Copper Precipitation in the Human Body Wilson's Disease R. P. Cslntalan and N. M. Senozan Callfornla State Universlty. Long Beach, CA 90840 In 1902 Bernard Kayser, a German ophthalmologist, reported greenish yellow opaque rings in the cornea of a patient who was thought t o suffer from multiple sclerosis. One year later Bruno Fleischer, a physician in Munich, noted similar circular lesions in the eyes of another neurological patient. The chemical nature of the pigmentation, however, remained a mystery until the mid-'30's when the startling composition of the corneal rings was revealed: They were made of fine granules of a copper precipitate (1-4). Kayser-Fleischer rings, as the yellow-green circles of the cornea eventually were named, are one of several symptoms of a disorder in copper metabolism known as Wilson's disease. In 1912 Samuel Wilson, an American neurologist working in Great Britain, published a 211-page monograph that gave a nearly complete description of a degenerative ailment characterized mainlv bv the destruction of the liver and nerve tissue, including the so-ralled lentirular regions of the brain (5). Wilson named his disease "nrowressive lenticular degeneration". He did not realize its rklacon to copper metabolism, but suspected a "morbid agent.. . probably.. . a toxin"to be responsible for the neural and hepatic degeneration. A year later the toxin was identified as copper when excessive amounts of this metal were found in the liver, although apparently no note of this fact was taken in the literature again until 1929 when large quantities of copper were discovered in the brain as well as in the liver of patients with Wilson's disease. Wilson's treatise does not mention Kayser-Fleischer rings; that these pigmented lesions are a frequent symptom of the progressive lenticular degeneration was realized gradually. The rings, we now know, are present in all Wilson's patients who display neurological disorders; they are also observed in most cases when the aymptoms are primarily due to liver malfunctioning (1,4,6). a
Dichloro(2-chlorov1nyl)arsine
H
I
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See Figure 1 fw the svuctures of the compounds mentioned in the article.
H
l
H
l
H-C-C-C-H I l l
SH SH OH
British anti-lewlslte or
2.3-dimercaptopropanol
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Penlclllamlne and the Treatment ot Wllson's Disease Prior to the '50's Wilson's disease was uniformly fatal. The progress of the illness varied among the patients; sometimes liver damage and its manifestations, such as jaundice, fatigue, abdominal pain, preceded neurological disorders, while, just as often, tremors, defective speech, and clumsiness heralded the onset of the disease. What changed the hopeless outlook for Wilson's patients was the discovery in 1956 that penicillamine could arrest the accumulation of comer, reverse the course of the disease, and restore normal ~ i fkxpectancy i (1,4, A glimmer of hope in the treatment of Wilson's disease first appeared in 1948 when a chemical developed by the British military laboratories against lewisite, CzH2AsCI3, a highly poisonous arsenic compound, was shown to promote excretion of copperl. T o be effective, however, BAL, as the British anti-lewisite agent 2,3-dimercaptopropanol is commonly known, had to be administered daily as intramuscular injections. I t was impractical, painful, and toxic, and the patients often refused treatment (1,4,8).
'
CTCHZCH-AsCI2 Lewlslte or
Penictllamine or
p,p-dimethylcystetne Figure 1. Structures of lewisite, British anti-lewisiteagent IBAL), and penlcliia-
mine.
Meanwhile, as ex~erimentationswith BAL continued, a British physician naked J. M. Walshe noted the presenceof penicillamine in the urine of the patients on penicillin. As a metabolic end product of a common antibibtic, penicillamine seemed to pass through the body painlessly and without harm. Like BAL, it contained a sulfhydryl group (Fig. I), and Walshe was struck by the idea it might be used as an oral sequestering agent for copper without the impracticality and painful side effects of 2.3-dimercaptopropanol. T o prove its safety, he swallowed several grams of the crystalline compound, then gave some to a Wilson's patient. The copper level in the patient's urine rose markedly. Penicillamine was, apparently, binding copper and facilitating its removal. Further tests with a young and very ill woman left no doubt about the drug's remarkahle effectiveness. Within several weeks the woman's neurological symptoms disappeared, Kayser-Fleischer rings faded, and the liver function returned to normal (I, 9,lO). Penicillamine today remains the drug of choice for Wilson's disease. If the diagnosis is made before the damage to the liver and nerves is too advanced and the treatment is promptly started, most patients recover. Even mental disorders brought on indirectlv . bv. the imnairment of the brain tissue disappear with penicillamine. For example, one devastating ~svchiatricasnect of Wilson's disease in voune neop~e--id&t patients-the symptoms first appear between
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Volume 68 Number 5 May 1991
365
the ages of 10 and 30-is the loss of "sexual attractiveness" as a result of the disturbances in the neurological system. ill-kempt ap"Open-mouthed, drooling, masklike face pearance and dirtiness of the entire body" stemming from the ~-~~lack of muscular coordination put an enormous burden on the ego and psyche of a patient whose sexual urges and intelligence remain intact. Penicillamine, restoring neural harmony and muscular agility, helps the victim regain his or her attractiveness. self-esteem, and mental health i l l ) . Normal therap&tic dosage of penicillamine is about 1g a dav taken on an emptv stomach to enhance absorption. ~ l k o u the ~ hchelatin; capability of the D- and L-isomers of penicillamine are expected to be the same, the drug is supplied in the D- form. In children and young adults the Lisomer interferes with the action of pyridoxine, a Bs vitamin (12). of medicine recommend that the drug ~ - -Textbooks , ~ should be augmented by avoiding copper-rich foods such as liver. chocolate. nuts. mushrooms. molasses. peas, broccoli. some authorities also sugge;t taking small and ;hell fish (1i). amounts of notassium sulfide orallv LO block the absorption of copper, gut they do not mention how the patientLcope with HIS-laden gases likelv to evolve from the hydrolysis of the sulfide in the gastric environment (14).
