Developmental
changes in hepatic copper proteins in the guinea pig
We have suggested that Wilson’s disease is caused by failure to adapt from the fetal to adult mode of copper metabolism and that the study of liver copper ontogeny might provide clues to the pathogen& of Wilson’s disease. This study traces the developmental chauges in hepatic copper biding proteins in the guinea pig. During the last trimester, as fetal hepatic copper increases, metallothionein is the major copper binding peak in both the soluble and particulate supematant fixtions. After birth this peak decreasesin parallel with the fall in liver copper. Metallothionein is absent from adult soluble supematant and copper is associated with superoxide dismutase and a high molecular weight protein. A novel low molecular weight copper binding component is present in the particulate supematant of neonatal liver, but is absent from the adult. Unlike many other animals, but similar to men, the switch from the fetal to adult mode of copper metabolism occurs at birth. Comparison of the copper protein profiles of the fetus and neonate with Wilson’s disease are required to test the hypothesis tha: Wilson’s disease is caused by developmental arrest of copper ontogeny.
In all mammals, fetal liver copper concentrations increase in the last trimester and decline at varying intervals after birth (l-4). Paradoxicatty, serum copper and caeruloplasmin levels are substantially lower than adult levels at birth, but increase thereafter (2,5-6). The healthy human neonate has a copper profile indistinguishable from Wilson’s disease and it is possible that the disease is caused by perpetuation tabolism into childhwd ny may provide
of the fetal mode of copper me(7). The study of copper ontoge-
insight into both the control of hepatic
(10) and may act as a major store for copper and zinc in normal tissue (11). CuZn-SOD is involved in defence against toxic free radicals (12,13). The nature and funcdon of the high molecular weight fraction is unknown. There have been few attempts to study the ontogeny of all three hepatic copper-binding proteins simultaneously, although this is essential for a fuller understanding of cop. per homeostasis (9). In a previous study of neonatal and adult guinea pigs, we reported capper
binding
proteins
a difference
of newborn
in the hepatic
and adult animals
copper homeostasis and the pathogenesis of Wilson’s disease. Changes in hepatic copper concentration are associated with changes in the expression of hepatic and serum copper binding proteins. Hepatic copper is associated with
(14). The present study traces the qualitative and quantitative changes in the association of copper with hepatic bindingpmteins in the developing guinea pig.
three protein peaks following gel filtration; metallothionein (MT; M, M)oO), CuZn-superoxide dismutw (CuZn
Methods
SOD: M, 32 Ooa) and a poorly characterized hi molec. ular weight fraction (M, 150 MJO) (8,9). Cu-MT increases in copper overload states such as primary biliary cirrhosis
Experimental
Comrpondence: Calin D. Bin@. Academic Department NW3Ztx.“.K.
lrnimols
pigs,
Time-mated Dunkin Hardy guinea fed on GPFDl standard solid diet (SDS, Ltd.), mntainirtg 16 @kg Cu.
ofMedicine. Royal Free Hospital School othledicine. Rowland Hill Street, ,..,ndon,
139 were USed throughout. Normal gestation is 69 days and day 0 is defined as the first 24 hours after birth. All litters suckled normally. Fetal litters were obtained by cesarian section performed under halolhane anaesthesia.
All prmedms
gene perfontted
using copper-free
in-
Animals ww sacrificed and IWfs wereremovedand stored on ice or fnxen at -20 T, .ssIsquired. Bile was collected from the gall bladder and urine from the bladder. Blood was cokcted into hepaMt=d tubes and the plasma was femoved fo&x%tg sennifugalion. Red cells were washed three timer with icecold 6.9% NaCl and, following removal of the huffy coat, =*M=~P
and plsrtiwtrc.
the cells were lysed with deionized stored at -2B “C until required. Fractionation
waler. The Iysale was
in 2.5 vol.
