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The effects of certain chelating agents on some aspects of copper metabolism in rats and mice Wyse, David George 1966

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THE EFFECTS OF CERTAIN CHELATING AGENTS ON SOME ASPECTS OF COPPER METABOLISM IN RATS AND MICE by DAVID GEORGE WYSE B.S.P., University of B r i t i s h Columbia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY i n the D i v i s i o n of Pharmacology of the Faculty of Pharmacy We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1966 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and study . I f u r t h e r agree that pe rmiss ion f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n permiss ion Department o f PAJA RM f?C Y The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date M a y 13 , I'M THE EFFECTS OF CERTAIN CHELATING AGENTS ON SOME ASPECTS OF COPPER METABOLISM IN RATS AND MICE by DAVID GEORGE WYSE ABSTRACT P l i c a t i c a c i d and 5-isopropyltropolone, which are found i n the heartwood of western red cedar (Thu.la p l i c a t a . Donn), are known chelators of copper. A study has been made of t h e i r e f f e c t s on some aspects of copper metabolism i n r a t s and mice. As a basis of comparison s i m i l a r experiments were c a r r i e d out using penicillamine (PEN), a chelating agent with wide c l i n i c a l use. In the experiments the sodium s a l t of 5-isopropyltropolone (T-Na) and the potassium s a l t of p l i c a t i c a c i d (P-X) were used. Tflfhen administered d a i l y , P-K and PEN caused an increase i n urinary and f e c a l excretion of copper In rats on a normal di e t and an increase i n the l e v e l of copper i n the l i v e r and kidney and a decreased l e v e l of copper i n the heart and br a i n . The increased urinary excretion i s much more marked with PEN than with P-K. T-Na administered d a i l y had no e f f e c t on the excretion of copper i n rats on a normal di e t and causes a r i s e i n copper content of l i v e r i i i l l and kidney and a lowering of copper content of heart and brai n . I t appears that the copper l e v e l elevation i n kidney and l i v e r caused by P-K and PEN i s due to an increased u t i l i z a t i o n of the routes of excretion while the increased l e v e l due to T-Na i s probably due to deposition i n the c e l l s . When these compounds are administered d a i l y to copper-fed rats s i m i l a r observations were made with the difference that i n organs where copper leve l s are increased the increases are greater and i n organs where copper l e v e l s are decreased the decreases are smaller. With T-Na there i s one marked difference i n that the copper l e v e l i n the brain i s increased. I t i s f e l t that t h i s increase i s due to a greater penetrating a b i l i t y of the T-Na-copper chelate because of i t s l i p i d s o l u b i l i t y . P-K and PEN have very l i t t l e e f f e c t on the t o x i c i t y of a single large dose of copper. E a r l i e r experiments with T-Na showed that when T-Na i s given shortly a f t e r a single large dose of copper the t o x i c i t y i s greatly increased. The explanation of t h i s i s very l i k e l y that T-Na increases the penetration of the copper into the CNS and i t i s here that the toxic e f f e c t i s exerted. I t i s f e l t that t h i s i s due to a s o l u b i l i t y f a c t o r , T-Na and i t s chelate being l i p l d - s o l u b l e and P-K, PEN and t h e i r chelates being water-soluble. iv When T-Na, P-K and PEN are given to rats i n small d a i l y doses over a long period, the a t r i a from such rat s exhibited a reduced chronotropic response to tyramine. I f at the same time as the rats are being given the chelating agent, they are given excess copper i n drinking water the tyramine response remains close to normal. In t h i s regard T-Na possesses the most a c t i v i t y . These observations support the theory that chelating agents i n h i b i t dopamine-@-hydroxylase by rendering copper inaccessible to the enzyme and that subsequent to t h i s i n h i b i t i o n the biosynthesis of the catecholamines i s i n h i b i t e d at the dopamine stage preventing the formation of norepinephrine and epinephrine. As the lev e l s of endogenous norepinephrine and epinephrine f a l l , the r e s u l t i s a reduced tyramine response. I f T-Na, P-K, PEN and D-Na are given i n a large single dose the i n h i b i t i o n of dopamine-^ -hydroxylase i s not evident while i t appears that COMT may be being i n h i b i t e d . Signatures of Examiners TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES i x LIST OF FIGURES x INTRODUCTION . . . . 1 COPPER METABOLISM 2 Absorption of Dietary Copper 2 Interdependence of Copper and Other Heavy Metals 3 Plasma Copper 4 Routes of Excretion 5 Tissue Content 6 Copper Proteins 9 Ceruloplasmin 1 0 Tyrosinase 12 Monamine Oxidase 13 Dopamine- ^ -hydroxylase 13 Cytochrome C Oxidase 14 Cerebrocuprein 15 Erythrocuprein 1 6 Other Proteins 1 7 Copper Deficiency 1 7 v v i Page Phys i o l o g i c a l Role of Copper 1 7 Copper T o x i c i t y 20 WILSON'S DISEASE (HEPATOLENTICULAR DEGENERATION) 21 General 21 Etiology 22 Pathological Observations 2 3 General 2 3 Liver 24 Brain 2 5 Kidney 2 7 C l i n i c a l Observations 2 8 Treatment 3 ° THE EFFECTS OF CHELATING AGENTS ON ENZYME SYSTEMS 3 2 STATEMENT OF PROBLEM " . . . 3 4 DRUGS USED 3 6 5-Isopropyltropolone 3& P l i c a t i c Acid 40 Penicillamine 41 SOME METHODS OF COPPER DETERMINATION . . . . . . . 45 EXPERIMENTAL PROCEDURE AND RESULTS 47 Copper Determination 47 Sample Taking and Ashing 48 v i i Page Urine 48 Feces 49 Tissues 50 Deionization and Colour Development 51 Colour Extraction and Measurement 52 Standard Curve 53 Estimation of Accuracy of Method 55 Ef f e c t s of Prolonged Treatment with Chelating Agents i n Normal and Copper-fed Rats 5& Method 56 Results 59 E f f e c t s of P-K and PEN on Copper Deposition and T o x i c i t y of a Single Large Dose of Copper . . . 72 Method 72 Results 74 Response to Tyramine of A t r i a from Rats Treated with Chelating Agents 77 Chronic Treatment with T-Na, P-K and PEN . . . 77 Method 77 Results 79 Acute Treatment with T-Na, P-K, PEN and D-Na . 8 l Method 81 Results 82 v i i i Page DISCUSSION 84 Long Term Administration of Chelating Compounds 84 Single Dose of Chelating Compound 88 E f f e c t on the Response of Isolated A t r i a to Tyramine 92 SUMMARY AND CONCLUSIONS 94 REFERENCES 98 LIST OF TABLES Table Page I. Literature Reports of Copper Content of Various Tissues of the Rat 7 I I . E f f e c t of T-Na, P-K, and PEN on Urinary and Fecal Excretion of Copper i n Normal and Copper-fed Rats 63 I I I . E f f e c t of T-Na, P-K, and PEN on the Copper Content of Brain of Normal and Copper-fed Rats 66 IV. E f f e c t of T-Na, P-K, and PEN on the Copper Content of Heart of Normal and Copper-fed Rats 68 V. E f f e c t of T-Na, P-K, and PEN on the Copper Content of Liver of Normal and Copper-fed Rats 69 VI. E f f e c t of T-Na, P-K and PEN on the Copper Content of Kidney of Normal and Copper-fed Rats 71 VII. The E f f e c t s of T-Na, P-K and PEN on a Single Acutely Toxic Dose of Copper . . . 74 VIII. The E f f e c t of P-K and PEN on the Amount of Copper i n Mouse Organs a f t e r a Single, Acutely Toxic, I.P. Dose of Copper . . . . 76 IX. The i n vivo E f f e c t of Prolonged Administra-t i o n of Copper, T-Na, P-K and PEN oh the Tyramine Response of Isolated Rat A t r i a . 80 X. The i n vivo E f f e c t of D-Na, T-Na, P-K and PEN on "the Tyramine Response of Isolated Rat A t r i a A f t e r Large Doses . . . 83 i x LIST OF FIGURES Figure Page 1. Schematic Pathway fo r Conversion of Dopamine to Norepinephrine by Dopamine--hydroxylase 1 5 2. Structure of Three Isopropyltropolone Isomers Found i n Heartwood of Thu.la p l i c a t a , (Donn) 3 7 3« Proposed Structure of Metal Complexes of 5-Isopropyltropolone 3 9 4. Structure of P l i c a t i c Acid 41 5 . Structure of Penicillamine 42 6. Standard Curve f o r the Determination of Copper 5 4 7 . The E f f e c t of T-Na, P-K and PEN on the Urinary Concentration of Copper i n Normal Rats 60 8. The E f f e c t of T-Na, P-K and PEN on the Urinary Concentration of Copper i n Copper-fed Rats 6 l x ACKNOWLEDGEMENT The writer i s indebted to Dr. J.E. Halliday f o r the guidance he so f r e e l y contributed throughout the course of t h i s work. A note of thanks i s also tendered to Mr. B. Twaites of the Department of Pathology who did the histochemical preparations and to Dr. W.H. Chase who aided i n the in t e r p r e t a t i o n of the r e s u l t s . Thanks are al s o extended to Dr. L.H. Bock of Rayonier of Canada (B.C.) Ltd., who supplied the p l i c a t i c a c i d and 5-isopropyltropolone and to Merck, Sharp and Dohme of Canada Ltd., who supplied the penicillamine. F i n a n c i a l assistance from the Warner-Lambert Research Fellowship and a National Research Council Scholarship i s g r a t e f u l l y acknowledged. The author would e s p e c i a l l y l i k e to thank h i s wife, Bonnie, f o r her patience and encouragement and Mr. D. Leathern and Mr. A.D. B l a i r f o r t h e i r p r a c t i c a l assistance. x i INTRODUCTION The element copper i s widely d i s t r i b u t e d on the earth's surface and i t s t i n a l l o y , bronze has been i n use by man f o r thousands of years. Salts of copper were used therapeutically i n eye disease by physicians of some of the ancient Middle Eastern Empires. Copper was i d e n t i f i e d i n plant and animal material over a century ago and was considered to be a contaminant. Eighty years passed before i t was recognized as an e s s e n t i a l constituent of a l l l i v i n g things. More recently, derangements i n copper metabolism have been p o s i t i v e l y implicated i n human and animal diseases. (1) Studies on the metabolism of copper have lagged behind those of the well-documented cations such as iron and calcium. One of the main reasons f o r t h i s was the lack of an e n t i r e l y adequate assay of copper i n b i o l o g i c a l material. With the advent of radio-isotopes i n the mid-f i f t i e s t h i s problem was overcome. Recently a great number of empirical observations of the b i o l o g i c a l s i g n i f i c a n c e of copper have been made and f o r a comprehensive picture there i s a necessity for c r i t i c a l s e l e c t i o n of t h i s material. An attempt w i l l be made to confine the discussion to man and the r a t . 1 2 COPPER METABOLISM Absorption of Dietary Copper The d a i l y d i e t of a North American man contains 2 to 5 mg. of copper. (2) The usual laboratory d i e t of rats contains approximately 10 ^m. of copper per gram and the intake of copper as a r e s u l t of drinking water i s n e g l i g i b l e . (3) Observations i n t h i s lab place the d a i l y intake of copper by an adult rat between 200 and 500^gm. Studies using radioactive c o p p e r ^ <4,5) have indicated that i n the r a t only a small proportion of ingested copper i s absorbed and that as the amount of copper i n the d i e t i s increased the percentage absorbed decreases although the absolute amount increases. The s i t e of absorption, which i s at l e a s t p a r t l y a c t i v e i n man, has been a t t r i b u t e d to the upper i n t e s t i n a l t r a c t . (1,45) I*1 the r a t the upper jejunal area of the small i n t e s t i n e appears to be the s i t e of maximal absorption (4,24) Studies on absorption i n the mouse indicate that two mechanisms are involved, one active and one passive. (1) In v i t r o , studies i n the hamster int e s t i n e indicate that uptake of copper from the mucosal side i s probably passive and that further transport to the serosal side involves a mechanism dependant on metabolic energy. (6) This study also implicates the lower small intestine as the s i t e of maximal absorption i n the hamster. 3 Interdependence of Copper and Other Heavy Metals A number of studies have been c a r r i e d out on the eff e c t s of various other cations on the metabolism of copper. When given i n high doses, zinc was found to have a negative e f f e c t on copper metabolism i n the r a t and Its action was thought to be a decreased u t i l i z a t i o n of copper and increased excretion with l i t t l e e f f e c t upon absorption. ( 7 , 9 ) One author ( 9 ) suggested t h i s e f f e c t of zinc may be primarily on i r o n metabolism which i n turn a f f e c t s copper. Molybdenum was also found to decrease copper u t i l i z a t i o n within the tissues and i t too was thought not to in t e r f e r e with copper uptake from the digestive t r a c t . (8) A more recent i n v e s t i g a t i o n ( 10) found that zinc and eadmium did i n fact depress the uptake of radiocopper 0 4" from the r a t stomach and duodenum. Cadmium al s o changed the proportions of copper d i s t r i b u t e d to various organs. S i l v e r and mercury also changed the proportions d i s t r i b u t e d to various tissues but had no s i g n i f i c a n t e f f e c t on absorption. Manganese, on the other hand, has been shown to cause an increase i n the amount of copper absorbed. (11) The exact sign i f i c a n c e of the i n t e r - r e l a t i o n s i s not known but these few examples give an i n d i c a t i o n of the complexity of copper metabolism. 4 Plasma Copper A study i n 1953 (12) presented evidence that copper i s present i n human and rat plasma i n at least two d i f f e r e n t forms. One f r a c t i o n , which reacts with sodium diethyldithiocarbamate only a f t e r removal from the protein by treatment with hydrochloric a c i d , corresponds to the copper cx^-globulin, ceruloplasmin. This f r a c t i o n constitutes approximately 96 and 99 percent of the copper normally present i n human and rat plasma, r e s p e c t i v e l y . The remainder of the copper i n plasma i s i n a form which reacts d i r e c t l y with the carbamate reagent but, l i k e the ind i r e c t - r e a c t i n g f r a c t i o n , i s also non-dialyzable within the physiologic pH range. Explanation of t h i s phenomenon i s given by recent studies with radiocopper^". (l ,2 ,5>13» 49) Copper f i r s t appears i n the plasma as cupric ion which i s probably loosely bound to albumin and thus i s non-dialyzable and yet reacts with the carbamate. Within about two hours of administration the i n i t i a l r i s e i n t o t a l plasma copper concentration i s followed by a sharp f a l l . During both t h i s r i s e and f a l l there i s a continued uptake of copper by the l i v e r , and there appears i n the blood a gradually increasing concentration of copper which i s t i g h t l y bound to ceruloplasmin and thus does not react d i r e c t l y with the carbamate. Liver uptake may be a pre-r e q u i s i t e f o r copper to appear i n ceruloplasmin since the available evidence suggests that ceruloplasmin i s 5 synthesized by the l i v e r . (2) I t i s also f e l t that cerulo-plasmin may be a donor of copper to other t i s s u e s . (5) Workers who have examined factors which influence the amount of copper i n the various f r a c t i o n s of the plasma (14,15) found that turpentine, e s t r a d i o l , epinephrine, typhoid vaccine and b a c t e r i a l cultures increased plasma copper l e v e l while dietary r e s t r i c t i o n decreased plasma copper. Routes of Excretion I t appears that there are three major routes of excretion followed by ingested copper. (1,4,16) These three routes are the b i l e , urine and d i r e c t l y through the I n t e s t i n a l w a l l . A n e g l i g i b l e quantity of copper i s excreted i n sweat, and i t i s estimated that 20 micrograms per day i s l o s t through menstrual flow. (1) Following the intravenous administration of radiocopper D H" to dogs, approximately 0.6 percent of the administered a c t i v i t y was excreted i n urine, about 1.5 percent passed d i r e c t l y through the i n t e s t i n a l w a l l , and about 7 to 10 percent was excreted into the b i l e . In the mouse 60 to 80 percent of an i n t r a -venously administered dose i s excreted. 