Jounzal of ffeparolog)~, 1991: 15: 94-101 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 0168”e278/92/$05.00

94 HEPAT 01081

Copper metabolism in hypercupre Studies of its subcellular distribution, association with binding proteins and expression of mRNAs Colin D. Bingle’, ‘Department of Protein and Molecular Biology and

Surjit K.S. Srai’ and Owen Epstein’ ‘Academtc Department of Medicine, The Royal Free Hospital School of Medicine, London, United Kingdom

(Received 25 March 1991)

study we have used differential centrifudation, size exclusion chromatography, Western and Northern blotting to investigate the subcellular distribution of hepatic copper, the association of the metal with hepatic copper binding proteins and the expression of specific mRNAs for copper binding proteins in liver tissue from two patients with Wilson’s disease, two patients with chronic liver disease and two patients with normal hepatic copper levels. Unlike previous studies the present results fail to show any gross differences in subcellular distribution of copper between the livers, with most of the copper being found in the soluble supernatant where it is associated with metallothionein. Caeruloplasmin mRNA levels were reduced in the two patients with Wilson’s disease and also in a patient with fulminant hepatic failure. It remains to be confirmed if the reduction of caeruloplasmin mRNA is specific for Wilson’s disease. Levels of mRNAs for copper zinc superoxide dismutase and metallothionein were variable and not related to liver copper. In the present

The liver plays a central role in the metabolism of copper (1). Newly absorbed copper is preferentially accumulated by this organ and, depending on the prevailing circumstances, may be stored, secreted into the plasma or excreted in the bile (1,2). Mammalian copper metabolism is regulated at the level of biliary excretion, and therefore, any condition which preturbs the normal excretory mechanism results in hepatic accumulation of the metal (3). Secondary hepatic copper accumulation occurs in a many other forms of liver disease, for example, primary sclerosing cholangitis and primary biliary cirrhosis, as a direct result of the chronic cholestasis (3). Hepatic copper accumulation also occurs in the inborn error of copper metabolism, Wilson’s disease (4). The basic defect in Wilson’s disease remains unknown, but the accumulation of liver copper results from failure of normal biliary excretion (5) and impaired secretion of caeruloplasmin into the serum (6,7). In addition, during the latter stages of pregnancy neonatal mammals accuCorrespondence: Dr. Surjit K.S. Srai. Department

Street. London, NW3 2QG. U.K.

mulate large stores of hepatic copper (8), probably as a result of abnormal biliary copper and caeruloplasmin metabolism (9,lO). It has been suggested that changes in hepatic copper concentration are associated with changes in the subcellular distribution of copper and the expression of bepatic and serum copper binding proteins. In normal liver copper is primarily found in the soluble supernatant (1,2) where it is associated with copper-zinc superoxide dismutase (CuZn-SOD) (11). Previous studies using histochemistry, liver biopsies and post-mortem tissue in pathological copper oveiload and the neonate, have indicated that increasing amounts of the metal become associated with the subcellular particles, particularly the lysosomes and nuclei, as the hepatic copper levels rise (12,13). The hepatic accumulation of copper found in Wilson’s disease is considered to be hepatotoxic. However, in other situations where liver copper is elevated, such as in cholestasis, direct hepatotoxicity due to the metal has not been confirmed (14). It has been

of Protein and Molecular Biology, Royal Free Hospital School of Medicine, Rowland Hill

COPPER

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suggested that such differences may be due to alterations in the s.ubcellular localisation of copper and its association with hepatic copper binding proteins. The limited number of studies that have been performed to date with Wilson’s disease liver, have provided contradictory results. Some studies support the view that in Wilson’s disease much of the accumulated copper is associated with the nuclear, mitochondrial and lysosomal fractions (15,16), whereas other studies suggest that the accumulated copper is predominantly soluble (17,lS). In the present study we have taken advantage of the Royal Free Hospital liver transplant program to study freshly isolated human liver and re-investigare the subcellular llcalisation of hepatic copper in patients with Wilson’s disease, hepatic copper accumulation secondary to chronic liver disease and patients with normal hepatic copper levels. Furthermore we have investigated the distribution of soluble copper between hepatic copper binding proteins and used Northern blotting techniques to investigate the expression of specific mRNAs for hepatic copper binding proteins.