0.25 M sucrose al 4 “C.
‘Ihe homogenate was centrifuged at 105 Ooo x g for 1 hour and the soluble supematanl was removed and stored al 4 C. The p-&t was suspended in 1% (v/v) 2.mercaplo&anal in water, beeze&awed three limes and incubated at 37 ‘C for 3 hours lo disrupt membranes. The resuiting sample was centrifuged at 105 000 X 8 for 1 hour lo yield the ‘particulate supernalant’. Both soluble and particulate supematant fractions were rubiecled to gel filtration on a Sephadex G-75 column (Pbrrmacia) (2.6 X 95 cm) at 4 ‘C using 0.01 M Trig+ acetate buffer, pH 7.4, containing 0.1% (v/v) 2-mercaptcethanol. Fnctionr of 4.5 ml were collected. The column was calibrated with appropriate protein standards (M,
12.3 @Xl-66CU0).
Liver Cu. wg) 28.91 + 7.78 (7) 46.49 f 4.49 (5) 55.@3+ 19.15 (9) 63.55 + 11.m (8) 1M.Of 4l.40(30) 154.9* 60.3 (1.4) 74.73 + 21.4 (18) 42.87 * 20.24 (20) 38.66 * 8.34 (9)
mtifs/mgprofein. 1 unit is de65ed as ffte amount of enzyme causing hal! the maximum inhibitiott of Nitmbluc tetrazolium reduction. protein was determined u&g the Bio-Rad protein assay reagent (16) with b01 ‘e gmtma globulia as standard. Caerttloplasmin was measured in plasma by determining the rate of oxidation ofp-phenylendiamine at 37 “C and pH 6 (17). Rcsul~sme expressed ds absorbanceuniWml. Copper wncentrmions in plasma,
chromatography bations were de-
POrtiOns of fresh liver were homogenised
A8C VV) 49 55 M 67 ob 1 4 12 28
erythrcqte
bile, urine, liver and termined by electrothermal atomic sbsmprion spectmmetry using a Perkin Elmer 3Mo insmmtent equipped with a
of livers
umes of 3 mM Hepes containing
Activity of CuZnSOD was determined in chloroformlethanol extractsof lysates, in soluble supematant and also in column fractions (15). The reaction was performed for 6 tin in a foil-lined box (Xl x 25 x 20 cm) with an 8 wall tight source. Red cell activity is expressed as units@ Hb and soluble supernalant activity as
Plasma cu C@) 0.15 + 0.04 (8) 0.13 f 0.01 (5) Cl.19* 0.03 (9) 0.23 * 0.07(12) 0.25 ? 0.07(24) 0.35 + O.u7(11) 0.41 i 0.14(17) 0.39 * 0.09w) 0.52~o.tottt~
HGA4lO graphite fumrre and autosampler. Liver samples were digested with comxntrated
nitric acid al 90 ‘C
far 1 hour and diluted lo appropriate capper-free deionizedw&r.
concenlratians
with
RSldlS
Copperprojik Developmental changes in liver copper, plasma copper and caeruloplasmin let& are shown in Table 1. Liver copper peaked on the fim day after birth, al which time the con~enwation was tive limes higher than the 28&yold animal. There was tittte change in fetal plasma copper in the last trimester, but a dramatic increase occurred after birth. Before birth, carmloplasmin zctivity was unde-
Bile ti (1(8/101) N.D.1.59 t; 0.M (3) N.D. 2.75 t 0.93 (6) 1.93 f x29(31) 2.18 f 1.71(13) 1.52 f. 0.75 @_I000. The second peak (VJV. = 1.75) had an M, of approximately 30 OW. This peak is assumed to represent CuZn-SOD, in view of its molecular weight and the ohservatirms that pure bovine CuZn-SOD eluted in the same fractions. These fractions were also the only peak to elicit CuZn-SOD activity. The third peak (VJV, = 2.3) had an approximate M, of 10 Ow. This peak is assumed to represent MT, in “iew of its elution pr&le, its lack of abscnption at 280 nm and its cross-reactivity with an anti-rat MT antibody which is known to cuss-react with the guinea pig protein (Dr. Ian Bremner, personal communication).