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Ceruloplasmln and the Copper Metabolism In normal adults the average intake of copper is 2 to 5 mg per day. Of this amount, about one-third is absorbed. For normal balance to be maintained, losses of about 0.6 to 1.6 mg per day must occur in addition to fecal loss of unabsorbed copper. Biliary excretion accounts for 80% of the total copoer loss or 0.5 to 1.3 me-. per dav. The remaining 0.1 to 0.3 mg is expelled through urine a& sweat. The distribution of comer through " variousareas of the bodv is shown in Fiaure . 2 (1i;lfi). The absorbed copper enters eventually into one of the copper-containing compounds of the human body. More than half a dozen copper proteins in mammals have been characterized; their properties and function are given in the table. Since none of these proteins except ceruloplasmin displays unusual levels in the victims of Wilson's disease, the attention has been focused on this proteinas a prime suspect in the precipitation of copper in the body (I, 8). Ceruloplasmin, a deep blue compound first detected in swine blood where its concentration is high enough to impart a hluish tinge to the color of the plasma, has a molecular weieht of 132.000 and contains 6 to 89 carbohydrates. I t is cogposed of a single polypeptide chain with-1065 amino acids whose sequence is about half resolved. The copper content of ceruloplasmin is 0.3% which corresponds to six copper atoms per molecule (1,8,17-20). Ceruloplasmin exhibits oxidase activity toward a variety of compounds including aromatic amines, phenols, cysteine, ascorbic acid, and the ferrous ion. I t is not known if this general oxidizing capability is physiologically significant; what appears to be certain is the involvement of ceruloplasmin in the mobilization of iron-in its relocation from the storage s~ cells where it exists as ferritin and hemosiderin to transferrin, the protein that carries the iron in the plasma. For over 50 vears it has been known that lack of comer in the diet leads to anemia. Direct evidence for the role i f cerulonlasmin. however. came to light when its administration to iats lead to rapid discharge oliron from the liver. The metal was also released from the isolated preparations of this organ when they were perfused with a ioluiion of ceruloplas&n (17-20). In ferritin, hemosiderin, and transferrin the iron is in the ferric state. I t is believed that, to get across the membrane of the storage cell, the metal must be reduced to the ferrous state. Ferroxidase ability of ceruloplasmin comes into picture in the reoxidation of iron to the ferric state a t the surface of the storage cell so that it can be incorporated into transferrin (21-23). ~~
366
Journal of Chemical Education
Cerulo~lasminconcentration in normal adults range from 200 to 460 m g L of blood. Over 90% of the copper found in the plasma is bound to ceruloplasmin, the remaining 60-120 rg/L is attached mostly to albumin and some to histidine or glutamine. That ceruloplasmin carries most of the copper in the plasma makes thisprotein a most likely suhstance for transporting this metal between the liver and the target molecules. The conner is bound to cerulo~lasminvery tightly and is not exchanged easily, but, in thepresence ofproper enzymes it can readily be delivered to the target cells to be incorporated into molecules like superoxide dismutase and cytochrome oxidase. A number of experiments with labeled copper in fact strongly suggest tbat ceruloplasmin is the rotei in tbat transfers copper .. from the liver to all other cells (17,ZO). In 95% of the Wilson's patients the serum ceruloplasmin levels are well below the normal values quoted above. In some cases the copper protein is virtually absent. Lack or deficiencv of cerulonlasmin in Wilson's disease immediately suggests a mechanism for the development of hepatolenticular disorder. Normally about 0.5 mg of copper a day is used in p
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Figure 2. Disbibution and route of wpper lhrovgh various areas of the body. me numbers in boxes indicate milligramsof copper in each area Or organ. m e numbers on arrows refer to milligrams of copper that pass through lhe indicated route each day. Cu-Alb represents the copper bound to albumin. Ceru refers to copper in ceruloplasmin.