45 to 65 percent i n the b i l e and 0 to 35 percent i n the urine. (17) The most extensive study (4) i n rats using radio-copper 0^ shows a f i x e d percentage (2 to 4 percent) of an 6 intravenously administered dose appeared i n the feces and i n t e s t i n a l contents of rats that had no b i l i a r y connection to the gut. Regardless of dose, intravenously administered copper began to be excreted promptly i n the b i l e and more than h a l f of the eventual t o t a l appeared within the f i r s t 4 to 8 hours. The urinary excretion of o r a l l y administered radiocopper^" varied with dosage. As the dose of radio-copper0"* increased the percentage of the dose which appeared i n the urine decreased. Presumably part of the explanation f o r t h i s i s a decreased percentage of copper absorbed with the increasing dose. In general, i f the dose LA of administered radiocopper O H" i s near that found i n the normal rat ' s d i e t about 1 percent of the dose i s recovered i n the urine. From these studies i t would appear that the major route of excretion i s the b i l e . I t has been shown (4) that i f the b i l e duct i s l i g a t e d the amounts of copper excreted by the other pathways increases. Tissue Content have a higher copper concentration, the largest amounts of copper are found i n muscle and skin by v i r t u e of t h e i r s i z e . (3) Of the i n t e r n a l organs the one which contains the most copper i s the l i v e r . Other organs which contain The t o t a l amount of copper found i n an adult r a t While other tissues may 7 a high proportion of copper are the kidneys, testes, brain and heart. Since the reports of the values of copper i n the various organs are sparse and not wholly i n agreement, they have been summarized i n Table I. Table I. Literature reports of copper content of various tissues of the r a t . ORGAN, TISSUE COPPER COMMENT REFERENCE or BODY FLUID /Mgm./gm. or ml, Muscle Skin t i Marrow Bone t i Liver i t t i t i II 1.41 2 . 5 1 1 . 7 4 . 9 9 I . 8 7 3 . 0 4 . 3 8 1 5 . 6 9 12.91 12 . 8 9 lit 9 . 8 5 . 5 1 3 2 5 14 -wet weight -dried weight -dried weight washed by per-fusion i n s i t u -wet weight -dried weight -dried weight 3 tt 3 1 3 t« 3 1 3 1 1 1 9 20 21 (continued) 8 Table I. (Continued) ORGAN, TISSUE, COPPER or BODY FLUID ugm./gm. or m l . COMMENT REFERENCE Liver Blood Fat Kidney it Testes Brain tt n Heart Lungs Pancreas Spleen Saliv a r y Glands 6.26 5.45 10.8 1 .24 0.35 5.10 23.8 16.9 20 10.19 19.1 1.96 2.88 4.53 11.2 4.54 1.57 2.70 1.80 2.79 -wet weight -wet weight washed by per-fusion i n s i t u -wet weight •dried weight washed by per-fusion j,n situ, -dried weight -wet weight t i 22 23 24 3 11 21 22 30 3 ti 22 30 3 (Continued) 9 Table I. (Continued) ORGAN, TISSUE, COMMENT REFERENCE or BODY FLUID ^gm./gm. or ml.  Adrenals 2.06 -wet weight 3 Eyes 0.29 n 1 1 Thyroid 2.64 " " P i t u i t a r y 4.85 " " B i l e 0.56 " " Feces 33-65 " " Urine O.32 1 1 Copper Proteins Highly p u r i f i e d copper proteins have been obtained from many mammalian sources (1,2) and a number of copper enzymes have been i d e n t i f i e d which are widely d i s t r i b u t e d i n the body. (1,2) There are undoubtedly several forms i n which copper may ex i s t i n vivo and these include free cupric or cuprous ions, and combinations of copper with amino acids, purines, pyrimidines, nucleotides, DNA, RNA and proteins. (2) What follows i s a consideration of the pr i n c i p l e proteins and enzymes. 10 Geruloplasmin As previously mentioned, over 95 percent of the copper i n plasma i s associated with the cX2~gl°bulin, ceruloplasmin. Ceruloplasmin has a molecular weight of 151*000. (2) I t contains about 0.3 percent copper which corresponds to eight atoms of copper per molecule. (2,25) Of the eight, 4 are thought to be cuprous and 4 cupric. (27) Normal plasma contains about 30 mg. of the protein per 100 ml. so that ceruloplasmin constitutes roughly 0.5 percent of the plasma proteins. (25) The copper appears to be an i n t e g r a l part of the molecule and i s presumably responsible f o r i t s blue c o l o r , the absorption peak of whieh i s at 6100 angstroms. (25»27) In v i t r o i t has been shown that about h a l f of the 8 atoms of copper present i n each molecule of ceruloplasmin are capable of exchanging with radioactive copper 0 4". (26,27) The exchange has been produced only when the protein and copper 0 4 - are mixed i n the presence of s u f f i c i e n t ascorbic a c i d to reduce r e v e r s i b l y the blue ceruloplasmin to i t s c o l o r l e s s form. One author (28) states that a t r a n s f e r r i n - l i k e function for ceruloplasmin now appears u n l i k e l y i n view of recent f a i l u r e to demonstrate t h i s exchange of copper i n vivo. A more recent study (29) shows that a l l eight copper atoms of ceruloplasmin may be r e v e r s i b l y removed by sodium diethyldithiocarbamate i n the presence of ascorbate or 11 c y s t e i n . I t goes on to f i n d that when copper i s removed from ceruloplasmin, or restored to apoceruloplasmin, no stable intermediate species are found with more than zero and fewer than eight atoms of copper per molecule. The authors state that i t appears that the copper f i r s t released from ceruloplasmin or bound to apoceruloplasmin, r e s u l t s i n a conformation of the protein from which subsequent copper ions are much more r e a d i l y released i n the former case, or to which the a d d i t i o n of successive copper ions i s greatly f a c i l i t a t e d i n the l a t t e r case. Sulfhydryl groups are not involved i n the binding of the copper and t i t r a t i o n studies implicate h i s t i d y l , and l y s y l or t y r o s y l residues of the protein i n i n t e r a c t i o n with the metal. (27) Ceruloplasmin i s commonly thought to have low ascorbic a c i d oxidase a c t i v i t y (2,27) but there i s some doubt (2) as to whether t h i s a c t i v i t y i s a property of free cupric ions i n solution and has f a l s e l y been a t t r i b u t e d to ceruloplasmin. In any event, the c a t a l y t i c a c t i v i t y probably involves reduction of C u + + by substrate and reoxidation by oxygen. (27) Ceruloplasmin, l i k e hemaglobin, albumin, hapto-globin, and t r a n s f e r r i n , i s heterogeneous. I t has not yet been proven that t h i s heterogeneity i s g e n e t i c a l l y determined as i s the heterogeneity of the other proteins l i s t e d . Pour human ceruloplasmins have already been 12 described and two more have since been noted. ( 2 ) The si g n i f i c a n c e of t h i s chemical heterogeneity of ceruloplasmin remains obscure. An inherited abnormality of ceruloplasmin w i l l be considered i n the discussion of Wilson's disease. Tyrosinase Tyrosinase, a mammalian copper protein with considerable chemical resemblance to ceruloplasmin, has a molecular weight of 34,000 and contains one copper atom per molecule. (1,2) The oxidase a c t i v i t y of tyrosinase i s most marked toward DOPA, although, unlike ceruloplasmin, i t a l s o seems able to oxidize the monophenol tyrosine and some of i t s derivatives which possess a free amino group. The r e v e r s i b l e d i s s o c i a b i l i t y of the copper protein bond of tyrosinase i s s i m i l a r to that of ceruloplasmin. There Is some evidence that tyrosinase, l i k e ceruloplasmin, may be heterogeneous with as many as three forms. Rare indivi d u a l s exhibit an autosomally and rec e s s i v e l y inherited deficiency of normal tyrosinase. Thus the physiologic role of tyrosinase has been elucidated by comparing these i n d i v i d u a l s , who are complete albinos with normally pigmented subjects. The albinos possess no detectable tyrosinase a c t i v i t y , which i s thus confirmed as being responsible for the production of melanin i n the skin and uveal t r a c t of normal i n d i v i d u a l s . 13 The two proteins discussed so f a r , ceruloplasmin and tyrosinase, both have oxidase a c t i v i t y . Suitable sub-strates f o r ceruloplasmin are paraphenylenediamine and d e r i v a t i v e s , benzidine, DOPA, serotonin and epinephrine as well as some other metabolic r e l a t i v e s of these compounds. (2) As previously mentioned tyrosinase has a marked oxidase a c t i v i t y towards DOPA and also tyrosine. Tyrosinase oxidizes tyrosine at a slower rate than i t oxidizes DOPA and may require the presence of the l a t t e r to "spark" the oxidation of the former. (2) The involvement of copper-protein-enzymes i n the metabolism of the catecholamines has been further implicated i n some recent findings. Monamine Oxidase One of the p r i n c i p l e enzymes involved i n the b i o l o g i c a l degradation of the catecholamines i s monamine oxidase. For several years i t has been known that the MAO of beef plasma i s a copper protein (32) but just r ecently i t has been shown that human MAO i s probably also a copper protein. (27»33) It has also been found that one of the enzymes which i s involved i n the biosynthesis of the catecholamines i s a copper protein. (34,35) Dopamine-^ -hydroxylase Dopamine-^-hydroxylase i s the enzyme which converts dopamine to norepinephrine and t h i s reaction i s 14 believed to be the f i n a l step i n the biosynthesis of norepinephrine. (35) The enzyme i s a co l o r l e s s copper protein, containing a f a i r l y constant amount of cuprie ions ( 2 per molecule) and a variable amount of cuprous ions. I t appears that ascorbate i s also necessary f o r the action of the enzyme and treatment of the enzyme with ascorbate leads to a reduction of most of the cupric ions i n the enzyme to the cuprous state. On subsequent exposure of the reduced enzyme to substrate, a large part of the cuprous ions are oxidized to cupric ions. A proposed scheme f o r the reaction i s given i n F i g . 1 . Cytochrome C oxidase Cytochrome C oxidase has well-known importance i n aerobic oxidation. I t has been shown that one molecule of heme and one atom each of i r o n and copper are present i n each water-soluble monomer of cytochrome oxidase. Studies indicate that the copper of cytochrome oxidase i s reduced s p e c i f i c a l l y by cytochrome C, and probably by ascorbate as we l l , and that t h i s reduction may be connected with the function of the enzyme. ( 2 ) Cerebrocuprein Cerebrocuprein i s a copper protein i s o l a t e d from bovine and human bra i n . It contains 2 atoms of copper per molecule. Cerebrocuprein i s si m i l a r to ceruloplasmin i n Figure 1. Schematic pathway f o r conversion of dopamine to norepinephrine by dopamine- ^-hydroxylase. 16 i t s bluish-green colour and the r e l a t i v e s t a b i l i t y of i t s copper-protein bond to diethyldithiocarbamate. It d i f f e r s from ceruloplasmin i n having a molecular weight of only 30,000 to 40,000 and i n lacking any enzymatic a c t i v i t y toward paraphenylenediamine. (2) Erythrocuprein Erythrocuprein i s a copper-protein which has been iso l a t e d from human red blood c e l l s . This protein binds almost a l l the copper i n erythrocytes. I t d i f f e r s from ceruloplasmin i n being only f a i n t l y greenish-blue, i n having less than one-fourth the molecular weight, i n possessing a d i f f e r e n t i s o e l e c t r i c point, and i n lacking oxidase a c t i v i t y toward paraphenylenediamine. I t a l s o d i f f e r s i n having i t s own absorption spectra i n both v i s i b l e and u l t r a - v i o l e t l i g h t , i n immunochemical s p e c i f i c i t y , and i n being present i n i t s usual concentration i n patients with Wilson's disease. On the other hand, both proteins bind copper t i g h t l y enough to prevent, almost completely, i t s d i r e c t reaction with diethyldithiocarbamate, both are glycoproteins, both contain the same amount of copper by weight, and both lose t h e i r copper i n the presence of s u f f i c i e n t cyanide. (2) 17 Other Proteins Other copper proteins are of minor importance. Two proteins (butyryl coenzyme A dehydrogenase and S-aminolevulinic a c i d dehydrase) where copper at f i r s t was thought to be an i n t e g r a l part of the protein have sub-sequently been shown to be copper f r e e . (2) Copper Deficiency Unequivocal and s i g n i f i c a n t deficiency of copper has never been reported i n human beings (1,2) but both n a t u r a l l y occurring (e.g. swayback i n sheep) and a r t i f i c i a l l y induced copper deficiency have been described In many animal species. (1,2,7,8,9,10) From a r t i f i c i a l l y induced deficiency states much has been learned about the physiological role of copper and what follows i s a consideration of t h i s aspect of copper metabolism. Physiological Role of Copper There i s some evidence that absorption and u t i l i z a t i o n of Iron are abnormally low i n copper deficiency and i r o n deficiency seen i n copper-deficient r a t s cannot be corrected by feeding i r o n alone. Furthermore, i f i r o n i s supplied parenterally the e f f e c t s of i t s deficiency are not corrected unless copper i s also supplied. (2) 18 I t has also been shown that the enzymatic a c t i v i t y of the heme-protein, cytochrome oxidase, i s markedly diminished i n copper deficiency. Such deficiency i n cytochrome oxidase may be the r e s u l t of several f a c t o r s . F i r s t , i t has already been pointed out that each monomeric molecule of cytochrome oxidase contains one atom of copper. Second, there i s evidence suggesting that synthesis of heme can be diminished i n copper deficiency, as demonstrated i n red c e l l s i n v i t r o . Third, the interference with i r o n absorption found i n copper deficiency may diminish the i r o n available f o r synthesis of t h i s protein. ( 2 ) Anemia has been observed i n copper d e f i c i e n t pigs and dogs and i n the case of pigs t h i s anemia i s morphologically indistinguishable from that induced by i r o n deficiency. ( 2 ) I t has been demonstrated that a deficiency i n phosphatidic acids i n copper-deficient rats was probably due to a diminished capacity f o r coupling coenzyme A-activated f a t t y acids to glycerophosphate. This observa-t i o n may be important i n understanding the pathogenesis of the demyelinating disease, "swayback," which i s seen i n newborn lambs dropped by ewes which are copper d e f i c i e n t . ( 2 ) In copper-deficient dogs, swine and chickens a defect i n osteoblastic a c t i v i t y i s demonstrable, although chondroblastic a c t i v i t y and c a l c i f i c a t i o n of c a r t i l a g e are unimpaired. The defect c l o s e l y resembles that seen i n 19 deficiency of ascorbic a c i d . In natural copper deficiency of c a t t l e and lambs, and i n the experimental deficiency of dogs, s k e l e t a l changes similar to those seen i n scurvy often develop. ( 2 ) In sheep suffering from n a t u r a l l y occurring copper deficiency a defect i n wool-keratin has been noted. This has been thought to be rela t e d to an abnormality i n the cross-linkages of ker a t i n which normally occur through d i s u l f i d e bridges. The wool of such sheep, and the ha i r of rats made copper d e f i c i e n t may show achromotrichia and i t i s l i k e l y that t h i s i s due to deficiency of tyrosinase. ( 2 ) In summary, low-copper diets i n animals lead to anemia, bone disorders, central-nervous-system demyelination and degeneration, abnormalities i n the pigmentation and curl i n g of hair and wool, f i b r o s i s and hypertrophy of the myocardium, diarrhea and scouring, and reproductive f a i l u r e , with the resorption of the fetus. (1) The foregoing pathologic e f f e c t s of copper deficiency have not been seen i n human beings. There are, i t i s true, conditions where the concentrations of serum copper are below normal but i f there are simultaneous s i g n i f i c a n t decreases of t o t a l body copper, or c l i n i c a l consequences, they have yet to be defined. Nevertheless, i n addition to the evidence from the observations made i n animals, the existence of the unique copper-proteins i n human beings which have already been discussed i s presumptive evidence f o r the e s s e n t i a l 20 nature of copper i n man. To varying degrees the functions of ceruloplasmin, tyrosinase and cytochrome oxidase are known and i t hardly seems l i k e l y that the other copper-proteins would e x i s t i f they had no function. Copper T o x i c i t y Presumably copper deficiency does not occur i n human beings because of the facts that dietary copper i s abundant r e l a t i v e to body requirements and that loss of copper i s i n s i g n i f i c a n t . (1,2) This abundance of copper i n the diet leads one to inquire as to whether or not copper t o x i c i t y i s a problem. I t i s rather remarkable that despite the dietary copper, and the widespread use of copper f o r plumbing, kitchen u t e n s i l s , beer-brewing k e t t l e s and whiskey s t i l l s , and exposure of many kinds of workers to high concentrations of copper, poisoning by t h i s metal i s almost, i f not quite, as unusual as copper deficiency. (2) Acute t o x i c i t y with copper