95 ant homogenate was retained for estimation of liver copper levels. The remainder was centrifuged at 1000 x g for 10 min to separate the unbroken cells and tissues and the crude nuclear fraction. The resultant post-nuclear supernatant was recentrifuged at 10 000 x g for 10 min to yie!d the crude mitochondrial fraction. The supernatant was spun at 16 500 x g for 30 min to yield the crude lysosomal fraction. A final spin at 105 000 x g for 60 min gave the crude microsomal pellet and the soluble supernatant * Gel-filtration studies

Portions of soluble supernatant, cytosol and particulate supernatant were subjected to gel filtration on columns of Sephadex G-75 and Sepharose 6B (2.6 x 95 cm) as described, at 4°C using 0.01 M Tris/acetate buffer (pH 7.4), containing 0.1% (v/v) 2-mercaptoethanol (2-ME) as eluent. The columns were standardised with the appropriate molecular weight standards, including bovine erythrocyte copper-zinc superoxide dismutase and bovine caeruloplasmin, both of which were purchased from Sigma. Recovery of copper from the column varied between 8697%. SDS.PACE

Patients

All livers used in the present study were taken from patients who underwent liver transplantation at the Royal Free Hospital School of Medicine. The study utilised livers from two patients with Wilqpn’s disease, two p”tients with hepatic copper overload (one with primary sclerosing cholangitis, one with cryptogenic cirrhosis) and two with normal hepatic copper concentrations (one with fuiminant hepatic failure due to paracetamol overdose and one with oxalosis). Diagnosis of all patients was by standard clinical and biochemical methods including histological examination of liver biopsies. Sample collection

Human livers were collected in the operating theatre and immediately put on ice. Portions of tissue to be used for RNA extraction were snap frozen in liquid nitrogen and stored at -70°C until required. Fractionation of the liver was begun within 30 min of removal. Serum samples were taken prior to transplantation. Subcellular fractionation of livers

Portions of fresh liver (l-5 g) were minced using copper-free scissors and homogenised on ice in IO vol. of 3 mM Hepes containing 0.25 M sucrose, (pH 7.0) using a Potter-Elvehjem homogeniser (19). Ati subsequent procedures were performed at 4°C. A portion of the result-

and Western blotting

Protein samples were resuspended in SDS-sample buffer, 0.125 M Tris/I-ICl (pH 6.8) containing 10% (w/v) SDS, 10% (v/v) 2-ME, 50% (w/v) sucrose, a;~d 0.005% bromophenol blue, heated at 100°C for 5 min and resolved by SDS-PAGE o:i either 7.5 or 15% gels. Protein bands were visualised by Coomassie brilliant blue, or identified by Western blotting as desc -ibed below. Following electrophoresis proteins were transferred electrophoretically to nitrocellulose membranes (Bio-Rad) using a constant current of 60 mA for 1 h. One portion of the resultant blot was stained with amido black and the other was immunostained using the required antibody as follows. The blot was incubated in phosphate-buffered saline (PBS) containing 3% (w/v) de-fatted Marvel, 1% (w/v) Tween 20 for 2 h after which it was washed four times in PBS. The blot was incubated with the primary antibody (1:500-l: 1000) in PBS overnight, washed four times, incubated with a second antibody conjugated to horseradish peroxidase (HRP) (1:500-131000) for 1 h and then developed with hydrogen peroxide and HRI developer (BioXad). Antibodies us:d in the present study included sheep anti-human caeruloplasmin, sheep anti-human CuZn-SOD both from Serotec and sheep anti-human metallothionein-I (20). RNA extraction and Northern blotting

Total RNA was extracted from tissues by homogeni-

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TABLE 1 Patient details Diagnosis

Age (years)

(30-X) (p/U

Bilirubin (5-17) (iimoII1)

AST (5-4(I) (U/i)

Copper (==l()&g)

C’aeruloplasmin (20-40) (mg/dl)

36 31 40 34 45 41

748 YO 124 63 10 66

231 80 91 83 11 2436

122.5 247.2 132.5 95.5 8.8 4.5

8.28 11.7 30.2 24.7 25.3 18.0

Albumin

~~_~_~~____~_~~~~ Wilson’s disease Wilson’s disease Primary sclerosing chotangitis Cryptogenic cirrhosis Oxalosis Fulminant hepatic failure