Cower Protelna In the Human Bodv* PTOteln Ceruloplasmin TyrosinasaL Superoxide dismutase Cytochrome c oxidasa Amine oxidase'
Molacular Weightb
Locationd
132
6
Plasma
87 33
2 2
200-280
2
Skln Red cells. brain. liver All cells
170
2
Plasma
120-180 290 @nydroxylase
Copper Contentr
4
Cartilage Plasma
Functlon COpper transport, oxldation of Fez+ Melanin formation Dispmportionation of superoxide ion Reduction of D2 Oxidation of biogwnic amines Cross linking of collagen Synthesis of noradrenaline
BCornpiled from references 6.8, 17. and 32. In unlts of 1000 daltons. o N ~ m b eof r copper elm6 per molecule. 'Only major locations are indicated. e ~ o l e c u l aw r e l m and copper content vary wnh sour-. me valves given are for human ryroJinare. 'Thsre are s v e r s l varieties. T ~ valve3 E given are for bovine plasma mine oxldaae.
the sjnthesis and catabolism of ceruloplasmin; in Wilson's patients with little or no ceruloplasmin, the liver fails to rid itself of this amount of copper on a daily basis. F:ventually the accumulated copper exceeds the storage capability of the liver. soills into bluodstream. and orecioitates in suitable environments including the biain, tde kidneys, and the eyes. The . orecioitate is ~ r o b a b l va comolex of comer . . . with a sulfur-richcornpound.The metalcontent of thedepositscan be auite bieh. and in dried samoles of Kavser-Fleischer rines (he percentage of copper reaches 1%(I,>, 6). Lack of cerulodasmin as the orinci~alcause of comer accumulation falis shurt of explaining one important aspect of \Vilson's disease: about 570 of the patients have normal levels of the blue protein. A more plaisible explanation for the collection of copper in the liver and its subsequent diffusion to other tissues might be a faulty biliari excretion mechanism. Since 80% of the absorbed copper is expelled through the bile, even a slight impairment of this route can lead to a serious imbalance in the body's copper economy (8, 24). Genetlcs of Wllson's Disease Wilson's disease is an inherited disorder. The defective gene is recessive and occurs with a frequency of about one in 90 people with no particular preference for any racial or national group. The probability of two persons with defective genes to unite is (1/90)2. Since the disease manifests itself in homozygous offspring, the chances that a couple, both carriers of Wilson's gene, will have an affected child is 114. This brings the incidence of Wilson's disease to about 30 per million. What the defective gene does is far from being clear. It may lead to insufficient or faulty synthesis of cernloplasmin or another protein essential for the proper excretion of the copper through the bile. Metallothionein, a small protein with a great affinity for heavy metal ions, is believed to be An altered involved in the storage of copper in the liver (25). form of this or a similar protein may interfere with the normal excretion routes and lead to pathological accumulation. Perhaps more than a single gene is involved in the development of Wilson's disease with one, for example, being responsible for making a protein for the regulation of copper excretion and the other an enzyme for its mobilization and deliverv to the tareet oreans. The wide soectrum of symptoms seen in Wilson's disease may reflect variations in the exoression of the defective aenes (I.6.26). ~ h ~ d e f e c t i vgene e (or is locited on chromosome 13, one of the 22 pairs of autosomal, i.e., nonsex, chromosomes (27). The gene must be involved in the regulation of other h e a w metals too, because comer is not the only metal accumulated by ~ i l s o n ' patients. s w h e n ~ u r n ~first e l noted the presence of excessive copper in the liver in 1913,he also pointed out the presence ofsilver in this organ as well as in the Kayser-Fleischer rings. In fact he thought that the rings were silver deposits and Wilson's disease was due to silver poisoning. Apparently, the same factors responsible for the mismanagement of copper spoils the body's way of handling silver too and leads to its precipitation in the liver and the eyes. Wilson's disease in some ways resemble copper poisoning. Chronic copper poisoning is rare, but acute cases from accidental or st&idalingestion of copper salts or usingsoftwater that has been allowed to circulate too long in copper pipes have been reoorted (16. . . 28). A maior . svmntom . . of acute copper poiso&ng is hemolytic anemia-a rapid breakdown of red cells as a result of the cupric ions' penetration into them (21). Hemoglobin, in which the iron is normally in the +2 state, is readilv oxidized to the ferric state bv the c u ~ r i c ions. Ferric hemoglobin, also known as methemoilobin, ;an-
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not bind oxygen and tends to denature. Denatured protein precipitates, distorts the cell membrane, and leads to premature destruction of the red cells (29-31). Episodes of hemolytic anemia are common among the victims of Wilson's disease too. The overflow of copper from the liver into the bloodstream where it is loosely attached to albumin brines on bouts of anemia much like that in case of copper poisoning. Neurological and heparic symptoms associated with chronic . orecioitation of coooer . .. in Wilson's disease are, of course, absent in acute poisoning cases.