i s r e l a t i v e l y unknown c l i n i c a l l y . The reason i s that the ingestion of more than 10 to 15 mg. of copper at one time w i l l cause nausea and vomiting and perhaps diarrhea and cramps and as a r e s u l t l i t t l e i s l e f t f o r absorption. One subject, who apparently ingested 20 grams of copper sulphate, and had been doing so several times weekly f o r months, was admitted to a h o s p i t a l with hemolytic anemia. (2) In pigeons i t has been shown (36) that subarachnoid inje c t i o n s of copper cause rapid 21 onset of convulsions followed by death. Presumably copper does not normally reach concentrations In the c e n t r a l -nervous-system s u f f i c i e n t to give t h i s e f f e c t due to i t s r e l a t i v e exclusion by the blood-brain b a r r i e r . There Is some evidence that animals are more susceptible to chronic copper poisoning than man. (2) Chronic t o x i c i t y experiments have been done many times both by feeding excessive copper i n the di e t (11,18,37,38) and by parenteral administration. (39,40,41,42,43) The usual re s u l t s are loss of appetite and necrosis and pigmentation of hepatic and renal c e l l s with the elevation of copper concentration i n the organs e s p e c i a l l y the l i v e r . There i s , however, one very small group of individ u a l s i n whom there r e g u l a r l y develops progressive and f a t a l copper t o x i c i t y although they eat no more dietary copper than other i n d i v i d u a l s . They suffer from hereditary hepatolenticular degeneration (Wilson's disease). WILSON'S DISEASE (HEPATOLENTICULAR DEGENERATION) General Wilson's disease i s inherited i n autosomal recessive fashion. (44,46) I t has been reported from Europe and North America, from the Caribbean and South America, from China, Japan and Viet-Nam, from A u s t r a l i a , 22 from India and from I s r a e l . Estimates of the gene's frequency are approximate because of i t s recessive nature and vary from 1 i n 2000 i n the United States to about 10 times as high i n Japan. Respectively, these figures would indicate an incidence of the disease of about 12 and 1200 per 200 m i l l i o n people and the t o t a l number of patients i n the United States i s probably close to t h e i r arithmetic mean. (44) Etiology The r e l a t i o n of the plasma copper-protein, ceruloplasmin to the normal and abnormal a l l e l e s of the gene i s s t i l l obscure. Although long-standing deficiency or absence of t h i s protein i s found i n over 90 percent of patients with Wilson's disease, (25*44,47,48) more than a dozen patients have been reported i n whom a normal concentra-t i o n of ceruloplasmin and Wilson's disease coexisted at least t r a n s i e n t l y . (44) This decrease i n ceruloplasmin i s generally noted by an increase i n the f r a c t i o n of serum copper which d i r e c t l y reacts with diethyldithiocarbamate and a decrease i n the f r a c t i o n which reacts only a f t e r being treated with a c i d . (47,48) In view of the fact that the disease exists i n some eases even when ceruloplasmin i s present i n normal amounts, i t i s doubtful that ceruloplasmin i s the normal a l l e l e ' s primary product and 23 that a very low rate of synthesis of ceruloplasmin i s the primary inherited defect r e s u l t i n g i n the disease although t h i s was the o r i g i n a l hypothesis. This observation, however, does not preclude the p o s s i b i l i t y that there may be more than one abnormal "Wilson's disease a l l e l e " or that there may be a suppressor gene capable of modifying the e f f e c t s of an abnormal a l l e l e to account f o r these observations. (44) Recently evidence has been presented (30) that a l t e r a -tions i n the l e v e l of serum ceruloplasmin i s secondary to some phase of l i v e r damage. Pathological Observations General Proof of homozygosity f o r a "Wilson's disease gene" consists of a po s i t i v e family h i s t o r y and c l i n i c a l f indings, prime among which i s a Kayser^Fleischer r i n g ; and a decreased or zero concentration of ceruloplasmin i n serum, together with an increased concentration of copper i n the urine and t i s s u e s . The detection of heterozygous c a r r i e r s of a "Wilson's disease gene," who are always asymptomatic, i s more d i f f i c u l t and less c e r t a i n . On the one hand, they may be distinguished from homozygously normal indivi d u a l s when they exh i b i t hereditary deficiency of ceruloplasmin i n serum or when abnormal copper metabolism can be demonstrated by means of radioactive copper 0 4". On 24 the other hand, heterozygotes may be d i f f e r e n t i a t e d from homozygously abnormal patients by the absence, i n the former, of hypocupriuria and by t h e i r hepatic copper concentrations which, though somewhat above normal, average only one eighth the mean value found i n untreated patients. (44) The concentrations of copper i n several organs and tissues of patients with Wilson's disease, p a r t i c u l a r l y the l i v e r , b rain, and cornea are much higher than i n those of other i n d i v i d u a l s . (2,44,50) The l i v e r may have a hundredfold and the brain tenfold greater concentration of copper than normal, and the Kayser-Fleischer corneal r i n g , found only i n t h i s i l l n e s s , consists of copper. Increases of tissue copper to t h i s extent are not known i n any other pathological condition. As t h i s elevation of tissu e copper concentration almost i n v a r i a b l y precedes h i s t o l o g i c and c l i n i c a l manifestations of the disease, copper has come to be generally considered the toxic agent which causes the pathologic changes of Wilson's disease. (44) Liver In biopsy and autopsy specimens of l i v e r from patients, copper has been demonstrated histochemically i n the cytoplasm of hepatic c e l l s — b u t not i n the nucleus or i n Kupfer c e l l s — w i t h a tendency to greater concentration at the periphery of hepatic lobules. (44,51>52) Within 25 c e l l s copper granules are concentrated near the border adjacent to the b i l e canaliculus, which has led to the suggestion that copper i s present i n lyosomes i n Wilson's disease. (44,53>53a) Another study (54) found that i n patients with Wilson's disease 35 percent of the t o t a l l i v e r copper was found i n the mitochondrial f r a c t i o n and t h i s f i g u r e represents 40 times the amount normally found i n t h i s f r a c t i o n . This study found further that 27 percent was i n the soluble f r a c t i o n , 10 percent was i n the micro-somal f r a c t i o n and 28 percent i n the nuclear f r a c t i o n . The author f e e l s , however, that the disproportionately large amount of copper i n the nuclear f r a c t i o n i s due to con-tamination by mitochondria. Similar i n t r a l o b u l a r and i n t r a c e l l u l a r (except f o r a higher nuclear f r a c t i o n ) d i s t r i b u -tions of copper have been observed i n the l i v e r s of r a t s experimentally poisoned with copper. (44,51»52>57) In view of t h i s , the f a c t that necrosis, which plays a central role i n the probable evolution of the parenchymal hepatic changes i n Wilson's disease, has not been reproducibly obtained with copper i n experimental animals remains unexplained. (44) Brain In the brain, copper seems l i k e l y to be the primary cause of the complex neurologic and p s y c h i a t r i c disturbances. In h i s o r i g i n a l paper (50) Cumings found an 26 elevation of copper concentration i n both c o r t i c a l grey and c o r t i c a l white matter. There was also an increase i n the thalamus and caudate nucleus and the l e n t i c u l a r nucleus and thus the name, hepatolenticular degeneration. Later other workers (44) found elevated copper i n the cerebellum, brain stem and spinal cord as w e l l . Recently there has been a trend to e s t a b l i s h the name hepatocerebral degeneration i n place of the older a p p e l l a t i o n . (55) Popoff (55) has described a patient i n whom c l i n i c a l and pathological changes (except f o r the Kayser-Fleischer ring) were very si m i l a r to those normally associated with Wilson's disease although i n the absence of demonstrated abnormalities of copper metabolism. Consequently the d i s t i n c t i o n i s made between "copper" and "non-copper" hepatocerebral degenera-t i o n . I t has been shown that less than 15 percent of the copper i n f r a c t i o n I copper proteins from normal human brain reacted d i r e c t l y with diethyldithiocarbamate. Furthermore, i t has been shown that i n Wilson's disease 29 to 45 percent of the f r a c t i o n I copper reacted d i r e c t l y . This d i r e c t reacting copper, amounting to more than 7.5 m. per gm. of wet t i s s u e , i s more than 20 times the amount of di r e c t - r e a c t i n g copper i n normal brain f r a c t i o n I. It i s f e l t that t h i s may l a r g e l y represent copper bound to brain proteins which are normally copper-free. (56) 27 In brain, both from Wilson's disease and from normal subjects, the largest proportion of the tissu e copper i s found i n the subcellular soluble f r a c t i o n . Y i e l d of copper i n the f r a c t i o n from brain of patients with Wilson's disease i s about 18 times that obtained i n t h i s f r a c t i o n from normal brains. In normal brain soluble copper was found reproducibly i n a single chromatographic peak suggesting a l l the soluble copper i n normal human brain may be accounted f o r by cerebrocuprein. In Wilson's disease, however, soluble pathological copper was found reproducibly spread over a very wide range of chromatographic fra c t i o n s while the pattern of e l u t i o n of brain proteins appeared normal. This would also suggest that copper i s bound to a number of d i f f e r e n t normal brain proteins which are normally copper-free. (54) Kidney I t i s probable, too, that excess copper i s nephrotoxic i n Wilson's disease. In rats exposed experi-mentally to cupric ion i n toxic amounts, copper has been v i s u a l i z e d histochemieally i n association with h i s t o l o g i c degeneration p r i n c i p a l l y i n tubular epithelium. (42) Copper and necrosis have also been seen i n the cytoplasm of renal tubular epithelium of patients with Wilson's disease. (58) 28 The question as to where t h i s copper comes from remains unanswered. I t seems unquestionable that patients must have had a greater than normal p o s i t i v e copper balance since b i r t h . Investigators, however, are divided on whether i t i s the r e s u l t of increased absorption or decreased excretion. (44) C l i n i c a l Observations The c l i n i c a l onset of Wilson's disease i s r a r e l y seen before the s i x t h year, while some patients apparently remain normal u n t i l the f i f t h decade. Generally the diagnosis i s made between late childhood and early adult l i f e , and the i n i t i a l symptoms are as l i k e l y to r e f l e c t damage to the l i v e r as to the brain. (44) Neurologic manifestations, though often e a r l y characterized by subtle signs and symptoms, generally include eventually some c h a r a c t e r i s t i c s of Parkinsonism and others of multiple s c l e r o s i s . In some patients, intention and r e s t i n g tremors and dysarthria are the out-standing symptoms; i n others, hypertonia, or dystonia, sometimes with associated choreo-athetosis, are predominant; more commonly, some degrees of tremulousness, dysarthria, dystonia, and drooling are seen. The occasional convulsions reported are not correlated with electroencephalograph^ changes. (44) 29 The f i r s t c l i n i c a l signs of brain damage are some-times p s y c h i a t r i c . Among things which have been observed as the f i r s t sign of loss of health are: paranoid delusion with assault, bizarre behaviour leading to a r r e s t , and emotional maladjustment. Psychological and emotional disturbances have been reported i n many patients with Wilson's disease l a t e as well as early i n the course of the disease. However, as has been recently pointed out, the great majority of these which have been labled schizophrenia or hysteria were i n f a c t not such s p e c i f i c e n t i t i e s . (44) The l i v e r too shows signs of the toxic accumulation of copper. I t can manifest e i t h e r parenchymal dysfunction, such as h e p a t i t i s , or c i r c u l a t o r y complications of post-necrotic c i r r h o s i s , such as rupture of esophageal varices and hypersplenism. Certain signs of hematologic disorder may appear which are d i r e c t l y or i n d i r e c t l y hepatic i n o r i g i n . (44) Renal dysfunction i s usually only observed a f t e r the primary diagnosis has been made and i s mainly concerned with tubular damage although glomerular function may also be adversely affected. Hematuria, albuminuria, glycosuria, phosphaturia, generalized aminoaciduria, u r i c o s u r i a , and hypercalciuria have a l l been noted. (44) The Kayser-Fleischer r i n g i s the single most important diagnostic finding i n patients. It provides v i s i b l e evidence of the tissue copper deposits i n the 30 disease, and t h e i r disappearance with treatment, and i s remarkable i n the t o t a l lack of v i s u a l symptoms i t produces. Other c l i n i c a l symptoms sometimes seen but which are probably secondary are bone and j o i n t abnormalities and, i n women, menstrual i r r e g u l a r i t i e s . (44) Treatment Cumings (50) was the f i r s t to suggest that chelating agents may be useful i n the treatment of Wilson's disease. His suggestion of BAL (2,3-dimercaptopropanol) was followed through by Denny-Brown and Porter. (59) In 1951 they reported that BAL increased the urinary copper excretion of f i v e patients, with marked neurological improvement i n three. Walshe (60) proposed that o r a l administration of penicillamine ( ^ , ^ - d i m e t h y l c y s t e i n e ) , a metabolite of p e n i c i l l i n , might be more desirable than BAL, which was not an i d e a l drug f o r prolonged therapy since i t s administration, requiring i n j e c t i o n , was rather frequently accompanied by headache, nausea, vomiting, and dizziness as well as pain. (59) It soon became evident that removal of copper from patients with Wilson's disease by penicillamine was a remarkable advance. (1,2,44,61,62, 63,64,65) Where penicillamine administration has been accompanied by l i t t l e or no improvement, t h i s has been quite probably due to too low a dosage, to too short a period of therapy since months to years are often required 31 f o r s i g n i f i c a n t amelioration to occur, or to the existence of i r r e v e r s i b l e damage at the onset of treatment. The administration of penicillamine has been accompanied by reactions—presumably h y p e r s e n s i t i v e — p r i n c i p a l l y involving the skin, white blood c e l l s , p l a t e l e t s , and kidney, a l l of which are almost always reversible but can, r a r e l y , be f a t a l . T o x i c i t y of both the L- and D- isomers may be due to t h e i r antagonism toward vitamin B£. (44) Ethylenediaminetetraacetic a c i d s a l t (Versene) has also been used. Orally i t gives no c u p r i u r i a and although i t gives cupriuria parenterally no c l i n i c a l improvement has never been reported. (44) Sodium diethyldithiocarbamate given intravenously has been reported to give c l i n i c a l improvement with a s l i g h t increase of excretion i n the urine. (66) Orally i t increased f e c a l excretion of copper three times. (66) As well as these chelating agents c e r t a i n a d d i t i o n a l measures are undertaken to decrease the absorp-t i o n of copper. Examples are: a low copper d i e t (61,63) and administration of potassium sulphide (2,6l) or cation exchange resins (44) to help eliminate dietary copper i n feces. Measures to increase ceruloplasmin t i t e r have not been successful. (44) 32 THE EFFECTS OF CHELATING AGENTS ON ENZYME SYSTEMS In 1965 Friedman and Kaufman (34,35) published two papers dealing with the nature of the enzyme dopamine-s-hydroxylase, the enzyme which converts depamine to norepinephrine i n the f i n a l step of the biosynthesis of t h i s catecholamine. They noted that t h i s enzyme was copper-dependent and that i t could be i n h i b i t e d by diethyldithiocarbamate (sodium s a l t to be referred to as D-Na) and that t h i s i n h i b i t i o n was reversed by adding copper. On the other hand, i f the apoprotein i s formed by cyanide treatment the a c t i v i t y of the enzyme was reduced to zero and