18 30 47 45 17 25

4 M guanidinium isorhiocyanate followed by density-gradient centrifugation through caesium chloride (21). RNA was quantitated by its absorption at 260 nm. RNA samples (10 pug) were denatured in formaldehyde containing buffer and electrophoresed in agarose gels (O&1.6%), containing 2.2 M formaldehyde. Following electrophoresis, gels were stained with ethidium bromide to ensure that equal RNA was loaded onto each lane. RNA was transferred to nylon filters (Hybond-N. Amersham) overnight and fixed by U.V. cross linking. Blots were hybridised with the following probes, human caeruloplasmin (22), human CuZn-SOD (23), human albumin (24), rat glyceraldehyde phosphate dehydrogenase (GAPDH), rat actin (25) and mouse metalYothionein-I sation

in

8o1 WDi

(MT-I) (26), labelled with 32P either with the SP6/T7 polymerase transcription system (Boehringer) or with Multiprime DNA labelling system (Amersham) according to the manufacturers instructions. Hybridization was performed in standard solutions (25) at either 42°C (for cDNA probes) or 58S”C (for cRNA probes). After hybridisation the blots were washed under highly stringent conditions and autoradiography was performed at -7°C with an intensifying screen. Blots were stripped using boiling 0.1% (w/v) SDS which was allowed to cool to room temperature and rehybridised with further probes. Analytical procedrrres

Activity of CuZn-SOD was determined in column fractions using nitro blue tetrazolium (27). The reaction was performed for 6 min in a foil-lined box (30 x 25 x 20 cm) with an 8 watt light source. Protein was determined using the Bio-Rad protein assay reagent (28) with bovine y-globulin as standard. Caeruloplasmin oxidase was measured in serum and column fractions by determining the rate of oxidation of p-phenylenediamine at 37°C and pH 6 (29). Results are expressed as absorbance units/ml. Copper concentrations in liver and chromatography fractions were determined by electrothermal atomic absorption spectrometry using a Perkin-Elmer 3030 instrument equipped with a HGA-400 graphite furnace and autosampier. Liver samples were digested with concentrated nitric acid at 90°C for 1 h and diluted to appropriate concentrations with copper-free deionized water.

Results

Fig. 1. Subcellular distribution of copper in diseased human livers. Portions of post nuclear supernatant were subjected to differential centrifugation to investigate subceHular distribution. Results are expressedas a percentage of copper recovered. 10 x IO” g = crude mitochondrial fraction. 16.5 x 111”g = crude lysosomal fraction. 105 x 10-1g = crude microsomal fraction and 105 x IO-’g ssn = soluble supernatant.

Patient details are given in Table 1. The two patients with Wilson’s disease had markedly elevated liver copper levels. as did the patients with primary sclerosing cholangitis and cryptogenic cirrhosis. The patients with fulminant hepatic failure and oxalosis had liver copper levels within the normal range for human liver.

COPPER

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LlVERS

97

2400

:'I

2000

WD2

:; 1600

I)

:

100

ox

80 g

60

;

40 20 0 Peak 12

Peak

Peak 3

Peak 12

Peak

Peak 3

Fig. 3. The association of copper with soluble superrlatant proteins in human livers, following fractionation on Sephadex G-‘75. The amount of copper in each of the peaks is expressed as a percentage of total copper recovered, calculated from concentration and traction volume. Peak I = void volume, Peak 2 = copper zinc superoxide dismutase and Peak 3 = metallothionein.

homogenising the tissue and subsequently separating the nuclei from unbroken cells and unhomogenised tissue, For this reason we have expressed the results as a percentage of the post nuclear yupernatant copper (Fig. 1). In all livers the soluble supernatant was the largest copper containing fraction (Fig. 1). No clear differences could be seen between either of the three groups of patients despite liver copper covering a range from 4% 247 /igIg wet wt.

60 Ir IV 50

40

5 3

30

20

10

0 JO

40

50

60

70

60

90

lQ0

Fraction Number

Fig. 2. Distribution of soluble copper ii, hepatic soluble supernataut. fractionated on Sephadex G-75. Copper concentration per fraction (4.5 ml) is given in icg copper/l per g wet weight of tissue. Reccvery from the column varied between 86-97%. Note differing scales. Caeruloplasmin levels in the two patients with Wilson’s disease and the patient with fulminant hepatic failure were below the normal range. Liver function was impaired in ail of the livers other than the case of oxalosis which was essentially normal. Abnormal histology was present in all livers except for the oxalosis case.

