 1962. The occurrence of acetaldehyde in various parts of the rat brain afer alcohol injection and its effects on pyruvate oxidation. Extp. Cell Res. 27: 267-268. 103 Kitaon, T. 1976. The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver. Biochem. J. 155: 445-448. Rjelgaard, N. 1949. Inhibition of aldehyde oxidase from liver by (tetraethylthiuram disulphide) Antabuse. Acta Pharmacol. 5: 397-403. Knucsen, B. 1949. Tidsskrift fur den Norske Laegeforening 69: 436. Lang, M., Marselos, M., Torronen, R 1976. Modification of drug metabo lism by disulfiram and diethyldithiocarbamate. I. Mixed-function oxygenase. Chem. Biol. Interact. 15: 267-276. LeQuesne, P.M. 1975. Neuropathy due to drugs. In, Peripheral Neuopathy, eds. P.J. Dyck, P.K, Thomas, E.H, Lambert, pp. 1263-1280. W.B. Saunders, Philadelphia. Lettieri, J.T., Fung, H.L. 1979. Dose-dependent pharmacokinetics and hypnotic effects of sodium gamma-hydroxybutyrate in the rat. J. Pharmacol. Exp. Ther. 208: 7-L1. Levitt, M*, Spector, S., Sajoerdsma, A., tfadenfriend, S. 1965. Eluci dation of che race limiting step in norepinephrine biosynthesis in the perfused guinea pig heart. J. Pharmacol. Expt. Therap. 118: 1-8. Lewey, F.H. 1941. Experimental chronic carbon disulfide poisoning in dogs. J. Industr. Hyg. 23: 4-5. Lewey, F.H. 1941. Neurological, medical and biochemical signs and symptoms indicating chronic industrial carbon disulfide absorption. Ann. Int. Med. 15: 869. Li, T., Vallee, B. 1969. Alcohol dehydrogenase and ethanol metabolism. Surg. Clin. North Am 49: 577-582. Liddon, S.C., Satran, R. 1967. Disulfiram (Antabuse) psychosis. Am. J. Psychiatr. 123: 1283-1289. Lieber, C., DeCarli, L.M. 1968. Ethanol oxidation by hepatic microsomes: Adaptive increase after ethanol feeding. Science 162: 917-918, Linderholm, H . , Berb, K. 1951. A method for the determination of tetraethylthiuram disulphide (Antabuse, Abstinyl) and diethyldichio- carbamate in blood and urine. Some studies on the metabolism of tetra ethylthiuram disulphide. Scand. J. Clin. Lab* Invest, 3: 96-102. Linoilal, Haltia, A.M., Seppaiaine, A.M. j a £ a K , 1974. Experimental carbon disulphide poisoning. Morphological and neurophysiological studies. In, Proceedings of the Seventh International Congress of Neuropathology, eds. S.T. Kornyey,~~S.T. Tarisk, G. GoagtonyiT Excerpta Medica, Amsterdam. 104 Lundwall, L., Baekeland, F. 1971. Disulfiram treatment of alcoholism. J. Nerv. Meat. Dis. 153: 381-394. Lynn, R.K., Smith, R.G., Leger, R.M., Deinzer, M.L., Criffin, D., Gerber, N. 1977. Identification of glucuronide metabolites of benzomorphan narcotic analgesic drugs in bile from the isolated perfused rat liver by gas chromatography and mass spectrometry. Drug Metab. Dispos. 5: 47-55. MaCabe, E.S., Wilson, W.W. 1954. Dangerous cardiac effects of tetra ethylthiuram disulfide (Antabuse) therapy in alcoholism. Am. Med. Assoc. Arch. In. Med. 94: 259-263. MacLeod, L.D.', 1950. Acetaldehyde in relation to intoxication by ethyl alcohol. Quart. J. Stud. Ale. 11: 385-390. McBain, J., Mann, J. 1969. S-methylation, oxidation, hydroxylation and conjugation of thiophenol in the rat. Biochem. Pharmacol. 18: 2282- 2285. Majchrowics, E., Lipton, M., Meek, J., Hall, L. 1968. Effects of chronic ethanol consumption on the clearance of acutely administered ethanol and acetaldehyde from blood in rats. Quart. J. Stud. Ale. 29: 553-557. Major, L., Murphy, D., Gershon, S., Brown, G. 1979. The role of plasma amine oxidase, platelet monoamine oxidase nd red cell catechol-O-methyl transferease in severe behavioral reactions to disulfiram. Am. J. Psychiatr. 