only about 40$ could be recovered by the ++ —6 addition of Cu at a concentration of 5 x 10 M. and further addition of copper caused i n h i b i t i o n of the enzyme. Goldstein et a l . (85) had already shown i n 1964 that dopamine-^ -hydroxylase i s i n h i b i t e d i n v i t r o by chelating agents among which were EDTA and 4-isopropyl-tropolone. These r e s u l t s were confirmed i n vivo i n the rat heart and spleen using a single dose of lOOmg./Kg. of 4-isopropyltropolone. Goldstein argued that because tropolones also i n h i b i t catechol-O-methyl transferase and as dopamine i s a substrate f o r both enzymes, i n order to separate the e f f e c t s of the tropolone on the dopamine-^ -hydroxylase an a r t i f i c i a l system must be set up. To do 33 t h i s he used C labeled tyramine which i s converted to octopamine by dopamine-^ -hydroxylase but does not serve as a substrate f o r COMT. I t i s noteworthy that 4—isopropyl tropolone i s indeed a powerful i n h i b i t o r of dopamine--^ -++ hydroxylase as the additi o n of Cu ions did not reverse t h i s i n h i b i t i o n . (85) The i n h i b i t i o n of dopamine-^-hydroxylase has been noted elsewhere a l s o . In 19&5? C o l l i n s (111) found that i n vivo D-Na i n a dose of 500mg./Kg. S.C. lowered the amount of norepinephrine i n r a t ileum by 56$ and at the same time raised the dopamine l e v e l by 6 l%. In v i t r o he found that the e f f e c t was even greater. This indicates that as the enzyme i s i n h i b i t e d there i s an accumulation of dopamine. In 1966, Carlsson et a l . (112) also studied t h i s i n vivo e f f e c t of D-Na on the le v e l s of the two catecholamines i n various r a t t i s s u e s . The tissues studied by t h i s group include the heart, brain, ileum, femoral muscle and adrenals. On the heart they found no ef f e c t while i n a l l the other tissues a sim i l a r pattern to C o l l i n ' s observations was noted with the e f f e c t being most marked i n brain. This group also used a dose of 500mg./Kg. S.C. As has already been mentioned there are reports of the i n h i b i t i o n of COMT by the tropolones. (84,113) Belleau and Burba (84) indicate that t h i s i s a s p e c i f i c e f f e c t of the tropolones while other work (113) has 34 indicated that i t may be a general chelating e f f e c t as EDTA and T-Na have very s i m i l a r potentiating e f f e c t s on the response of is o l a t e d guinea pig a t r i a to the catecholamines and t h i s e f f e c t cannot be reversed by addition of magnesium which i s thought to be the metal involved. (84) In summary, dopamine-^ -hydroxylase i s i n h i b i t e d by excess copper and by chelating agents and thi s tends to decrease the amount of norepinephrine and increase the amount of dopamine. On the other hand, COMT i s i n h i b i t e d by chelating agents also and t h i s tends to increase the amount of norepinephrine. Two drugs have already been mentioned. D-Na has no e f f e c t on the norepinephrine l e v e l of rat heart although i t i s known to i n h i b i t dopamine-^-hydroxylase i n the r a t heart and thus tends to lower the norepinephrine l e v e l but i t also i n h i b i t s COMT i n the guinea pig heart and t h i s would tend to cancel out the ef f e c t on dopamine-B-hydroxylase. STATEMENT OF PROBLEM The p r i n c i p a l approach to the therapy of Wilson's disease i s the use of chelating agents. These drugs mobilize the toxic copper deposits and promote t h e i r excretion r e s u l t i n g i n amelioration of the symptoms. Although there i s some evidence (44) to suggest that 3 5 sulfhydryl groups are of necessity an i n t e g r a l part of a sa t i s f a c t o r y chelating agent i n the therapy of Wilson's disease, t h i s has not been unequivocally shown. As has already been pointed out, introduction of excess copper into experimental animals can imitate the pathological conditions of Wilson's disease to some degree. Wilson's disease i s unique i n that new drugs have never been screened i n experimental animals before use i n patients, probably because of the ( u n t i l recently) pernicous nature of the disease. The object of t h i s study i s to observe the ef f e c t s of two compounds which are constituents of the heartv/ood of western red cedar (Thuja p l i c a t a . Donn) and which are known to be chelators of copper. Experiments w i l l be concerned with t h e i r e f f e c t s on deposition or removal of copper from c e r t a i n organs and by c e r t a i n path-ways of excretion both i n normal rats and i n rats which have been fed an excess amount of copper over a period of several months. The e f f e c t s of the compounds on t o x i c i t y and d i s t r i b u t i o n of a single large dose of copper w i l l a l s o be investigated. Simultaneously, experiments w i l l be c a r r i e d out with the current drug of choice i n the therapy of Wilson's disease, penicillamine. Copper content of the organs, urine and feces w i l l be estimated c o l o r i m e t r i c a l l y and an attempt w i l l be made to demonstrate copper h i s t o -chemically. 36 I n l i n e w i t h the recent observation as t o the v u l n e r a b i l i t y of dopamine-^-hydroxylase t o c h e l a t o r s of copper and the r e p o r t s of i n h i b i t i o n of COMT by trop o l o n e s , the e f f e c t of these agents on the tyramine response of i s o l a t e d a t r i a from t r e a t e d r a t s w i l l be examined. DRUGS USED 5-Isopropyltropolone 5-Isopropyltropolone ( 2-hydroxy - 5-isopropyl-2,4 , 6,-cycloheptatrien-l-one) i s one of three isomers which are found i n the heartwood of western red cedar (Thuja p l i c a t a . Donn). Tropolone designates the seven membered carbon r i n g compound, 2 - h y d r o x y - 2 , 4 , 6 , - c y c l o h e p t a t r i e n - l -one, which i s r e l a t i v e l y r a r e i n nature. There are only three groups of n a t u r a l tropolones which are known: the tropolones of Cupressaceae ( i n c l u d i n g 5 - i s o p r o p y l t r o p o l o n e ) , the hydroxytropolonecarboxylic a c i d s , and the a l k a l o i d a l tropolones of L i l i a c a e ( c o l c h i c i n e ) . The three isomeric i s o p r o p y l t r o p o l o n e s which occur i n the heartwood of western red cedar have the s t r u c t u r e s shown i n F i g . 2. (67,68) Various s t u d i e s have been undertaken w i t h these compounds and a number of i n t e r e s t i n g f a c t s have been brought to l i g h t . Isopropyltropolones have been t e s t e d f o r t h e i r 37 2 F i g u r e 2. S t r u c t u r e s of three i s o p r o p y l t r o p o l o n e isomers found i n heartwood of Thu^la  p l i c a t a . (Donn). a n t i v i r a l e f f e c t on Japanese B. e n c e p h a l i t i s but a c t i v i t y was too l i m i t e d to recommend t h e i r use f o r t h i s purpose at the present time. (69) The sodium s a l t of 4- i s o p r o p y l t r o p o l o n e has been i n v e s t i g a t e d because of i t s a b i l i t y t o i n h i b i t glutamate o x i d a t i o n by R i c k e t t s i a t y p h i and i n t h i s regard i t i s more a c t i v e than the wide-spectrum a n t i b i o t i c s and p e n i c i l l i n . (70) Tropolones i n general have undergone very i n t e n s i v e study because of t h e i r a b i l i t y to exert a t o x i c e f f e c t on wood-dest r o y i n g f u n g i . (71,72) The sodium s a l t of 5-isopropyltropolone has even been screened f o r cancer i n h i b i t i n g p r o p e r t i e s . (73) The only pharmacological s t u d i e s of the i s o p r o p y l -tropolones are those done by the Japanese workers on the 4- i s o p r o p y l t r o p o l o n e and the st u d i e s of H a l l i d a y on the 5- i s o p r o p y l isomer, and Sanders and H a l l i d a y on 7-hydroxy-4-i s o p r o p y l t r o p o l o n e . The 4 - i s o p r o p y l isomer was found t o be l a r g e l y depressant i n nature though low concentrations .may stimu-l a t e some organs, e.g. i s o l a t e d heart and r a b b i t u t e r u s . (74,75*76) H a l l i d a y found that 5-isopropyltropolone exerted a combination of 38 stimulant and depressant actions on the mammalian c e n t r a l nervous system and only depression i n frogs. (77) Sanders noted that 7-hydroxy-4-isopropyltropolone resembled previously studied isopropyltropolones i n that i t produces both stimulant and depressant e f f e c t s on the ce n t r a l nervous system of mice. (78) A l l three of the isopropyltropolones exhibit one and the same green f e r r i c reaction. With copper acetate green copper complexes are obtained which are r e a d i l y soluble i n chloroform. This i s a c h a r a c t e r i s t i c and sensitive t e s t f o r the presence of isopropyltropolones. The proposed structure for the metal complexes of t h i s type i s shown i n F i g . 3 . (67,68) The green cupric complexes have been investigated extensively. They are insoluble i n water but dissolve i n , and may be c r y s t a l l i z e d from, chloroform, benzene, ethanol etc. These copper chelates are very stable. The copper chelate i s more stable than the corresponding chelates of beryllium, lead, z i n c , n i c k e l or cobalt and metal tropolone compounds are more stable than the corresponding acetylacetone compounds. (79»8o,8l,82) Isopropyltropolones have also been found to be potent i n h i b i t o r s of c e r t a i n enzyme systems. They i n h i b i t the e f f e c t of catechol-methyltransferase and although t h i s a ction was at f i r s t a t t r i b u t e d to chelation (83) i t i s now 39 ( C H 3 ) A C H C H ( C H 3 ) X Figure 3» Proposed structure of metal complexes of 5-isopropyltropolone. believed to be competitive I n h i b i t i o n wherein the tropolone r i n g , which i s i s o s t e r i c with the catechol r i n g , forms a complex with enzyme active s i t e s normally occupied by the substrate. (84) The copper-containing dopamine-^-hydroxylase enzyme system i s also i n h i b i t e d by tropolones. (85) The interference with these enzymes may explain c e r t a i n empirical observations (74,75*76,77,78,86) on the e f f e c t s of tropolones 14 and the f a c t that tropolones increase amounts of c e r t a i n C -labeled catechols found i n the brains of pre-treated r a t s . (87) to be referred to as T-Na, was used i n the experiments described here. This was p r e c i p i t a t e d from a concentrated solution of the tropolone i n alcohol by the addition of a f r e s h l y prepared a l c o h o l i c solution of sodium ethoxide. The p r e c i p i t a t e was washed with successive portions of anhydrous ether, and dried and powdered. The sodium s a l t of 5-isopropyltropolone, hereinafter 40 Pll c a t i c Acid A second compound isolated from Thu.ia plica^a is the strongly acidic (pK& 3) polyoxyphenol, pl i c a t i c acid. This compound is the major constituent of the hot water extract of the heartwood. The structure which i s shown in Fig. 4 i s named 2,3,6-trihydroxy-7-methoxy-2-hydroxymethyl-4-(3 *> 4 1-dihydroxy-5 *-methoxyphenyl)-tetralin - 3-carboxylic acid. This lignan is novel in that i t is the f i r s t known to have a monomethylated pyrogallol ring. It i s also the f i r s t lignan to have a free carboxylic acid group in the side chain. The compound was of Interest in this work as i t was known to be effective at complexing copper and holding i t in solution. At pH 8 i t w i l l hold 36 to 42 percent of i t s weight in copper. (91) Solutions of pl i c a t i c acid are unstable to sun-light, rapidly turning to dark red-brown in color. Heating the solid or aqueous solution causes a conversion to the 7 -lactone. The potassium salt, hereinafter to be referred to as P-K, i s f a i r l y stable however, and this i s the form in which i t was obtained. (88,89,90,91) The powdered P-K as well as i t s solutions were kept in the dark under refrigeration and solutions were freshly made every 5 days. As far as i s known, no biological tests of this compound have been made. O H F i g u r e 4. The s t r u c t u r e o f p l i c a t i c a c i d . P e n i c i l l a m i n e P e n i c i l l a m i n e i s the common name f o r 3 - m e r c a p t o v a l i n e (5) (Cuprimine 4 *, M . S . & D.) I t i s a c h a r a c t e r i s t i c d e g r a d a t i o n product of p e n i c i l l i n s b o t h i n v i v o and i n v i t r o . Commercial p r e p a r a t i o n i n v o l v e s h y d r o l y s i s o f mold-produced p e n i c i l l i n g i v i n g the D - i s o m e r . The L- i somer i s u n d e s i r a b l e because of i t s potent antagonism of p y r i d o x i n e . The s t r u c t u r e o f p e n i c i l l a m i n e i s g i v e n i n F i g . 5 . The a b i l i t y o f p e n i c i l l a m i n e t o c h e l a t e copper i s w e l l known and of great c l i n i c a l use i n W i l s o n ' s d i s e a s e . The exact nature of the p e n i c i l l a m i n e - c o p p e r l i n k a g e i s unknown. The copper atom might be h e l d as a b r i d g e between the s u l f h y d r y l groups o f two p e n i c i l l a m i n e molecules or bound i n a r i n g between the s u l f h y d r y l group and e i t h e r the amino or c a r b o x y l i c a c i d group. (92) 42 CH 3 H S — C - C H - C O O H I I CH 3 NH A Figure 5« The structure of penicillamine. Penicillamine has also been found useful i n a number of other c l i n i c a l conditions. The pathological macroglobulin of Waldenstrom's macroglobinemia can be re v e r s i b l y depolymerized by penicillamine i n v i t r o and c l i n i c a l improvement has been seen i n patients treated with penicillamine. (93»94) Another abnormal serum protein whose t i t e r i s reduced by penicillamine i s Rheumatoid Factor, and treatment of patients with penicillamine gives favourable r e s u l t s . (95) Penicillamine's chelating properties have also been applied to tre a t poisoning by heavy metals other than copper. (96,97»98) Used i n children with c y s t i n o s i s , penicillamine gave noticeable c l i n i c a l Improvement and t h i s i s thought to be due to i t s a b i l i t y to supply a sulfhydryl group and reactivate or maintain t h i o -dependant systems. (99) There have also been reports of the use of penicillamine i n the prevention of stone formation i n c y s t i n u r i a . (99a) A report of the regrowth of hair i n a patient with acrodermatitis enteropathica caused by penicillamine i s most i n t e r e s t i n g . (101) Two recent and most provocative a r t i c l e s (102,103) with regard to penicillamine are worthwhile examining i n 43 d e t a i l because of the h i n t of copper involvement i n sc h i z o p h r e n i a . I t has been observed t h a t melanogenesis ( i . e . production of melanin) i s increased i n schi z o p h r e n i c p a t i e n t s . At the time, the p a t i e n t s observed had been on prolonged phenothiazine therapy and i t was assumed t h a t the increased melanogenesis was due t o the phenothiazine medication. I n an e f f o r t t o support t h i s h y pothesis, necropsy m a t e r i a l of schi z o p h r e n i c p a t i e n t s f o r the p e r i o d 1947-49> during which phenothiazines were not i n use, was reviewed. I t was found that melanin was deposited i n the same areas as i n the phenothiazine-treated cases, although i n l e s s e r amounts. As has a l r e a d y been pointed out, melanin i s produced from t y r o s i n e by t y r o s i n a s e (DOPA oxidase) i n the presence of oxygen. This r e a c t i o n i s c o n t r o l l e d by the s o - c a l l e d darkening and l i g h t e n i n g f a c t o r s . Darkening f a c t o r s are o<^ - and ^ - m e l a n o c y t e - s t i m u l a t i n g hormones (M.S.H.) androgens, and estrogens. The most potent f a c t o r i s o(j-M.S.H. The l i g h t e n i n g f a c t o r s are melatonin and catecholamine, the most important being melatonin. Increased melanogenesis i s produced by a r e l a t i v e increase i n darkening f a c t o r s , i . e . , e i t h e r a r i s e i n darkening or f a l l i n l i g h t e n i n g f a c t o r s . An ex p l a n a t i o n i s r e q u i r e d f o r the melanosis o c c u r r i n g i n schi z o p h r e n i c p a t i e n t s not t r e a t e d w i t h phenothiazines and the f o l l o w i n g i s the hypothesis put 44 forward by these authors. (102,103) Melatonin, which i s the most potent lightening f a c t o r , i s produced i n the pineal gland from serotonin. Serotonin i s aeetylated and, i n the second step, methylated to form melatonin. Normally, melatonin i s metabolised to 6-hydroxymelatonin. It can be metabolised by cyclodehydration into 10-methoxyharmalan, which i s re l a t e d to harmaline, a known hallucinogenic agent. I t