Subcellular fractionation of livers Fibrosis of the diseased liver caused some difficulty in

Gel-filtratiow studies Following gel filtration on Sephadex G-75 all soluble supernatants contained three copp:~ peaks (Fig. 2). The first peak was at the void volume {V.&V, = 1) corresponding to proteins with an apparent M, of > 60. The second peak (VJV,, = 1.75). had an M, of approx. 30. This peak is assumed to represent CuZn-SOD, in view of its molecular weight. the observation that this peak was the only one to exhibit CuZn-SOD activity and also it was the only peak that contained immunoreactive CuZnSOD by Western blotting. The third peak (I/,/V, = 2.3) has the elution characteristics of metallothionein. and is assumed to represent this protein as metallodiionein could be identified in the peak by Western blotting. The metallothionein containing peak was the n?ajor copperbinding peak in all livers studied. In livers with abnormally high copper the percentage of copper in this peak

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et al.

CP

SOD 30

40

50

60

70

60

90

-.

FractioclNumber Ag. 4. Elation profile hepatic soluble supernatant from primary scierosing cholangitis liver fractionated on Sepharose 6B. Copper concentration per fraction (4.5 ml) is given in {ig copper/l per g wet weight of tissue. Horizontal bar represents fractions which contained caeruloplasmin protein identified by Western blotting.

was higher than in the livers with normal copper levels (Fig. 3). In livers with normal liver copper concentrations the CuZn-SOD-containing peak contained a greater percentage of total copper than in the copper-loaded livers (Fig. 3). The amount of copper in the void volume peak varied between 0.9-11%. This peak was further characterised by fractionating samples on Sepharose 6B,

CP

.-.

~-

_- _-. .-

_. _

MT

Alb

GAPDH

Fig. 6. Steady-state expression of specific mRNA for caeruloplasmin (CP), copper zinc superoxide dismutase (SOD). metallothionein (MT), albumin (Alb) and glyceraldehyde phosphate dehydrogenase (GAPDH) in human livers. Northern blots were prepared as described using 10 yg of total RNA and hybridized with specific cRNA/cDNA probes. Autoradiographic exposure time varied for each probe.

a gel with a larger fractionation range than Sephadex G-75. Using this gel some copper still remained in the void volume peak but a distinct peak with an apparent M, of 125-140 was evident (Fig. 4). This peak is assumed to represent caeruloplasmin in view of its molecular weight and the demonstration of immunoreactive caeruloplasmin by Western blotting (results not shown).

SOD

MT

Fig. 5. Localization of caeruloplasmin (Cp). copper zinc superoxide dismutase (!XID) and metallothionein (MT) in hepatic soluble supematants by Westernblotting following SDFS-PAGE of 100 peg of soluble supernatant protein on 7.5 or 15% gels. The position of molecular weight markers is indicated at the side.

Western blodng Following SDS-PAGE of hepatic cytosols it was possible to immunolocalise caerulopla,min, superoxide dismutase and metallothionein in all samples. The two Wilson’s disease livers both contained caeruloplasmin of the normal molecular weight (Fig. 5), however, levels were lower than in the other livers studied. The smaller immunoreactive bands are due to degradation of the protein during manipulation. Superoxide dismutase protein was present in similar amounts in all livers studied. Metallothionein was present in all of the livers and it appeared that levels of immunoreactive protein were sim-