136: 679-684. Mardonea, J. 1963. Physiological Pharmacology, eds, W.S. Root, F.G. Hofmann, Vol. 1, Chapter B. Academic Press, New York. Markham, J.D., Hoff, E.C. 1953. Toxic manifestations in the Antabuse- alcohol reaction. Study of electrocardiographic changes. J.A.M.A. 152: 1597-1600. Marselos, M., Lang, M., Torronen, R. 1976. Modifications of drug metab olism by disulfiram and diethyldithiocarbamate. II. D-glucuronic acid pathway. Chem. Biol. Interact. 15: 277-287. Martensen-Larsen, 0. 1951. Psychotic phenomena provoked by tetra ethylthiuram disulphide. Quart. J. Stud. Ale. 12: 206-216. Matteis, F. 1974. Covalent binding of sulfur to microsomes and loss of cytochrome P-450 during the oxidative desulfuration of several chemicals. Mol. Pharmacol. 10: 849-854. Maw, G. 1954. Sulfate formation from dimethylthetin in rat liver. Biochem. J. 58: 665-670. 105 Milleri S. 1977. Disulfiram: An unusual side effect. J.A.M.A. 237: 2602-2603. Moddle, G., Bilbao, J., Payne, 0., Ashby, P. 1978. Disulfiram neuro pathy. Arc. Neurol. 35: 658-660. Mottin, J.L. 1973. Drug induced attenuation of alcohol consumption.' Quart. J. Stud. Ale. 34: 444-472. Neims, A., Coffey, D«, HeHerman, L. 1966. Interactions between tetra ethylthiuram disulphide and the sulfhydryl groups of D-amino and oxidase and of hemoglobin. J. Biol. Chem. 241: 5941-5948. Nora, A.H., Nora, J.J., Blue, J. 1977. Limb-reduction anomalies in infants born to disulfiram-treated alcoholic mothers. Lancet 2: 664. Nygaard, A., Sumner, J. 1952. D-glyceraldehyde 3-phosphate dehydro genase: A comparison with liver aldehyde dehydrogenase. Arch. Biochem* Biophys. 39: 119-128. Olesen, O.V. 1966. Disulfiram (Antabuse) as inhibitor of phenytoin metabolism. Acta Pharmacol. Toxicol. 24: 317-322. Olesen, O.V. 1967. The influence of disulfiram and calcium-carbamide on the serum diphenylhydantoin. Arch. Neurol. 16: 642-644. O'Reilly, R. 1973. Interaction of sodium warfarin and disulfiram (Antabuse) in man. Ann. Int. Med. 78: 73-76. Podgainy, H., Bressler, R* 1968. Biochemical basis of the sulfonyl urea induced Antabuse syndrome. Diabetes 17: 679-683. Popper, H., Schaffner, F. 1957. Liver: Structure and Function, p. 103-106, McGraw-Hill, New York* Price, T., Silberfarb, P. 1976. Convulsions following disulfiram treatment. Am. J. Psychiatr. 133: 235. Prickett, C.S., Johnston, C.D. 1953. The in vivo production of carbon disulifide from tetraethylthiuram disulfide (Antabuse). Biochim. Biophys. Acta 12: 542. Prue, C.A., Warner, C.R., Kho, B.T. 1972. Pulse polarographic deter mination of disulfiram. J. Pharra. Sci. 61: 249. Pullar-Strecker, H. 1955. Notes on substances other than TED causing distaste, disinclination or dislike for alcohol. Int. J. Ale. Alcsm. 1: 98-102. 106 Raby, K. 1956. The antabuse-alcohol reaction. Summary of clinical and experimental investigations. Danish Med. Bull. 3: 168*171. Rainey, J. 1978. Disulfiram and CS2 toxicity. Am. J. Psychiatr. 