seems quite possible that under cer t a i n circumstances, such as when 5-nydroxyindole -0 -methyl transferase, which i s necessary i n the second step of melatonin formation, i s congenitally absent, harmaline alkaloids could be produced d i r e c t l y from 5-methoxy tryptamine. I f t h i s happens, the patient would have increased melanogenesis due to a lower l e v e l of melatonin and hallucinations due to the presence of harmaline a l k a l o i d s . In the authors' words the hypothesis i s "purely speculative and i t i s an o v e r s i m p l i f i c a t i o n of the complex problem of schizophrenia," but i t does o f f e r a d i r e c t i o n f o r further research into the biochemical basis of schizophrenia. At present work i s going on i n the d i r e c t i o n of pineal dysfunction. In treating the melanogenesis a low copper d i e t and penicillamine were t r i e d with the r a t i o n a l that t h i s would i n h i b i t tyrosinase and decrease the amounts of melanin and subsequently reduce the pigmentation. This therapy proved e f f e c t i v e and i t was noted at the same time 45 that there was an amelioration of schizophrenic symptoms which i n some cases was quite s t r i k i n g . (102,103) These observations support the hypothesis that hallucinogenic substances re l a t e d to these normal pigments may be the cause of schizophrenia i n some patients. The penicillamine used i n t h i s lab, hereinafter to be referred to as PEN, was supplied as a capsule. Solutions were made by di s s o l v i n g the PEN i n d i s t i l l e d water and subsequently f i l t e r i n g off the insoluble " f i l l e r . " SOME METHODS OF COPPER DETERMINATION An e n t i r e l y s a t i s f a c t o r y method f o r the determination of copper i n b i o l o g i c a l material has not yet been found. As previously mentioned, t h i s has been one of the primary problems i n the study of copper metabolism. The four most commonly used methods are absorptiometry and use the copper-selective organic reagents neocuproine, dithizone, (104) bis-cyclo-hexanone-oxalydihydrazone, (104, 105) and s a l t s of diethyldithiocarbamate. (106,107) How-ever, agreement between various methods has not been p a r t i c u l a r l y good. (104) This can be seen i n the v a r i e t y of copper concentrations found by d i f f e r e n t workers and methods f o r the various rat tissues given i n Table I. Perhaps the method which w i l l evolve as the best i s one 46 recently t r i e d with very good r e s u l t s which uses neo-cuproine, followed by subsequent displacement of the neo-cuproine with diethyldithiocarbamate. This gives a method which i s both copper-specific and s e n s i t i v e . (108) With the advent of radioisotopes i n the mid-f i f t i e s the use of radiocopper 0 4 has become very popular. However, i t s usefulness i s li m i t e d by the facts that i t has a very short h a l f - l i f e ( 12.8 hrs.) and i t gives no i n d i c a t i o n of endogenous copper. The l a t e s t technique to be used i n copper determination i s the electron probe microanalyzer. (53a) This i s an instrument capable of d i r e c t analysis of elements present i n a few cubic microns of t i s s u e . The p r i n c i p l e involved i s simple, an electron beam, 1 i n diameter, i s focused on a specimen and the atoms i n the path of the beam emit c h a r a c t e r i s t i c X-rays which are analyzed by an X-ray spectrometer and recorded. The method has been used more q u a l i t a t i v e l y than quanti-t a t i v e l y but i t s great usefulness l i e s i n the f a c t that i t can be applied to i n d i v i d u a l sub-cellular p a r t i c l e s a few microns i n s i z e . (53 a) 47 EXPERIMENTAL PROCEDURE AND RESULTS Copper Determination With s l i g h t modifications the copper analysis which was used was the diethyldithiocarbamate method of Eden and Green. (106) In our experiments the wet ashing procedure was replaced by dry ashing i n a muffle furnace. The chief disadvantage to the use of diethyldithiocarbamate colouring agent i n b i o l o g i c a l material i s the fac t that t h i s reagent also forms a colour complex with i r o n which has an absorption peak approximately i n the same area as copper. However, f e r r i c ammonium c i t r a t e i s completely unionized i n the presence of s u f f i c i e n t ammonium and c i t r a t e ions and thus i n t e r f e r i n g i r o n i s e f f e c t i v e l y removed by adding excess of these substances. During the colour development i t i s important to avoid so-called " l o c a l excess f a c t o r s , " that i s , i f the colouring agent i s added r a p i d l y without s u f f i c i e n t a g i t a t i o n , an area i n the solu t i o n w i l l develop wherein the amounts of ammonium and c i t r a t e are not s u f f i c i e n t to hold the i r o n i n an unionized form. (106) Sodium diethyldithiocarbamate has been found to be a very sensitive reagent f o r the determination of copper. The golden-brown coloured copper s a l t can be rapi d l y and qua n t i t a t i v e l y extracted from aqueous 4 8 solution by amyl alcohol and the colour i s i n t e n s i f i e d i n the organic solvent. The depth of colour i s d i r e c t l y proportional to the amount of copper present, provided the range of copper concentration i s not too great. The colour complex i s stable f o r at least 2 hours and the pH of the solution has l i t t l e e f f e c t on the colour Intensity between pH 5«7 and 9 . 2 . (107) Consideration has been given to these factors i n the evolution of the following method. Sample Taking and Ashing Urine Urine was c o l l e c t e d i n polyethylene containers from r a t s placed overnight i n metabolism cages. During the time that the animals were i n the cages they were deprived of food but had free acess to water. The metabolism cages were metal and any parts which were l i k e l y to come into contact with the urine were coated with p a r a f f i n wax. The design of the cages was such that the urine and feces would be e f f e c t i v e l y separated and that there was no danger of the water supply d i l u t i n g the urine. Any i n d i v i d u a l r a t was placed i n a metabolism cage no oftener than once a week. On the following day, the overnight c o l l e c t i o n of urine was pooled and from 3 to 5 twenty ml. aliquots were removed f o r a n a l y s i s . Each of 49 the twenty ml. aliquots were placed i n a 30 ml. VYCOR® cru c i b l e and evaporated to dryness at 100°C. The residue was subsequently ashed i n the muffle furnace at 400°C. The ash was extracted f o r 45 to 60 minutes with 5 mis. of 1:1 hydrochloric a c i d . The a c i d was then placed i n a large t e s t tube and the crucible was rinsed three times with successive 2 ml. portions of d i s t i l l e d water and the rinsings were added to the acid extract i n the t e s t tube. Feces Feces were obtained from the r a t s at the same time as the urine. Because of the r e l a t i v e l y large amount of copper present, the analysis was attempted on i n d i v i d u a l stools but t h i s proved to give too large a standard deviation. Consequently larger samples had to be taken and a d i l u t i o n step included. From the overnight c o l l e c -t i o n of feces 3 to 5 samples were taken with a dry weight from 1.5 to 2.0 grams. The drying, ashing and a c i d extraction were c a r r i e d out as before. Instead of placing the a c i d extract d i r e c t l y into the t e s t tube f o r analysis i t was placed i n a 50 ml. volumetric f l a s k . The crucible was washed out with several successive portions of d i s t i l l e d water and the washings were added to the a c i d extract i n the volumetric f l a s k . A f t e r making up to volume with: d i s t i l l e d water suitable aliquots were removed and placed 50 i n a large test tube f o r a n a l y s i s . Because of t h i s large d i l u t i o n , a further 2 . 5 mis. of concentrated hydrochloric acid was added to the aliquot and s u f f i c i e n t d i s t i l l e d water to make the sample volume up to eleven mis. Tissues The procedure f o r a l l tissues was the same except i n the case of the i n t e s t i n a l t r a c t of mice where a d i l u t i o n step, i d e n t i c a l to the one used f o r feces, was used. The animals were deeply anesthetized with ether and then decapitated. Tissues were removed and rinsed b r i e f l y i n d i s t i l l e d water and blotted dry. I f the tissue was not to be analyzed immediately i t was frozen u n t i l used. The tissues were placed i n cru c i b l e s and dried, ashed, extracted and the aci d extract placed i n the te s t tubes as previously described. The samples of brain included the o l f a c t o r y bulb, cerebral hemispheres, cerebellum, b r a i n stem, medulla, and spinal cord to about the l e v e l of C^. The hypophysis was also included and the very large trigeminal nerve was severed where i t emerges from the pons. A l l other c r a n i a l nerves were also severed close to t h e i r point of emergence. For an a l y s i s , 1 to 2 r a t brains and 2 to 3 mouse brains made up an i n d i v i d u a l sample. In the cases where part of a brain was used, the brain was divided by a mid-saggital section. 51 When washing the heart, a gentle r o l l i n g action between the thumb and index finger was used to remove blood from the v e n t r i c l e s and the coronary vessels as much as possible. Two rat hearts were used to make up an in d i v i d u a l sample. The hearts were trimmed of a l l attached vessels so that each heart consisted of only a t r i a and v e n t r i c l e s . Three kidneys i n the case of the mouse and one of the r a t were used to make a sample. While i n the mouse the whole l i v e r made up one sample, i n the r a t , f o r the sake of consistency, samples were always taken from the l e f t l a t e r a l lobe. Samples of rat l i v e r were about 0.5 grams of dry material. In the mouse care was taken to remove the g a l l bladder. In taking samples of i n t e s t i n a l t r a c t the cuts were made just r o s t r a l to the p y l o r i c sphincter and through the rectum as caudal as possible. No attempt was made to remove the attached mesentery. The outside of the i n t e s t i n a l t r a c t was washed i n d i s t i l l e d water but care was taken not to wash out any of the contents. One i n t e s t i n a l t r a c t made one sample. Deionization and Colour Development For the purpose of deionization of any i r o n present i n the samples 2 mis. of 50$ ammonium c i t r a t e was added from a burette to the 1 1 mis. already i n the 52 t e s t tube. This was followed by 5 mis. of ammonium hydroxide sp. gr. 0.880 which neutralizes the a c i d , renders the solution strongly basic, and supplies the necessary ammonium ions f o r the deionization. Next, 7 mis. of d i s t i l l e d water i s added to make the f i n a l volume up to 25 mis. To develop the colour, 2 mis. of 0.5$ recently f i l t e r e d sodium diethyldithiocarbamate was added dropwise from a burette with constant shaking. This procedure guards against the development of any " l o c a l excess fac t o r s . " The diethyldithiocarbamate solution was prepared by d i l u t i n g a stock 2% aqueous solution which was kept i n the dark. Any deposit formed was f i l t e r e d o f f before d i l u t i n g aliquots f o r d a i l y use. Colour Extraction and Measurement Exactly 5 mis. of n-amyl alcohol i s then added from a pipette and the tube i s closed with a clean rubber cork and vigorously shaken fo r 30 seconds, a period s u f f i c i e n t to ensure extraction of the yellow copper compound. In the presence of the e l e c t r o l y t e s i n sol u t i o n , the n-amyl alcohol r a p i d l y separates to the top and i t was then pipetted o f f and centrifuged f o r 5 minutes to remove any suspended water. To measure the colour, 3 mis. of the n-amyl alcohol was placed i n the cuvette and the absorbance 53 determined using a Bausch and Lomb Spectronic 20 colorimeter with the wavelength set at 460 mU_. Standard Curve Standard solutions were made by d i s s o l v i n g 0.3928 gms. of CuS04 ,5H 20 i n 1 l i t e r of water containing was di l u t e d 1 i n 10 and several standard solutions were prepared ranging from 5 to 20 micrograms per sample. These samples were treated i n the same manner as the other samples by adding 5 mis. of 1:1 HC1 and making up to 11 mis., then adding 2 mis. of 50% ammonium c i t r a t e , 5 mis. of ammonium hydroxide sp. gr. 0.880, and 7 mis. of d i s t i l l e d water. The colour was developed, extracted and measured as previously described. At the same time a t o t a l blank was prepared to exclude the p o s s i b i l i t y of traces of copper i n the reagents i n t e r f e r i n g . A t o t a l of 17 samples from 3 standard solutions were used to give the standard curve shown i n F i g . 6. From these data, using the method of least squares, (109) the equation y = 0.0290x + 0.009 was obtained, where x equals the amount of copper i n y^gms., y equals the absorbance and 0.009 i s a correction factor to allow f o r the f a c t that the l i n e does not quite pass through the o r i g i n . a drop of HgSO^ to give 0.1 mg. of copper per ml. This 54 55 Estimation of Accuracy of Method At random i n t e r v a l s during the course of the experiments copper standards were also analyzed using a " b l i n d technique" wherein the analyst had no knowledge of the amount of copper i n a standard sample. For t h i s purpose two d i f f e r e n t types of standard solu t i o n were used. One as previously described using CuSO^JjB^O and one where 0 . 2 6 9 9 gms. of CuCl 2,2H 20 were dissolved i n 1 l i t e r of water containing a drop of HC1 to give 0.1 mg. of copper per ml. This was done on nine occasions and the average error was found to be 6 . 3 % . Part of t h i s error i s the r e s u l t of the decreased accuracy of the colorimeter near the extremes of the scale. I f two of the readings which were below 0.100 on the absorbance scale are excluded, the average error becomes 4.2%. This gives a better estimate of the error as a l l samples from animals were taken so that the absorbance readings f e l l between 0.100 and 0 . 6 0 0 on the colorimeter scale. The largest source of error i s contamination of samples from outside sources. This was scrupulously avoided by keeping a set of equipment s p e c i f i c a l l y f o r t h i s purpose. The equipment was washed thoroughly before use each time using dichromate cleaning solution, followed by tap water, followed by many rinsings with copious amounts of d i s t i l l e d water. 56 As has already been noted there i s great disagree-ment i n the l i t e r a t u r e on the amounts of copper i n the various rat tissues and, therefore, published r e s u l t s cannot be used as a r e l i a b l e reference standard. Neverthe-l e s s , the conditions of the copper analysis have been r i g i d l y defined and i t was f e l t that the method i s e f f e c t i v e f o r showing differences between various groups which i s the main purpose of these experiments. E f f e c t s of Prolonged Treatment with Chelating Agents i n Normal and Copper-fed Rats Method For t h i s experiment a t o t a l number of about 350 male rats of the Wistar s t r a i n , ranging i n weight from 110 to 160 grams were used. I t was necessary that one-half of the rats be given copper i n a d d i t i o n a l d a i l y amounts to that present i n the normal d i e t f o r a period of eleven weeks. Since i t has been reported by others (7,11,38) that o r a l l y administered copper increases the copper content of body organs i t was decided to administer the excess copper by t h i s route i n the drinking water. For t h i s purpose the water contained CuClg^RVjO i n s u f f i c i e n t amount to give a solution containing 100 «^-gm. of copper per ml. Preliminary observations on the volume of water consumed d a i l y by rats i n t h i s lab indicated that the 57 above concentrations of copper i n the water would r e s u l t i n a d a i l y intake of copper of 3.5 mg. which i s approxi-mately 7 times the d a i l y intake of rats whose drinking water contains no added copper. The rats were divided into eight (8) groups of approximately 4-5 rats each. Four groups were provided with drinking water containing 100 jJgm./ml. of copper. One of these groups received no further treatment and served as a "copper c o n t r o l " group. Of the other three groups one was given T-Na, 2mg./day, another P-K, 5mg./day, and the t h i r d PEN, 1.6mg./day. A l l doses of these agents were administered by intraperitoneal i n j e c t i o n on six days per week. The other four groups were kept on a normal copper intake i n that no excess copper was added to t h e i r water supply. Of these groups, three were treated with d a i l y doses of the chelating agents the same as those given to the copper-fed r a t s . The fourth group received no treatment and served as a "normal control" group. As has already been mentioned the intake of copper by the copper-fed groups i s about 3.5mg./day. According to Owen's figures (4) about 8% of t h i s would be absorbed, or 0.28 mg. giving a maximum d a i l y copper absorption of 0.0044 moles which i s less than h a l f of the 0.108 moles of each drug given d a i l y . 