COPPER

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ilar in all livers. There was no clear elevation of metallothionein in the livers with copper overload compared with those with normal copper levels. Expression of mRNAs of hepatic copper proteins The steady-state levels of caeruloplasmin, superoxide dismutase, metallothionein, albumin and GAPDH mRNA are shown in Fig. 6. It is clear that there are major differences in levels between the livers. The leve! of caeruloplasmin mRNA in the two Wilson’s disease livers was lower than in the oxalosis liver and the two with elevated copper levels. Caeruloplasmin mRNA was also markedly reduced in the liver from the patient with fulminant hepatic failure. The two known caeruloplasmin mRNA species (22) were present in all livers and there was no differential expression of the mRNA species. The pattern of expression of CuZn-SGD mRNA was similar, with mRNA levels being reduced in the two Wilson’s disease livers and the one with fulminant hepatic failure. Expression of metallothionein mRNA was markedly different with the highest levels being found in the Wilson’s disease livers and the primahy sclerosing cholangitis liver, however marked exp;es;ton was also found in the livers with normal hepatic copper concentrations. Albumin mRNA expression was clearly reduced in the patient with fuulminant hepatic failure and in one of the Wilson’s disease livers. The GAPDH probe, used as control, revealed that mRNA was expresssd in all of the livers, with measurable expression in the patients with Wilson’s disease and marked expression in the patient with fulminant hepatic failure. A similar pattern of expression was found using the actin probe (results not shown), suggesting that the reduced levels of caeruloplasmin, CuZn-SOD and albumin were not the result of underloading of the gel. However, it is clear that expression of both actin and GAPDH are variable in diseased human liver and do not represent the amount of RNA loaded onto the gel as estimated using ethidium bromide staining.

Discussion In the present study we have used fresh liver removed from liver transplant recipients to study the subcellular distribution of hepatic copper and its association with hepatic copper proteins. Using large portions of liver, we have been unable :o show a clear difference in the subcellular distribution of copper in Wilson’s disease, liver disease with elevated copper levels and liver with normal copper levels. These results differ from two previous reports which suggest that in Wilson’s disease, copper

99

accumulated preferentially in the particulate fraction (15,16). However, OUT results are in broad agreement with a more recent study w ich showed in a single transplant patient with Wilson’s disease that hepatic copper was locaslised predominantly in the soluble fraction (18). Reasons for these differences remain unclear and may to some extent relate to the use of different methods for producing subcellular fractions and the state of the diseased tissues. We also studied possible differences in the association of copper with known hepatic copper-binding proteins, In all livers the major copper peak corresponds to a protein with the physical and chemical features of metallothionein. This is in agreement with previous studies using similar gel-filtration techniques either alone (16) or in conjunction with ion-exchange chromatography (18). immunohistochemistry has also shown marked accumulation of metallothionein in Wilson’s disease livers (18,31). The Western blot study provided surprising findings. Abundant metallothionein was present in the two livers with normal copper levels and there appeared to be no major difference between any of the livers studied. This suggests that metallothionein levels are relatively high in human liver with normal copper levels, either as metal-free apoprotein or perhaps binding another metal, such as zinc. The increase in the amount of copper associated with metallothionein in livers with elevated copper levels may be the result of differences in the affinity of the protein for different metals. Copper is known to displace zinc bound from the protein, due to higher binding affinity (1). Metallothionein is believed to play a role in the detox;fication of metals, including copper, and metallothionein-bound copper is considered to be nontoxic (1,2). Indeed in Wilson’s disease, metallothionein has been demonstrated histochemically in late stage, but not early stage disease (31). It has been suggested that copper toxicity occurs in the early phase when liver copper is > 20-times normal and metallothionein can not be readily demonstrated by histochemistry (4,30-32). The patients with Wilson’s disease which we studied were seriously ill requiring a life-saving liver transplant. It might have been expected that if copper toxicity was the primary cause of the liver failure, less metallothioneinbound copper would have been present in these livers. However, no obvious difference in metallothionein could distinguish Wilson’s disease liver from chronic liver disease, f&ninant liver failure or ‘normal’ liver (oxalosis). Scheinberg and Stemlieb (4) have described a paradox that asymptomatic patients, often discovered when screening families, tend to have the highest liver copper levels and no copper or copper-associated protein can be demonstrated histochemically. There appears to be no