135: 623-624. Ranek, C., Andreasen, P. 1977. Disulfiram hepatotoxicity. Brit. Med. J. 2: 94-96. Raskin, N., Sokoloff, L. 1968. Brain alcohol dehydrogenase. Science 162: 131-132. * Remmer, H., Bock, K., Rexer, B. 1975. Relationship between microsomal hydroxylase and glucuronyltransferase. Adv. Exp. Med. Biol. 58: 335. Remy, C. 1963. Metabolism of thiopyrimidines and thiopurines. S-methyl- ation with S-methylation with S-adenylmethionine transmethylase and catabolism in mammalian tissues. J. Biol. Cehm. 238: 1078-1084. Richert, D., Vanderlinde, R., Westerfeld, W. 1950. The composition of rat liver xanthine oxidase and its inhibition by antabuse. J. Biol. Chem. 183: 261-274. Ridge, J.W. 1963. The metabolism of acetaldehyde by the brain in vivo. Biochem. J. 88: 95-100. Rothstein, E. 1968. Warfarin effect enhanced by disulfiram. J.A.M.A. 206: 1574-L575. Rothstein, E. 1970. Use of disulfiram (Antabuse) in alcoholism. N. Eng. J. Med. 283:936. Rothstein, E. 1970, Combined use of disulfiram and metronidazole in treatment of alcoholism. Quart. J. Stud. Ale. 3i:c446-447. Royer, R., Derby, C., Lamarche, M. 1962. Recherchea experimentales sur les reactions vaaomotrices a l'alcool apres administration de quelques sulfamides hypoglycemiants. Terapie 17: 989-997. Sarcione, E., Sokal, J. 1958. Detoxification of thiouracil by S- methylation. J. Biol. Chem. 231: 605-608. Sarcione, E., Stulzman, L. 1960. A comparison of the metabolism of 6-mercaptopurine and its 6-methyl analog in the rat. Cancer Res. 20: 387-392. Sauter, A.M., Wiegrebe, W., von Wartburg, J.P. 1976. Determination of disulfiram and its metabolites in human blood. Arzneimittel-Forschung 26: 173-177. 107 Saucer, A.M., von Warcburg, J.P. 1977. Quantitative analysis of disul firam and its metabolites in human blood by gas-*liquid chromatography. J. Chromatogr. 133: 167-172. Schlaepfer, W.W., Hager, H. 1964. Ultrastructural studies of INH-induced neuropathy In rats: I. Early axonal changes. Am. J. Path. 45: 209-220* Schlaepfer, W.W., Hager, H. 1964. Ultrastructural studies of INH-induced neuropathy in rats: II. Alterations and decomposition of myelin sheat. Am. J. Path. 45: 423— 434. Sharkavri, M., Cianflone, D., El-Hawari, A. 1978. Toxic interactions between disulfiram and some centrally acting drugs in rats. J. Pharm. Pharmacol. 30: 63. Sheinin, E.B., Benson, W.R., 1978. J. Assoc. Off* Anal. Chem. 61: 55. Smith, R.L., Williams, R.T. • 1966. In, Glucuronic Acid, Free and Combined, p. 457, Ed., G.J. Dutton. Academic Press, New York and London. Snow, G. 1957. The metabolism of compounds related to ethanethiol. Biochem* J. 65: 77-82. Solomon, H.M., Schrogie, J., 1966. The effect of phenylramidol on the metabolism of bishydroxycoumarin. J. Pharmacol. Expt. Therap. 154: 660-666. Stege, T., Hanby, J., Lusio, N. 1977. Acetaldehyde-induced hepatic lipid peroxidation. Currents in Alcoholism, ed. F. Seixas, Vol. 1, Biological, Biochemical and Clinical Studies, pp. 139-159. Grune and Stratton, New York. Steiner, J.W., Carruthers, J.S. 1961. Studies on the fine tree. 1. The morphology of normal bile canaliculi, pre-ductules and bile ductules. Am. J. Pathol. 38: 639-661. Stoppani, A., Schwarcz, M., Fred, C. 1966. Action of zinc-complexing agents nicotamide adenine dinucleotide-linked aldehyde dehydrogenases from yeast and liver. Arch. Biochem. Biophys. 113: 464-477. Stripp, B., Greene, F., Gillette. 1969. Disulfiram impairment of drug metabolism by rat liver microsomea. J. Pharmacol. Expt. Therap. 170: 347-354. Stromme, J.H. 1963. Inhibition of hexokinase by disulfiram and diethyldithiocarbamate. Biochem. Pharmacol. 12: 157-166. Stromme, J.H. 1963. Effects of diethyldithiocarbnamate and disulfiram on glucose metabolism and glutathione content of human erythrocyes. Biochem. Pharmacol. 12: 705-715. 