58 Treatment of the groups as outlined above was car r i e d on f o r eleven weeks. Urine and feces from each group were co l l e c t e d at approximately one week i n t e r v a l s . At three and eleven weeks a f t e r the s t a r t of the experiment, a number of rats from each group were s a c r i f i c e d and the kidneys, heart, brain and a portion of the l i v e r were removed fo r analysis and histochemical examination. In ad d i t i o n to the determination of copper i n the organs, the kidney and the l i v e r were also examined histochemically by the diethyldithiocarbamate method of Howell. (51) The f r e s h l y removed organs were stored i n buffered formalin u n t i l used. For the histochemical test a f r e s h l y prepared and f i l t e r e d , saturated aqueous solution of sodium diethyldithiocarbamate was used. The section was flooded with t h i s f i l t r a t e , l e f t f o r 15 to 30 minutes, washed and mounted. Copper stains as d i s t i n c t yellow-brown granules. Sometimes when only a very small quantity of copper i s present the discrete granules are not found, but a f a i n t yellow colour i s produced throughout the section. This i s due to s l i g h t s o l u b i l i t y of the complex with consequent d i f f u s i o n . Although very s e n s i t i v e , t h i s method has the s l i g h t disadvantage that the yellow granules may resemble other n a t u r a l l y occurring pigments. The counterstains that have been t r i e d tend to over-s t a i n and obscure small granules of copper, and are 59 therefore, better omitted from purely q u a l i t a t i v e studies. (51) I t was hoped that the histochemical t e s t would locate copper i n such s i t e s as the b i l e duct i n l i v e r or tubular lumen i n kidney and thus confirm routes of excretion. Results The e f f e c t s of the chelating agents on the concentration of copper i n the urine of rats on a normal diet are summarized i n F i g . 7 . These l i n e s were determined by use of the method of least squares. (109) I t can be seen that a l l three compounds increased the urinary concentration of copper although T-Na seems to at f i r s t cause a f a l l i n copper concentration. The greatest e f f e c t was obtained with PEN, followed by P-K and f i n a l l y T-Na. I t i s also evident that t h i s increased urinary copper concentration becomes greater as the treatment with the chelating agents i s prolonged. In the case of the copper fed rats a s l i g h t l y d i f f e r e n t picture emerges. The r e s u l t s are shown i n F i g . 8. Once again PEN has the greatest e f f e c t but T-Na has a greater e f f e c t than P-K and indeed P-K gives a urinary copper concentration which i s lower than the group which received copper alone. Another s l i g h t difference i n t h i s case i s that the effectiveness of PEN seems to 6o o.qo L-Figure 7. The e f f e c t of T-Na, P-K and PEN on the urinary concentration of copper i n normal r a t s . 61 0.<?0 D A Y S Figure 8 . The e f f e c t of T-Na, P-K and PEN on the urinary concentration of copper i n copper fed rats given 100 j^gm of copper per ml. i n drinking water. / 62 decrease s l i g h t l y as treatment i s prolonged. However, t h i s i s inconsequential i n view of the great s u p e r i o r i t y shown by PEN i n increasing urinary excretion of copper. When urine samples were taken the r a t s were i n the metabolism cages overnight, a period of about 16 hours. It was noted that the rats which had the copper water drank less water than the other rats and as a consequence the overnight urine volume was smaller than that obtained i n the other groups. Further i t was noted that PEN caused s l i g h t l y more urine to be excreted both when given to normal rats and copper-fed r a t s . Also that P-K caused a s l i g h t decrease i n the amount of urine produced i n normal rats and an increase i n copper-fed r a t s . And f i n a l l y , that T-Na had very l i t t l e e f f e c t on the volume of urine i n both normal and copper-fed r a t s . I f the overnight volume of the normal control group i s taken as 1, the r a t i o of the volumes of the other groups i s roughly as follows: T-Na 0.9, P-K 0.7, PEN 1 . 2 , Cu 0 .4 , Cu plus T-Na 0.3, Cu plus P-K 0.6, and Cu plus PEN 0.7. It can be appreciated that t h i s volume f a c t o r would change the picture given by the urinary concentration of copper i f the volume and concentration were combined to calculate the absolute amount of copper excreted per r a t per 16 hour period i n the metabolism cages. This has been done and the r e s u l t s are summarized i n Table I I . 63 Table I I . E f f e c t of T-Na, P-K, and PEN on urinary and f e c a l excretion of copper i n normal and copper-fed r a t s . TREATMENT URINE Copper Mgm./rat/l6 hr. / + S.D. FECES Copper Mgm./gm. dry weight ± S.D. 1. NORMAL CONTROL 1.70 ± 0.52 55.29 ± 2.86 2. T-Na 2mg./day 1.84 + 0.24 54.14 + 4.44 3 . P-K 5 mg./day 2.10 + 0.28* 70.45 ± 2.65** 4 . PEN 1.6mg./day 6.10 ± 0.66** 74.06 + 3.25** 5 . COPPER CONTROL 100 /*gm./ml. of copper i n water 1.39 ± 0.13 -6. 100 /^m./ml. of copper i n water T-Na 2mg./day 1.42 ± 0.25 -7. 100 ^ugm./ml. of copper i n water P-K 5mg./day 1.56 + 0.45 -8. 100 y^m./ml. of copper i n water PEN 1.6mg./day 7.05 ± 1.25** -* S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p < 0.05 • • S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p <^0.01 64 These re s u l t s were subjected to the t-Test (110) to determine t h e i r s i g n i f i c a n c e . As can be seen the r e s u l t s are d i f f e r e n t . P-K causes a s l i g h t increase of urinary-copper excretion i n normal rats and possibly i n copper-fed rats although i n t h i s case the increase i s not s i g n i f i c a n t . T-Na has no s i g n i f i c a n t e f f e c t on either normal or copper-fed rats as f a r as urinary copper i s concerned. S u r p r i s i n g l y copper-fed rat s excrete less urinary copper than normal r a t s . Again PEN has the most dramatic e f f e c t , increasing copper i n the case of normal rats 3»5 times and i n the case of copper-fed rats 5 times. As also shown i n Table I I , T-Na had no e f f e c t while both PEN and P-K caused a s l i g h t increase i n the copper content of the feces of normal r a t s . The e f f e c t s of the various treatments on the brai n , heart, l i v e r and kidney are summarized i n Tables II I to VI. The r e s u l t s here were also subjected to the t-Test (110) and the sig n i f i c a n c e i s indicated on the tables. In the case of the normal control group samples were taken on Days 1 and 49 i n addition to Days 21 and 77 and i f there was no s i g n i f i c a n t difference between these i n d i v i d u a l days the r e s u l t s were combined to give a more composite average. The copper content of the l i v e r s of the normal control group rats remained constant throughout the course of the experiment, I.e., there was no 65 s i g n i f i c a n t difference between samples taken on the four separate occasions. A l l of the other organs showed a gradual increase with time. The greatest increase was i n the kidney followed by the heart and f i n a l l y the b r a i n . A random sampling of the brains of rats just p r i o r to the sta r t of the experiment indicated a copper concentration of 8.53 ± 0.03 |*gms. per gm. of dry weight. The r e s u l t s of the analysis of brains of rats i n the various groups i s summarized i n Table I I I . I t can be seen that the copper content of the brains of rats i n the normal control group increased s l i g h t l y over the period of the experiment. T-Na at f i r s t caused an increase i n copper i n the brain of normal rats followed by a decrease at the end of 77 days. On the other hand, P-K i n i t i a l l y caused a decrease i n the copper content and a subsequent increase at 77 days although i n the l a t t e r case t h i s was not s i g n i f i c a n t . At both 21 days and 77 days PEN caused a decrease but this decrease was s i g n i f i c a n t only at 77 days. In copper-fed rats T-Na caused a s i g n i f i c a n t increase over the copper control both at 21 and 77 days. P-K had no apparent e f f e c t at 21 days, but by the end of 77 days had caused a s i g n i f i c a n t decrease i n the copper content of copper-fed r a t s . Again i n the case of copper-fed rats PEN caused a decrease i n the copper content of 66 Table I I I . E f f e c t of T-Na, P-K, and PEN on the copper content of brain of normal and copper-fed r a t s . TREATMENT COPPER ^gm./gm. of dry weight + S.D, Day 21 Day 77 1. NORMAL CONTROL 9.47 + 0.07 10.39 + 0.73 2. T-Na 2mg./day 10.90 + 0.44** 8.86 + 0.26** 3 . P-K 5mg./day 7*70 + 1.04* 11.29 ± 1.05 4. PEN 1.6mg./day 8.04 + 1.22 7.80 + 2.57* 5. COPPER CONTROL 100 Mgm./ml. of 9.73 + 0.18 12.01 + 0.50 copper i n water 6. 100 >Agm./ml. of copj/er i n water 10.73 + 0.05** 12.95 ± 0.57** T-Na 2mg./day 7. 100 Mgm./ml. of copper i n water 9.71 + 1.01 11.23 ± 0.26** P-K 5mg./day 8. 100 Mgm./ml. of copper i n water 8 . 8 l + 0.25** 9.80 + O.56** PEN 1.6mg./day • S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p <^  0.05 * * S i g n i f l e a n t l y d i f f e r e n t from corresponding control group p <^0.01 67 brain both at 21 and 77 days and t h i s was a s i g n i f i c a n t decrease i n both instances. Preliminary samples of hearts of rats taken from a l l groups indicated a copper l e v e l of 19»5l ± 0.18 ^Xgms. per gm. of dry weight before the st a r t of the experiment. Copper content of the hearts of rats i n the experimental groups i s summarized i n Table IV. The copper content of the hearts of rats i n the normal control group showed a sharp increase between the 21st and 77th days. Both T-Na and P-K caused an i n i t i a l r i s e of the copper content of heart above that found i n the normal control group but t h i s was followed by a f a l l i n copper content at 77 days. This change i n a l l cases was s i g n i f i c a n t . PEN had no s i g n i f i c a n t e f f e c t on the copper content of hearts of normal rats at 21 days but a f t e r 77 days i t caused a s i g n i f i c a n t decrease i n the copper content. In copper-fed rats none of the drugs had any s i g n i f i c a n t e f f e c t on the copper content of heart except i n the case of PEN a f t e r 77 days. In t h i s instance PEN again caused a s i g n i f i c a n t decrease i n the copper l e v e l . Random sampling of the l i v e r s of rats p r i o r to the s t a r t of the experiment Indicated a copper concentra-t i o n of 14.41 + 1.26 yU-gms. per gm. of dry weight. Liver copper content i s summarized i n Table V. The l i v e r was the only organ examined where the copper l e v e l i n the normal control group was constant for the duration of the 68 Table IV. E f f e c t of T-Na, P-K, and PEN on the copper content of heart of normal and copper-fed r a t s . TREATMENT COPPER ^gm./gm. of dry weight + S.D. Day 21 Day 77 1. NORMAL CONTROL 18.46 + 1.35 24.89 + I .69 2. T-Na 2mg./day 21.47 ± O.56** 21.00 + 1.06** 3 . P-K 5mg./day 21.76 + 2.68** 21.79 ± 2.14** 4 . PEN 1.6mg./day 20.20 + 2.37 20.18 + 3.19** 5. COPPER CONTROL 100 JL«gm./ml. of copper i n water 20.10 + O.34 25.95 ± 0.74 6. 100 Mgm./ml. of copper i n water 19,76 + 0.71 25.82 + 1.60 T-Na 2mg./day 7. 100 Ugm./ml. of copper i n water 20.24 + 1.48 23.09 + 2 . 1 8 P-K 5mg»/day "~ 8. 100 JLxgm./ml. of copper i n water 19.17 + 1 .6 l 22.01 + 1.34** PEN 1.6mg./day • S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p <C0.05 * * S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p <^0.01 69 Table V. E f f e c t of T-Na, P-K, and PEN on the copper content of l i v e r of normal and copper-fed r a t s . TREATMENT COPPER pgm./gm. of dry weight + S.D. Day 21 Day 77 1. NORMAL CONTROL 13.63 + 1.47 13.63 + 1.47 2. T-Na 2mg./day 12.62 + O.26 17.19 ± I . 7 8 * * 3 . P-K 5mg./day 13.00 + 1.11 15.97 ± 1.00** 4. PEN 1.6mg./day 15.21 ± I . 6 3 * 20.51 + 2.58** 5. COPPER CONTROL 100 [Igm./ml. of 16.72 + 2.09 16.14 + 0.74 copper i n water "~ 6. 100 flgm./ml. of copper i n water 16.24 + I . 17 16.44 + I .89 T-Na 2mg./day 7. 100 |igm./ml. of copper i n water 13.95 ± 2.65 19.09 + 0.25** P-K 5mg./day 8. 100 |4gm./ml. of copper i n water 15*52 ± 2.94 15.11 + 0.94 PEN 1.6mg./day • S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p < 0.05 * * S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p < 0.01 70 experiment. At the end of 21 days PEN was the only drug which had had any s i g n i f i c a n t e f f e c t on the copper l e v e l of l i v e r s of normal rats and i n t h i s instance PEN caused an increase. At the end of 77 days a l l three of the drugs had raised the l e v e l of copper i n the l i v e r over that found i n the normal control group rats and i n each cas© t h i s increase was s i g n i f i c a n t . On the other hand, i n the copper-fed rats the only agent which had any s i g n i f i c a n t e f f e c t was P-K. P-K caused a s i g n i f i c a n t increase i n the l e v e l of copper i n the l i v e r of copper-fed rats a f t e r 77 days. In the case of the kidney, preliminary samples Indicated a copper l e v e l of 27.56 ± 2.57 j-lgms. per gm. of dry weight. The copper content of the kidney i n the various groups i s summarized i n Table VI. T-Na and P-K s i g n i f i c a n t l y increased the copper content of kidneys of normal rats at both 21 and 77 days but had no apparent e f f e c t at 21 days. In the copper-fed groups none of the drugs had any s i g n i f i c a n t e f f e c t on the copper content of the kidneys. The reason for t h i s i s probably the fa c t that such large standard deviations were found i n these groups. It does appear, however, that the copper content of the kidneys i n a l l cases may be higher at 77 days than i t was at 21 days. 7 1 Table VI. E f f e c t of T-Na, P-K, and PEN on the copper content of kidney of normal and copper-fed r a t s . TREATMENT COPPER (igm./gm of dry weight + S.D. Day 21 Day 7 7 1. NORMAL CONTROL 25.16 + 4.63 33-01 + 3 . 6 l 2. T-Na 2mg./day 34-38 + 5.96* 48.15 ± 9.48** 3 . P-K 5mg./day 19.85 + 1.68* 40.47 ± 7.94* 4 . PEN 1.6mg./day 28.28 + 2.00 43.27 + 15.49* 5 . COPPER CONTROL 100 flgm./ml. of 46.62 + 9.40 5 8 . 7 7 ± 7 . 3 9 copper i n water ~ 6. 100 Mgm./ml. of copper i n water 40 . 3 3 ± 5 . 7 7 53.46 + 6 . 1 9 T-Na 2mg./day 7 . 100 jUgm./ml. of copper i n water 34.53 + 8 . 5 9 43.95 + 8 .84 P-K 5mg./day 8 . 100'flgm./ml. of copper i n water 30.23 + 3.8O 52.67 + 7.94 PEN 1.6mg./day • S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p < ^ 0 . 0 5 * * S i g n i f i c a n t l y d i f f e r e n t from corresponding control group p <^  0 . 