C.D. BlNGLE

100 simple explanation for these findings, and it would appear that other factors, such as inflammation might be important in the progression of the disease. Our results are in agreement with others which suggest that in livers with normal copper levels, CuZn-SOD is a major copper binding component (1,2). It is of interest that the amount of copper in this peak as well as the levels of immunoreactive protein do not increase when liver copper levels are greatly elevated. This suggests that liver copper levels do not regulate levels of the protein. We have made similar observations in the developing guinea pig liver (33). Many studies have shown a copper peak in the void volume of Sephadex G-75 columns. although this has never been well characterised (1.2.11). In our study, gel filtration and Wectern blotting have identified caeruloplasmin as a component of this peak. This observation is supported by Western blotting of all the soluble supernatants. However, it is possible that the caeruloplasmin in this peak may be serum contaminant as it was not possible to perfuse the liver prior to homogenisation. The reduction in caeruloplasmin mRNA levels in the patients with Wilson’s disease is similar to that described previously (34) and appear to correlate both with the reduced serum levels and reduced hepatic protein levels. However, hepatic caeruloplasmin mRNA was also reduced in fulminant liver failure. Expression of CuZnSOD mRNA was also reduced in Wilson’s disease and fulminant liver failure and was not related- to hepatic copper levels. Expression of metallothionein mRNA was elevated in Wilson’s disease and primary sclerosing cholangitis (cholestatic) liver. However, in the copperloaded ‘cryptogenic’ liver metallothionein mRNA levels were reduced. This suggests that there is no simple relationship between copper levels and metallothionein mKXA. It is recognised that metallothionein mRNA may be induced by many stimuli such as cytokines, steroids and other trace metal particularly zinc (1.13). ln an atReferences 1 Cousins RJ. Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and caeruloplasmin. Physiol Rev 19885:6% 238-309. 2 Ettinger MJ. Copper metabolism and diseases of copper metabolism. In: Lontie R. ed. Copper Protei::s. Vol III. Cleveland: CRC Press, 1984: 175-229. 3 Danks DM. Hereditary disorders of copper metabolism in Wilson’s disease and Menkes’ disease. In: Stanbur$ JB. et al.. eds. The Metabolic Basis of Inherited Disease. New York: McGraw-Hill. 1983: 1251-68. 4 Scheinberg IH. Sternlieb I. Wilson’s Disease. Philadelphia. WB Saunders, 1984. 5 Gibbs K. Walshe JM. Biliary excretion of copper in Wilson’s disease. Lancet 1980; ii: 538-9.

ta al.

tempt to see if a generalised impairment of hepatic function was responsible for the reduced levels of caeruloplasmin message in Wilson’s disease we also studied the expression of albumin mRNA. Albumin mRNA levels were reduced in both Wilson’s disease and fulminant liver failure. This finding differs from the study of Cazja et al. (34), who reported that albumin mRNA levels were increased in Wilson’s disease whilst caeruloplasmin mRNA levels were reduced. It is possible that these differences reflect differences in the severity of the disease between the patients. In conclusion, in the present study using fresh tissue removed at transplantation. we have been able to observe no major differences in the subcellular distribution of hepatic copper in the post nuclear supernatant of livers from patients with Wilson’s disease, hepatic copper overload secondary to chronic liver disease and livers with normai copper levels. In all livers the soluble supernatant was the major copper containing fraction and furthermore within this fraction we have shown that metallothionein is the major copper-binding protein. We have confirmed that caeruloplasmin mRNA is reduced in Wilson’s disease. However, reduced expression was also found in fulminant hepatic failure and it remains to be shown that such a reduction in mRNA is specific to Wilson’s disease and not just a reflection of the severity of the liver disease.

Acknowledgements This study was supported by the Sir Jules Thorn Charitable Trust. We are grateful to Dr. AK. Burroughs for making available the livers used in this study. We thank the following for gifts of probes and antibodies; Dr. J .D. Gitlin (caeruloplasmin. CuZn-SOD and actin probes), Dr. G.K. Andrews (metallothionein probe) and Professor R.J. Cousins (metallothionein antibody).

6 Earl CJ. Moulton MJ. Selverstone B. Metabolism of copper in Wilson’s disease and in normal subjects: studies with ‘%.I. Am J Med 1954: 17: 305-13. 7 Scheinberg IH. Gitlin D. Deficiency of caeruloplasmin in patients with hepatolenticular degeneration (Wilson’s disease). Science 1952: 116: 484-S. 8 Mason KE. A conspectus of research on copper metabolism and requirements of man. J Nutr 1979: 109: 1979-2066. 9 Srai SKS. Burroughs AK, Wood B. Epstein 0. The ontogeny of liver copper metabolism in the guinea pig: clues to the etiology of Wilson’s disease. Hepatology 1986; 6: 427-32. 10 Bingle CD. Srai SKS. Epstein 0. Developmental changes in hepatic copper proteins in the guinea pig. J Hepatol 1990; IL: 138-43. I1 Tero T. Owen CA. Nature of copper compounds in liver supernatant and bile of rats: studies with “‘CU. Am J Physiol 1973;