108 Stroone, J.H. 1965. Interaction of disulfiram and diethyldithiocarbamate with serum proteins studied by means of a gel-filtration technique. Biochem. Pharmacol. 14: 381-391. Stromme, J.H. 1965. Metabolism of disulfiram and diethyldithiocargamate in rats with demonstratin of an in vivo ethanol induced inhibition of the glucuronic acid conjugation of the thiol. Biochem. Pharmacol. 14: 393-410. Stromme, J.H. 1966. Distribution and chemical forms of diethyldithiocar- bamate and tetraethylthiuram disulphide (Disulfiram) in mice in relation to radio-protection. Biochem. Pharmacol. 15: 287-297. Svendsen, T . , Kristensen, M., Hansen, J., Skovsted, L. 1976. The influence of disulfiram on the half-life and metabolic clearance rate of di- phenylhydantoin and tolbutamide in man. Eur. J. Pharmacol. 9: 439-441. Taylor, A.F., 1964. Determination of tetraethylthiuram disulphide and elemental sulphur in organic extracts using cathode-ray polarography. Talanta 11: 894-896. Tompsett, S.L. 1964. The determination of disulfiram (Antabuse, Tetraethyl thiuram disulphide) in blood urine. Acta Pharmacol. Toxicol. 21; 20-22. Truitt, E.B., Duritz, G., Morgan, A.M., Prouty, R.W, 1962. Disulfiram-like actions produced by hypoglycemic sulfonylurea compounds. Quart. J. Stud. Ale. 23: 197-207, Truitt, E., Walsh, M. 1971. The role of acetaldehyde in the actions of ethanol. In, Biology of Alcoholism, eds., B. Kissin, H. Begleiter, Vol. 1, Biochemistry, pp. 161-196. Plenum Press, New York. Twiss, D.F., Bazier, S.A., Thomas, F. 1922. The dithiocarbamate accele rators of vulcanization. J. Soc. Chem. Industr. 41: 81T-88T. Vasak, V., Kopecky, J. 1967. On the Role of Pyridoxamine in the Mechanism of the Toxic Actions of Carton Disulfide, ed., H. Bneger, J. Teisinger, [>pr &-41. Excerpta-Medica, Amsterdam. Vasak, F,, Kopecky, J. 1967. On the Role of Pyridoxamine in the Mechanism of the Toxic Actions of Carbon Disulfide, ed.,H. Brieger, J. Teisinger, pp. 70-75. Excerpta Medica, Amsterdam. Vessel, E.S., Passananti, G.T., Cynthia, H. 1971. Impairment of drug metabolism by disulfiram in man. Clin. Pharmacol. Therap. 12: 785- 792, Vessel, E.S., Passananti, G.T., Greene, F.E. 1970. Impairment of drug metabolism in man by allopurinol and nortriptyline. N. Eng. J. Med. 283: 1484-1488. 109 Vial, J.D. 1958. The early changes in che axoplasn during Wallerian degeneracion. J. Biophys. Cytol. 4: 551-555. Wallerstein, R.S., Choclos, J.W., Friend, M.B., ec al., 1959. Hospital treatment of alcoholism: A comparative experimental study. Basic Books, New York. Weiner, M., Siddiqui, A.A., Bostanci, N., et al., 1965. Drug interactions. The effect of combined administration on the half-life of coumarin and pyrazolone drugs in man. Fed. Proc. 24: 153. Weisman, M.N. 1968. The use of disulfiram in the treatment of alcoholism. Md. St. Med. J. 17: 83-84. Williams, R.,T. 1959. Detoxification Mechanisms, p. 129. Chapman and Hall, London. Wilson, A. 1975. Disulfiram implantation in alcoholism treatment. Quart. J. Scud. Ale. 36: 555-565. Yodaiken, R.E. 1978. Ethylene bromide and disulfiram- A lethal combin ation. J.A.M.A. 239: 2783. Zendzikovski, S., Sletkiewicz, J., et al;, 1974. Pathomorphology of the experimental lesion of the peripheral* nervous system in white rats chronically exposed to carbon disulfide, In, Hausmanowa-Tetivsewics, eds., H. Tedjowska, Structure and function of normal and nri diseased muscle and peripheral nerve, pp. 319-326. Polish Medical Publisher, Warsaw.