0 1 72 The r e s u l t s with the histochemical treatment were very disappointing. In a l l cases except two r e s u l t s were negative. In one s l i d e of kidney of normal r at treated with PEN and one of kidney of a copper-fed r a t treated with PEN the blood vessels showed a pos i t i v e diethyldithiocarbamate s t a i n . E f f e c t s of P-K and PEN on Copper Deposition and T o x i c i t y  of a Single Large Dose of Copper Method E a r l i e r experiments i n t h i s lab with T-Na had shown that i t greatly increased the t o x i c i t y of a single large dose of copper i n mice when the T-Na was given shortly a f t e r the copper. I t was decided to do s i m i l a r experiments using P-K and PEN. In add i t i o n to observing the e f f e c t s of these compounds on the t o x i c i t y of the copper, determinations of the copper content of br a i n , kidney, l i v e r and i n t e s t i n a l t r a c t were also made. Due to a shortage of T-Na i t was not possible to repeat the acute experiments using t h i s compound. Male Swiss albino mice, ranging i n weight from 25 to 30 grams were divided into 6 groups. The treatments used are outlined i n Tables VII and VIII. Copper solutions were made using CuCl 2 , 2 H 20 and d i s t i l l e d water. Although the dosages of the drugs are given i n mg./Kg. 73 they were calculated on a molar basis so that each dose i s O .G97 moles/Kg. The dose of copper i s 1.15 moles/Kg. and i s , therefore, s l i g h t l y i n excess of each of the drugs on a molar b a s i s . On the day preceding the experiment and during the experiment i t s e l f food and water were f r e e l y a ccessible. A f t e r administration of the copper the animals were observed continuously f o r 4 hours and during t h i s period the drugs were administered at the appropriate times. Animals which died a f t e r the copper but before the drug were discarded and t h i s p a r t l y accounts for the v a r i a b i l i t y i n the size of the groups. At the end of 4 hours from the time of administration of copper the animals were s a c r i f i c e d and samples of l i v e r , kidney, i n t e s t i n a l t r a c t and brain were taken f o r a n a l y s i s . Results The r e s u l t s of t h i s experiment are summarized i n Tables VII and VIII. For purposes of comparison, r e s u l t s of a previous similar experiment using T-Na are included i n Table VII. Before the i n j e c t i o n of the copper there was the normal spontaneous a c t i v i t y of mice which included eating, drinking, intermittent f i g h t i n g and the usual i n q u i s i t i v e n e s s . Immediately a f t e r the i n j e c t i o n of the copper spontaneous a c t i v i t y ceased and there was a ce r t a i n amount of writhing as a r e s u l t of l o c a l i r r i t a t i o n at the 74 Table VII. The eff e c t s of T-Na, P-K and PEN on a single acutely toxic dose of copper. ( A l l i n j e c t i o n s intraperitoneal.) TIME BETWEEN INJECTION OF COPPER AND INJECTION DEATHS AFTER TREATMENT OF DRUG 4 HOURS 1. CONTROL - 0/9 2. COPPER 7.5mg./Kg. - 1/7 3 . *C0PPER 7.5mg./Kg. 10 min. 7/7 T-Na 40 mg./Kg. 4. COPPER 7.5mg./Kg. 10 min. 1/8 P-K 97 mg./Kg. 5. COPPER 7.5mg./Kg. 10 min. 0/8 PEN 32mg./Kg. 6. *C0PPER 7.5mg./Kg. 2 hr. 1/8 T-Na 40mg./Kg. 7. COPPER 7.5mg./Kg. 2 hr. 0/9 P-K 97mg./Kg. 8. COPPER 7.5mg./Kg. 2 hr. 0/6 PEN 32mg.Ag. •These r e s u l t s obtained i n previous experiments and presented here f o r comparison. 75 s i t e of i n j e c t i o n . A f t e r a short i n t e r v a l the writhing ceased and the animals appeared extremely l e t h a r g i c . Following i n j e c t i o n of P-K and PEN" there was no change i n t h i s appearance. In the previous experiments where T-Na was given 10 minutes a f t e r the copper, the animals started to die within 45 minutes and at the end of 1 hour and 45 minutes a l l seven were dead. Of the seven mice, 4 died amid various degrees of what appeared to be clonic-type convulsions. Although during the observed period only one of the mice i n the copper-treated group died, past observations over a longer period of time (72 hours) showed that t h i s dose of copper may be greater than the L D ^ Q (no. of deaths 5/7). The large standard deviations obtained i n the tissue analysis make i t impossible to make observations other than i n general terms. As noted on Table VIII, the error i n determining the amount of copper i n the brain i s considerably larger than the error encountered elsewhere. Surprisingly there seems to be a tendency toward lower amounts of copper i n the brain i n a l l the test groups. In the kidney the copper i n j e c t i o n causes a large increase i n the amount of copper and although the PEN was not able to increase t h i s further there i s an in d i c a t i o n that P-K causes a d d i t i o n a l accumulation of copper i n t h i s organ. Table VIII. The effect of P-K and PEN on the amount of copper in mouse organs after a single, acutely toxic, I.P. dose of copper. (Copper in y\ gms. /gm. of dry weight +_ S. D. ) TREATMENT TIME INTERVAL BRAIN* KIDNEY LIVER INTESTINAL BETWEEN INJECTION T R A C T OF COPPER AND No. of Copper No. of Copper No. of Copper No. of Copper INJECTION OF DRUG Samples Samples Samples Samples 1. CONTROL 14.56 +0. 72 22.23 + 1.92 20.64 +4. 37 14. 78 +2. 14 2. COPPER 7.5mg. /Kg. 7. 63 +2. 14 107. 89 +52. 85 40. 11 + 15. 12 55. 39 +22. 95 3. COPPER 7. 5mg. /Kg. P-K 97 mg. /Kg. 10 min. 10.56 +3. 36 163.28 +23. 35 50. 10 +20. 10 81. 14 +42.55 4. COPPER 7.5mg./Kg. PEN 32mg./kg. 10 min. 11. 13 +1.42 110.32 +62.00 61. 16 +33.48 52.24 +21. 32 5. COPPER 7.5mg. /Kg. P-K 97mg. /Kg. 2 hr. 8. 68 +0. 95 186.96 +26. 87 49.59 +4.62 55. 99 + 12. 13 6. COPPER 7. 5mg. /Kg. PEN 32 mg. /Kg. 2 hr. 10.07 +0.46 109. 60 +104. 50 41.41 + 15.67 70. 67 +40. 87 The small sample size of brain prevents keeping the readings on the colorimeter scale above 0. 100 and therefore by means of the previously presented accuracy test it is estimated that the error increases from 4. 2% to approximately 20%. 77 In the i n t e s t i n a l t r a c t and l i v e r the copper i n j e c t i o n also Increases the amount of copper to a marked degree. The P-K seems to cause a further s l i g h t increase of copper i n the l i v e r as does the PEN when i t i s given 10 minutes a f t e r the copper. On the other hand, the PEN causes no change i n the amount of copper i n the l i v e r when given 2 hours a f t e r the copper. In the case of the i n t e s t i n a l t r a c t there i s a further difference between the two drugs. The P-K seems to increase the copper i n the i n t e s t i n a l t r a c t when i t i s given 2 hours a f t e r the copper. Exactly the reverse s i t u a t i o n appears i n the case of PEN. Response to Tyramine of A t r i a from Rats Treated with  Chelating Agents Chronic Treatment with T-Na, P-K and PEN Method Tropolones and other chelating agents have been reported to influence the a c t i v i t y of enzymes concerned with the synthesis and metabolism of norepinephrine. (34-,35,84,85,111, 112,113) It was, therefore, thought l i k e l y that continued treatment with such compounds, as has been c a r r i e d out on the rats used i n these experiments, may bring about some a l t e r a t i o n of adrenergic function. This p o s s i b i l i t y was investigated pharmacologically by 78 measuring the response to tyramine of i s o l a t e d a t r i a from some of these r a t s . Intravenous injections of tyramine increase the amount of norepinephrine i n the venous outflow of several organs i n vivo and i n the e f f l u e n t of i s o l a t e d perfused organs, and infusion of large amounts of tyramine reduces the organ content of norepinephrine. The effects of tyramine are s t r i k i n g l y reduced i n organs where norepinephrine stores have been depleted either by removal of the adrenergic nerve supply or by treatment of the animal with reserpine. The e f f e c t s of tyramine are also reduced i n animals treated with cocaine, which prevents release of stored norepinephrine. The l o c a t i o n of the stored norepinephrine released by tyramine i s not yet f u l l y c l e a r , but the adrenal medulla i s not involved. The available evidence suggests that the main storage s i t e f o r norepinephrine i s within granules inside post-ganglionic adrenergic nerves. The mechanism involved i n the release of norepinephrine i s not yet known but there i s suggestion of the p o s s i b i l i t y of a competitive displacement. There i s also evidence to suggest that there may be more than one type of storage s i t e f o r norepinephrine. (114) The rats used i n t h i s experiment had been receiving the treatment as outlined i n Table IX f o r a 79 period of from 85 to 95 days. The animals were stunned by a blow to the base of the s k u l l and exsanguinated by cutting the jugular veins. The a t r i a were removed and suspended i n a bath of Kreb's solu t i o n containing double the amount of glucose at 35°C and gassed with 95% 0,, and 5% CO,,. The a t r i a were l e f t i n the bath f o r between 2 to 3 hours, washing at 40 minute i n t e r v a l s , before beginning the experiment. A f t e r that time the e f f e c t of a bath containing 1 V/ml. of tyramine on the rate of beating was measured on a Grass Model 5 Polygraph and FT .03B s t r a i n gauge. The e f f e c t to be measured was the maximum posi t i v e chronotropic e f f e c t of the tyramine. I t i s expected that i f the l e v e l of norepinephrine i n the storage s i t e s i s increased, an increased tyramine response may be seen and i f the stores of norepinephrine have been depleted, the response to tyramine w i l l be decreased. The maximum chronotropic e f f e c t was chosen as i t seemed to be more consistently reproducible i n control r a t a t r i a . Results The r e s u l t s of these experiments are shown i n Table IX. In a l l groups there was a normal basal rate except i n the one which was given copper water plus a d a i l y i n j e c t i o n of PEN. This group showed a f a i r l y marked increase i n the basal l e v e l . A l l the groups which 80 Table IX. The i n vivo e f f e c t of prolonged administration of copper, T-Na, P-K and PEN on the tyramine response of is o l a t e d r a t a t r i a . BASAL PEAK No. of beats/min. beats/min. PERCENT TREATMENT TESTS + S.D. + S.D. INCREASE 1. CONTROL 12 212 + 17 379 + 36 79 2. T-Na 2mg./day 5 208 + 12 304 + 30** 46 3 . P-K 5mg./day 3 216 + 19 337 + 26** 56 4 . PEN 1.6mg./day 7 217 + 15 334 + 36* 54 5. 100/>«gm./ml. of copper i n water 4 207 + 26 323 + 55* 56 6. 100 Mgm./ml. of copper i n water T-Na 2mg./day 5 197 + 12 338 + 15* 72 7. 100 Mgm./ml. of copper i n water P-K 5mg./day 3 204 + x 3 353 + 34 73 8. 100 ^gm./ml. of copper i n water PEN 1.6mg./day 4 251 + 387 + 19 54 • S i g n i f i c a n t l y d i f f e r e n t from control p ^ 0 . 0 5 • • S i g n i f i c a n t l y d i f f e r e n t from control p <^0.01 8 1 were given a drug alone and the group which was given copper water alone showed a decrease i n the tyramine response. The groups which were given copper water as well as the drugs showed a tendency for the tyramine response to return toward normal although i n no case was the tyramine response restored to the o r i g i n a l l e v e l . T-Na seemed to have the greatest e f f e c t on the tyramine response. Results were tested for si g n i f i c a n c e by the t-Test. (110) Acute Treatment with T-Na, P-K, PEN and D-Na Method Since chronic administration of the chelating agents appeared to a f f e c t responses to tyramine i t was decided to next investigate the influence of large doses of these comppunds given over a short period of time. In these experiments sodium diethyldithiocarbamate (D-Na) was also included since i t has been shown to i n h i b i t dopamine- ^ -hydroxylase both i n v i t r o and i n vivo. For t h i s purpose we used male rats of the Wistar s t r a i n ranging i n weight from 310 to 400 grams. Using the 500mg./Kg. dose of D-Na already proved e f f e c t i v e i n the l i t e r a t u r e equivalent doses of the other drugs were calculated. These were: T-Na 410mg./Kg., P-K lOOOmg./Kg. and PEN 330mg.Ag. The dosage was then divided into 82 5 equal parts and given S.C. every hour f o r 5 hours and at the end of the 5th hour the animals were s a c r i f i c e d and the tyramine response tested as before. The reason for dividing the dose i s that such a large dose of T-Na i s greater than the L D ^ Q and f o r the sake of consistency a l l the doses were divided. Animals were fasted overnight before each experiment. Results The r e s u l t s using these drugs i n a large divided dose are summarized i n Table X. Again the r e s u l t s were analyzed using the t-Test. (110) In t h i s experiment the shortage of supply of T-Na l i m i t e d the number of tests which could be done with t h i s drug. D-Na caused the rat s which received i t to have an extremely lethargic appearance as did the T-Na. The P-K and PEN did not have t h i s e f f e c t . D-Na also caused the animals to have a very loose s t o o l . T-Na and P-K caused l o c a l i r r i t a t i o n and development of an edematous wheal at the s i t e of i n j e c t i o n . P-K caused an elevation of the basal rate of contraction as did T-Na. In the case of T-Na the elevation was not quite s i g n i f i c a n t . I f t h i s same average f o r the basal rate with T-Na had been obtained with a few more tests i t would have been s i g n i f i c a n t . 83 Table X. The In vivo e f f e c t of D-Na, T-Na, P-K and PEN on the tyramine response of i s o l a t e d r a t a t r i a a f t e r large doses. BASAL PEAK No. of beats/min. beats/min. PERCENT TREATMENT TESTS ± S.D. ± S.D. INCREASE 1. CONTROL 12 212 + 1? 379 ± 36 79 2. D-Na 500mg./Kg. 7 200 ± 22 349 ± 39* 75 3 . T-Na 410mg./Kg. 3 231 + 16 383 + 26 66 4. P-K lOOOmg./Kg. 7 233 ± 14-** 393 + 32 69 5. PEN 330mg.Ag. 7 223 ± 19 397 ± 36 78 • S i g n i f i c a n t l y d i f f e r e n t from control p ^ 0 . 0 5 • • S i g n i f i c a n t l y d i f f e r e n t from control p ^ 0 . 0 1 84 The only drug which had a s i g n i f i c a n t e f f e c t on the peak rate was D-Na which caused a s l i g h t lowering. DISCUSSION Long Term Administration of Chelating Compounds The results of the excretion studies show that both PEN and P-K cause an increase i n the amount of copper excreted i n the urine while T-Na had no e f f e c t . The increase caused by PEN was by f a r the most marked. At the same time a l l three drugs caused an increase i n copper content i n kidneys from rat s on a normal d i e t . An explanation of t h i s may be that the Increase of copper i n the kidney caused by PEN and P-K represents copper i n the process of excretion, while i n the T-Na treated rats the copper may be deposited i n the c e l l s . Histochemical confirmation of t h i s could not be made. A si m i l a r s i t u a t i o n may ex i s t i n copper-fed r a t s . None of the drugs decreased or increased the amount of copper found i n the kidneys of copper-fed rats and only PEN markedly increased the excretion of copper. Excretion of copper was only s l i g h t l y increased by P-K. The difference i n the volumes of urine i n each group can be p a r t l y explained as follows. In the groups 8 5 which received copper the water intake was much less than with normal rats and the r e s u l t i n g hydropenia causes an increased production of ADH and subsequent concentration of urine. For t h i s reason the urinary concentration of copper was higher i n these groups while the actual amount being excreted was the same. This holds with a l l of the drugs except PEN which greatly increased urinary copper whether concentration or absolute amount i s considered. The fact that PEN gave a s l i g h t l y increased volume of urine both i n normal rats and copper-fed rats as did P-K i n copper-fed rats may be due to the fact that these agents as well as removing copper, remove other cations such as calcium and i r o n and i n order to preserve e l e c t r i c a l n e u t r a l i t y anions move into the tubular lumen along with the cations and these " s a l t s " exert a s l i g h t osmotic d i u r e t i c e f f e c t . It i s i n t e r e s t i n g to note that T-Na has no e f f e c t either on copper excretion or urinary volume. That P-K causes an increased copper excretion i n normal rats and at the same time a decreased urinary volume remains unexplained.. A somewhat s i m i l a r s i t u a t i o n exists i n r a t s on a normal di e t with regard to f e c a l excretion of copper and the concentration of copper i n the l i v e r . Both PEN and P-K cause an increase i n f e c a l excretion of copper. As has already been noted, i n man PEN has no e f f e c t on excretion of copper i n the feces and i t appears that i n 86 t h i s respect man and the rat d i f f e r . T-Na has no e f f e c t on f e c a l copper. The three sources of copper i n the feces are ingested copper which i s not absorbed, copper excreted i n the b i l e and that copper excreted d i r e c t l y through the i n t e s t i n a l w a l l . I t i s expected that ingested unabsorbed copper remains constant i n the normal rats which received only the parenterally administered drugs and t h i s leaves two sources as the main va r i a b l e s . In these experiments the two cannot be separated. The increased copper i n the l i v e r of rats treated with P-K and PEN may be due to increased b i l i a r y copper being excreted but t h i s could not be shown histochemically and, therefore, i t could just as well be due to increased deposition i n the l i v e r c e l l s as i s probably the case with the T-Na treated group as t h i s group showed no change i n the feces. I f the increase i n the l i v e r i s due to deposition i n the l i v e r c e l l s then the increase i n the feces must be due to an increased excretion of copper d i r e c t l y through the i n t e s t i n a l w a l l . P-K also increased