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Conclusion With onlv 150.000 cases estimated worldwide. Wilson's disease is not & urgent public health Yet the disease Drovides a fine examnle of the link between metals and medicine and draws scientists with varied backgrounds, including inorganic chemists, geneticists, and physicians, to the study of copper precipitation in the human body. I t has been remarked that the number of papers on Wilson's disease probably exceeds the number of known patients (4). Still, the underlying metabolic defect leading tometal accumulation remains elusive. Failure to discover the biochemical basis of the disease has not hamnered. however. the develooment of an effective drug. At time'when chemistry is heldin some quarters to be responsible for degrading the quality of life, i t is worthwhile to reflect on Wilson's disease. Walshe discovered penicillamine by expanding upon his knowledge of copper-sulfhydry1 complexes. His discovery is a triumph of chemistry-a victory of science over death and misery.
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Literature Cited 1. Scheinberg, 1. H.: Stornlieb. I. Wiison's Disrore: Saundem: Philadelphia, 19U. 2. Danks. D. M. In The Metobolir Basi6 of inherltsd Direoae, 6th ed.;Seriver. C. R.; Besudet,A.L.;Sly, W. S.:Valle, D., Ed% McGnw-Hill: NevYork, 1989:Val. 1 , p p 1411.1411 .... .....
3. Seymour, C. A. In Copper in Anirnols ond Man: Howell, 3. McC: Gawthorne. J. M., Eds.: CRC: Boca Raton. FL, 1987; Vol. 2, pp 79-106. 4. Owen, C.A. Wi1son'sDiseosr;Noye: Park Ridge, NJ, 1981. 5. Wi1son.S. A. K.Broin 1912.34.295-507. 6. Sarale. D. C.; Chan. W-Y.; Rennert, 0. M. In Metiiboliam a/ Trace Metals in Mon; Rennert, 0. M.: Chan, W-Y., Eds.; CRC: Boca Raton, FL. 1984: Vol.2. Chapter 4. 7. Walahe, J. M. Q.J. Med. 1989. 70.253-263. 8. Scheinberg, I. H. In Inorganic ChamistiyinBiologyand Medicine:Martoll, A. E.. Ed.: American Chemical Society: Wsahington. DC. 1980: Chaptor 21. 9. Wailhe. J. M. Lancer 1956.1.25-26. 10. Wslrhe. J. M. Am. J . M d 1956.21.487-495.
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13. Ref& o 141. 14. Tourian. A. In Cecil Textbook of Medieins, 16Lhed.: Wyngaarden, J.B.:Smith,L. H.. Jr., Eda.: Ssunders: Philedelphia, 1982; pp 1126-1128. 15. Wintrobe, M. M.; Lee, G. R : Bopps. D. R.: Bithell, T.C.; Athen., J. W.; Foemter, 3. Clinical H~rnolology.7th ed.: Lea and Fobiger: Philadelphia. 1971; ~p 150-152. 16. Williams, D. M. In Cvimnt Topics in Nulrilian and D i m s a ; Prasad. A. S., Ed.: Lisa: --v7?, ,-o. -. New York, 1982:Vol. ",vv IT. Owen. C A. BiocAemiea: I A s p r l s of Copper: Noyes: ParkRidge. NJ. 1982. 18. Friod;n. E. Sci. A;. 191i8.218. 102-114. ir1 fhsEnuironmmi:. Nrissu. 19. Frieden. E. In Conoar . . JO.. . Ed.:. Wilev: . New Yoik.. 1979:. Part 2, pp 241-284. 20. F~ieden.E. In M m l Ions in Bioloeieal Sy8frm: Sigel. H., Ed.: Dekker: New York. 1981:Vol. 13, pp 117-142. 21. Wintrobe. M. M.; Lee, G. R.; Boggs, D. R.: Bitheil. T. C.: Foemter, J.: Athens, J. W.; Lukena. J. N.Clhical Hzmotolagy. 8th d . : Les and Febiger, Philadelphia, 1981: p ~
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