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224: 682-6. 12 Goldfischer S. Popper H. Sternlieh I. The significance of variations in the distribution of copper in liver disease. Am J Pathol 1980; 99: 715-30. 13 Bremner I. Involvement of metallothionein in the hepatic metabolism of copper. J Nutr 1987: 117: 19-X. I4 Epstein 0. Arborgh B. Sagiv M. Wr*>blewski R. Scheur PJ. Sherlock S. Is copper hePatotoxic in primary biliary cirrhosis. J Clin Pathol 1981: 34: 1071-5. 15 Porter H.: Tissue copper proteins in Wilsons disease. Arch Neurol 1964; 11: 341-9. 16 Sternlieb I. Van den Hamer CAJ, Morel1 AG. Alpert S. Gregoriadis G, Scheinberg IH. Lysosomal defect of hepatic copper excretion in Wilson’s disease (hepatolenticular degeneration). Gasteroenterology 1973; 64: 99-105. 17 Morel1 AG, Shapiro JR. Scheinberg IH. Copper binding proteins from human liver. In: Walshe JM, Cummings JN. eds. Wilson’s Disease: Some Current Concepts. Springhelds: CC Thomas, !96!: 36-41. 18 ?Qartey NO. Frei JV, Cherian MG. Hepatic copper and metallothionein distribution in Wilson’s disease (hepatolenticular degeneration). Lab Invest 1987: 57: 397-401. 19 Mehra RK. Bremner I. Species differences in the occurrence of copper-metallothionein in the particulate fractions of the liver of copper-loaded animals. Biochem J 1984: 219: 539-16. 20 Grider A. Kao K-J. Klein PA, Cousins RJ. Enz:rme linked immunosorbent assay for human metallothionein: correlation of induction with infection. .I Lab Clin Med 1989: 113: 221-S. 21 Chirgwin JM. Pry?byla AE. MacDonald RJ, Rutter WJ. Isolation of bio!ogically active ribonucleic acid from sources enriched with ribonuclease. Biochemistry 1979: i8: 5294-9. 22 Gitlin JD. Transcriptional regulation of ceruloplasmin gene expression during inflammation. J Biol Chem 19%: 263: 6281-7. 23 Sherman L. Dafni N. Lieman-Hurwitz J. Groner Y. Nucleotide sequence and expression of human chromosome 21-rncoded superoxide dismutase mRNA. Proc Natl Acad Sci USA 1983:

101 80. 5465-9. 24 Urano v. Sakai M, Watanabe K, Tamaoki T. Tandem arrangement of the albumin and alpha-fetoprotrin genes in the human genome. Gene 19%: 32: 255-61. 25 Fleming RE. Gitlin JD. Primaty structure of rat caerulopiasmin and analysis of tissue specific gene expression during development. J Biol Chem 1990: 265: 7701-i. 26 Durnam DM. Palmiter RD. Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. J Biol Chem 1981; 256: 2268-72. 27 Winterbourn CC. Hawkins RE, Brian M. Carrel1 RW. The estimation of red cell superoxide dismutase activity. Lab Clin Med 1975: 85: 337-41. 28 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976: 72: 248-54. 29 Henry RI, Chiamori N, Jacobs SL. Segalove M. Determination of caeruloplasmin oxidase in serum. Proc Sot Exp Med Biol 1960; 104: 620-4. 30 Goldfischer S. Sterrtlieb I. Changes in the distribution of hepatic copper in relation to the progression of Wilson’s disease (hepatolenttcular degeneration). Am J Pathol 1968: 53: 883-901. 31 Jain S. Scheur PJ. Archer B, Newmann SP. Sherlock S. Histological demonstration of copper and copper associated protein in chronic liver diseases. J Clin Pathol 1978; 31: 78-l-90. 32 Elmes ME, Clarkson JP, Mahy NJ, Jasani B. Mrtailothionein and copper in liver disease with copper retention - a histopathological study. J Pathol 1989: 158: 13L-57. 33 B

Copper metabolism in hypercupremic human livers. Studies of its subcellular distribution, association with binding proteins and expression of mRNAs.

In the present study we have used differential centrifugation, size exclusion chromatography, Western and Northern blotting to investigate the subcell...
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