the copper content of l i v e r i n copper-fed r a t s . In the hearts of r a t s on a normal di e t a l l three drugs had the net e f f e c t of lowering the copper content. However, i n the case of the copper-fed rats PEN was the only one which lowered the copper content of the heart, the other two having no e f f e c t . This i s probably due to the greater chelating a b i l i t y of PEN and i t s a b i l i t y to 87 increase removal of copper by way of the urine. P-K showed a s l i g h t tendency to lower copper content of heart of copper-fed rats but t h i s i s not s t a t i s t i c a l l y s i g n i f i c a n t . In the brains of both normal and copper-fed rats PEN showed a tendency to lower copper l e v e l s . P-K showed a s i m i l a r tendency but th i s was not as marked as with PEN. In rats on a normal d i e t , T-Na at f i r s t increased copper l e v e l s i n the brain but l a t e r decreased the l e v e l of copper and i n copper-fed rats i t caused an increase at both determinations. I t i s l i k e l y that t h i s increase may be due to the greater l i p i d s o l u b i l i t y of the T-Na-copper chelate, (67,68) thus f a c i l i t a t i n g entrance of copper into the brain. Further evidence to support the supposition of entry into the CNS i s given by the f a c t that the main pharmacological e f f e c t s of the tropolones are i n the CNS. (77,78) The f a i l u r e of the histochemical s t a i n i n g procedure to reveal the presence of copper i n the organs examined i s probably due to the very small amounts of copper present. Howell (5D stated that i n order to obtain a p o s i t i v e t e s t , copper concentrations of around 100 |Agm./gm. of wet tissues are needed. This l e v e l of eopper concentration was not even approached i n t h i s experiment. An added source of d i f f i c u l t y may be that the diethyldithiocarbamate was not able to displace the copper from the other chelating agents. In the two s l i d e s 88 which did show a p o s i t i v e diethyldithiocarbamate s t a i n i t cannot be p o s i t i v e l y stated that i t was due to copper. In looking through l i v e r cross-sections i t was noted that the red c e l l s a l so give a yellow-brown s t a i n with t h i s reagent. This i s probably due to a reaction with the iron of hemaglobin as i t i s known that diethyldithiocarba-mate forms a yellow-brown complex with t h i s metal. (107) As the stains showed only i n the blood vessels i t cannot be said i f i t i s due to the red c e l l s or to copper. I t i s noteworthy, however, that the two s l i d e s which were pos i t i v e f o r copper were from groups which had about the highest l e v e l s of copper obtained i n t h i s experiment. Single Dose of Chelating Compound Neither PEN nor P-K appeared to influence the effects of the single dose of copper within the period of observation whether given at 10 minutes or 2 hours a f t e r the copper. This i s i n marked contrast with the effects observed e a r l i e r when T-Na, given 10 minutes a f t e r the copper, caused the deaths of 7 mice out of a group of 7> even though the dose of T-Na was f a r below a l e t h a l dose. The explanation of t h i s i s thought to be increased penetration of the copper into the c e n t r a l -nervous-system. Evidence to support t h i s explanation i s circumstantial because, f o r reasons already given, the 89 amount of copper i n the brain i n t h i s case was not a c t u a l l y determined. F i r s t l y , there i s the extreme r a p i d i t y with which the animals died. The swiftness of death can only be accounted f o r by the cessation of function i n some v i t a l area, such as, r e s p i r a t i o n , the heart or the c e n t r a l -nervous-system. Secondly, i t i s known that the copper chelates of tropolones are much more l i p i d soluble than water soluble (67,68) and t h i s would be expected to f a c i l i t a t e penetration of the blood-brain b a r r i e r . Con-versely the copper chelates of P-K and PEN are water-soluble (91,92) and do not exhibit t h i s increased t o x i c i t y . And f i n a l l y , there i s the appearance of convulsions i n these animals which may be similar to the convulsions and death which occur i n pigeons given a subarachnoid i n j e c t i o n of copper. (36) The e f f e c t , i f any, of P-K and PEN on the amount of copper i n the brain i s not evident. The d i f f i c u l t y i n the determination of copper i n the brains of mice i s t h e i r small s i z e . Even using a pool of three brains, there was not s u f f i c i e n t copper present to give a reading on the colorimeter above 0.100. Past experience has shown that because of the mechanical l i m i t a t i o n s of the instrument readings on the scale i n the area of 0.050 to 0.080 may give errors as high as 20^. This, coupled with a standard deviation of the order of 10$, makes i t impossible to claim a difference between any of the i n d i v i d u a l groups. 90 The e f f e c t of these two drugs on the kidney i s a b i t puzzling. The increase of copper i n t h i s organ should be an index of the r e l a t i v e amount of copper being excreted by the urinary route. As i s expected, the copper i n j e c t i o n causes a very marked increase i n the amount of copper i n the kidney. Also as expected, P-K causes an even further increase i n the amount of copper i n the kidney which may indicate an increase i n urinary excretion of copper. On the other hand, although the increased urinary excretion of copper i n man caused by PEN has been well-documented (59>60,61,62,63,64) and PEN given to rats over a long period increased the urinary copper l e v e l and the l e v e l of copper i n the kidney the figures i n t h i s experiment show that the amount of copper i n the kidney of mice given 7.5mg./Kg. of copper i s not further increased by PEN. There are two possible explanations f o r t h i s . F i r s t , that PEN does not have the same mechanism i n man and the r a t as i t does i n the mouse or second, that PEN i s so r a p i d l y cleared by the kidneys that at the end of 4 hours when the samples were taken the peak a c t i v i t y has already past. In view of the w e l l -known rapid clearance of p e n i c i l l i n i t s e l f i t i s f e l t that the l a t t e r explanation i s more probably correct. As previously noted, besides the urine there are two other routes of excretion of copper. One i s the b i l e and the other i s d i r e c t l y through the i n t e s t i n a l w a l l . 91 Analysis of the l i v e r may give some i n d i c a t i o n of the b i l i a r y copper while analysis of the i n t e s t i n a l t r a c t and contents gives an i n d i c a t i o n of the f e c a l copper which comes from ingested copper, b i l i a r y copper and copper excreted d i r e c t l y through the i n t e s t i n a l w a l l . Under the conditions of t h i s experiment the l a t t e r two sources are the main variables i n the r e l a t i v e contribution to f e c a l copper. P-K causes a s l i g h t increase i n the amount of copper i n the l i v e r which may indicate increased b i l i a r y excretion. This increased b i l i a r y excretion i s r e f l e c t e d i n an increase i n the amount of copper i n the i n t e s t i n a l t r a c t when the P-K i s given 10 minutes a f t e r the copper. When the P-K i s given 2 hours a f t e r the copper the amount of copper i n the i n t e s t i n a l t r a c t i s not increased but th i s could possibly be due to a time f a c t o r such that the 2 hours (the time which has elapsed from the administration of the drug to the time of s a c r i f i c i n g ) i s not s u f f i c i e n t f o r the b i l e to have reached the i n t e s t i n a l t r a c t . The res u l t s with PEN are contradictory i n that when the PEN i s given 10 minutes a f t e r the copper the amount of copper i n the l i v e r increases while there i s no change i n i n t e s t i n a l copper and when the PEN i s given 2 hours a f t e r the copper the l i v e r copper i s unchanged while the i n t e s t i n a l copper i s increased. I t may be that when PEN i s given 10 minutes a f t e r the copper i t holds copper i n the l i v e r u n t i l i t can be removed by urinary excretion. 92 On the other hand when i t i s given two hours a f t e r the copper some copper has already passed through the b i l i a r y route into the i n t e s t i n e . In man i t has been reported that PEN has no e f f e c t on the f e c a l excretion of copper. (63) E f f e c t on the Response of Isolated A t r i a to Tyramine There was a reduction of the tyramine response of a t r i a from rats from a l l of the groups which had been on long-term treatment with the chelating agents. This may indicate that the drugs lowered the l e v e l of norepinephrine i n the heart and by the argument given e a r l i e r t h i s could be due to an In vivo i n h i b i t i o n of dopamine-^-hydroxylase. COMT may also be i n h i b i t e d but this i s not s u f f i c i e n t to mask the e f f e c t r e s u l t i n g from dopamine-^-hydroxylase i n h i b i t i o n . Excess copper also seemed to have a similar e f f e c t . It i s noteworthy that a l l these drugs also lowered the copper content of the heart. (Table IV) The addition of copper to the drinking water of rats treated with these chelating agents prevented the reduction i n the tyramine response somewhat which seems to support the theory that the i n h i b i t i o n of dopamine- ^ -hydroxylase Is due to the removal of copper by chelation with the compounds administered. T-Na would appear to be the most potent i n h i b i t o r as even with 93 the a d d i t i o n a l copper the tyramine response was s t i l l s i g n i f i c a n t l y lower than the control and also T-Na alone gave the greatest lowering of the tyramine response. The fact that PEN raised the basal contraction rate when the rats were fed copper could be due to the fa c t that i n t h i s case COMT i s being i n h i b i t e d as the peak response was also s l i g h t l y higher although t h i s i s not s i g n i f i c a n t . In the r a t s which were given a large divided dose of the drugs the r e s u l t s were not so s t r i k i n g . In only one case, with D-Na, was the tyramine response reduced and even then th i s reduction was only s l i g h t . On the other hand, a l l the drugs except D-Na seemed to raise the basal rate although only i n the case of P-K was t h i s s i g n i f i c a n t . The explanation f o r t h i s may be that the high concentration of drugs present are i n h i b i t i n g both COMT and dopamine-@-hydroxylase but that i n order f o r the i n h i b i t i o n of dopamine-^-hydroxylase to be evident a longer i n t e r v a l of time must pass i n order f o r the stores of norepinephrine which are already present to be depleted. In t h i s case the COMT i n h i b i t i o n shows up as an increased basal rate due to reduced destruction of endogenously released norepinephrine. Another factor may be the size of the dose as i n the two experiments the dosages as well as the length of experiments were d i f f e r e n t . 94-The appearance of l e t h a r g y f o l l o w i n g the a d m i n i s t r a t i o n of T-Na and D-Na may i n f a c t be a heavy sed a t i o n . C a r l s s o n et a l . (112) d i d i n f a c t c a l l t h i s reduced a c t i v i t y " s e dation." The f a c t t h a t t h i s was only observed i n r a t s t r e a t e d w i t h T-Na and D-Na could be explained once again on s o l u b i l i t y f a c t o r s . The l i p i d s o l u b i l i t y of these two drugs and t h e i r c h e l a t e s a l l o w s them to penetrate the CNS where t h e i r c e n t r a l a c t i o n may be due t o i n t e r f e r e n c e i n some aspect of catecholamine metabolism. I t has been reported that r a t s which have been pr e t r e a t e d w i t h tropolones show an increased l e v e l of some ^ C - l a b e l e d catecholamines. D-Na i s probably excreted through the feces as i t i s known to cause an increased;copper e x c r e t i o n i n the feces of man. (44) The loose s t o o l caused by D-Na may then be due to l o c a l i r r i t a t i o n i n the i n t e s t i n a l t r a c t as i t i s being excreted. The i r r i t a n t nature of the ' i n j e c t i o n s of T-Na and P-K are understandable i n view of t h e i r f a i r l y b a s i c and a c i d i c p r o p e r t i e s r e s p e c t i v e l y . SUMMARY AND CONCLUSIONS Two c h e l a t o r s of copper found i n the heartwood of western red cedar (Thu.ia p l i c a t a ? Donn) are 5-isopropyltropolone and p l i c a t i c a c i d . A study of t h e i r e f f e c t s on some aspects of copper metabolism i n r a t s and 95 mice has been undertaken. As a basis of comparison si m i l a r experiments were carr i e d out using penicillamine, a chelating agent with wide c l i n i c a l use. 1. When administered d a i l y , P-K and PEN caused an increase i n urinary and f e c a l excretion of copper i n rats on a normal diet and an increase i n the l e v e l of copper i n the l i v e r and kidney and a decreased l e v e l of copper i n the heart and brain. The increased urinary excretion i s much more marked with PEN than with P-K. T-Na administered d a i l y had no e f f e c t on the excretion of copper i n rats on a normal di e t and causes a r i s e i n copper content of l i v e r and kidney and a lowering of copper content of the heart and brain. I t appears that the copper l e v e l elevation i n kidney and l i v e r caused by P-K and PEN i s due to an increased u t i l i z a t i o n of the routes of excretion while the increased l e v e l due to T-Na i s probably due to deposition i n the c e l l s . 2 . When these compounds are administered d a i l y to copper-fed rats s i m i l a r observations were made with the difference that i n organs where copper l e v e l s are Increased the increases are greater and i n organs where copper l e v e l s are decreased the decreases are smaller. With T-Na there i s one marked difference i n that the copper l e v e l i n the brain i s increased. I t i s f e l t that t h i s increase i s due to a greater penetrating a b i l i t y of the T-Na-copper chelate because of i t s l i p i d s o l u b i l i t y . 96 3 . P-K and PEN have very l i t t l e e f f e c t on the t o x i c i t y of a s i n g l e l a r g e dose of copper. E a r l i e r experiments w i t h T-Na showed that when T-Na i s given s h o r t l y a f t e r a s i n g l e l a r g e dose of copper the t o x i c i t y i s g r e a t l y i n c r e a s e d . The e x p l a n a t i o n of t h i s i s very l i k e l y t h a t T-Na increases the p e n e t r a t i o n of the copper i n t o the CNS and i t i s here t h a t the t o x i c e f f e c t i s exerted. I t i s f e l t t h a t t h i s i s due t o a s o l u b i l i t y f a c t o r , T-Na and i t s chelate being l i p i d - s o l u b l e and P-K, PEN and t h e i r chelates being w a t e r - s o l u b l e . 4. When T-Na, P-K and PEN are given t o r a t s i n s m a l l d a i l y doses over a long p e r i o d , the a t r i a from such r a t s e x h i b i t e d a reduced chronotropic response to tyramine. I f a t the same time as the r a t s are being given the c h e l a t i n g agent, they are given excess copper i n d r i n k i n g water the tyramine response remains c l o s e t o normal. In t h i s regard T-Na possesses the most a c t i v i t y . These observations support the theory that c h e l a t i n g agents i n h i b i t dopamine- ^ - h y d r o x y l a s e by rendering copper i n a c c e s s i b l e t o the enzyme and t h a t subsequent t o t h i s i n h i b i t i o n the b i o s y n t h e s i s of the catecholamines i s i n h i b i t e d at the dopamine stage preventing the formation of norepinephrine and epinephrine. As the l e v e l s of endogenous norepinephrine and epinephrine f a l l , the r e s u l t i s a reduced tyramine response. 97 5. If T-Na, P-K, PEN and D-Na are given in large single doses the inhibition of dopamine-@-hydroxylase is not evident while i t appears that COMT may be inhibited. The a b i l i t y of 5-isopropyltropolone and pli c a t i c acid to mobilize copper and increase i t s excretion is not as marked as that of penicillamine. The experiments carried out would not appear to form a satisfactory method for testing chelating agents for use in Wilson's Disease. However, they appear to have conclusively shown that PEN, the present drug of choice in Wilson's Disease, is remarkable in i t s a b i l i t y to remove copper by urinary excretion when compared with P-K and T-Na. 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