The developmental changes in the soluble supematant copper binding proteins are shown in Fig. 314.During the last 3 weeks of gestation. copper associated with MT rose from a mean of 1.6 #g/g wet wt. liver on day 45 of gestation to a peak of 23.1 #g/g wet wt. liver on the first day afterhirth,fallingtoameanofZ.l/rglgwetwt.byday4and
35 r
of liver on the 67tb day of gestation. In the 4 days after birth, this peak accounted for 10-11 pg Cu/g wet wt. of liver, thereafter
falling to almost undetectable
levels.
CuZnSOD activity in liver and red cells !kpatic CuZn-SOD activity did not correlate with age and on day 4, activity was markedly decreased (Table 2). There was no con&don between bepstic CzZtwSOD activity and liver copper levels. This contrasts with red cell CuZn-SOD activity which increased sigcdftcantly over the same period (Table 2) and was carrelated positively with plasma copper levels (r = ct.%,’ = 0.84).
0.3 ,I& wet wt. by day 28 (Fig. 38). Over the same period, there was no significant change in the amount of cop per associated with the CuZn-SOD and void volume peaks (Fig. 3a).
The developing guinea pig, like the human neonate, has a copper profile similar to that found in Wilson’s disease (14) and study of copper ontogeny might provide insight into the cause of the disease. Gel filtration of the soluble supematard reveals sign&icant developmental differences in copper binding peaks.
Gel filtration of the Bmercaptoethanol solubilised particulate’ fraction (particulate supentatant) resolved dues peaks (Fig. 2b). The first peak eluted with the void volume, the second with MT and the third was of very low molecular weight (LMW; M, SM)[), VJV,, = 3). There was no peak mnesponding to the CuZo-SOD peak.
During the last 3 weeks of gestation there is a dramatic increast in the amount of copper associated with MT. A sirnils; increase has also been reported in rats and mice (18-U) The amount of copper asswiated with MT falls immediately after birth unlike in the rat where both liver copper actci MT continues to increase for upto 2 weeks
The developmental changes of the copper binding proteins in the particulate fraction are represented in Fig. 3b. By day.5.5of gestation, a meanof i.SflgCulgwet wt. liver was associated with MT, increasing to l8jzg Cd8 wet wt. at birth and then falling after birth. In fetal liver, the LMW peak accounted for a mean of 17.6/lg Cu/g wet M.
(22). Fewi hepatic MT increases in parallel with increasing liver copper cmtcentratians, but it remains unclear whether MT is induced by hepatic copper accumulation (II) oi *b&d the mpper kvc: bicraser as a consequence of MT synthesis (21). Our results suggest that in fetal liver, induction of MT is not simply a function of total
copper
142 liver copper concentration; for whilst liver copper coocentrations BE similar in the &day-old fetus and the Z&dayold adult, MT is the major hepatic copper binding protein in the 45.day-old fetus, whereas it is undetectable in 2& day-old animals. It is possible that liver copper sequestration in the fetus is secondary to induction of MT synthesis by glucocorticoids (21). It is htrtber possible that Zn may have a role in MT metabolism in the developing guinea pig as Z” is a potent inducer of the protein (ZZ), however Zo levels were not determined in the present stody. In adult mammals, hepatic soluble supemataot copper is associated almost exclusively with CoZo-SOD. This protein is believed to protect cells from free radical damage (13), and has been suggested also to wf as a storage
The nature of the void volume peak in both the soluble and particulate supematant remains unresolved. It may represent caeruloplasmin, polymer&d MT or en ““defined protein. As &-MT is not detectable in adult liver. it is unlikely to be derived from polymer&d MT. S&es have shown that in many tissues, including liver, newly absorbed copper appears rapidly in the void volume and subsequently transfers to other copper proteins (8), sug. gesting that the peek is ““t caeruloplasmin. ‘I%e peak might comprise more thas one pmtein and it still remains to be established whether the void volume peaks io the soluble and particulate soperoatents represent the st,me protein. It is dear from these studies that during develop
is not apparent. Despite major changes in liver copper concentration during development, CuZn-SOD activity does not change. Similar observations have been made in chicks and young rats (26,27). In wpper overload states, such as chronic cholestasis and erperimental copper overload, copper accmnulates preferentially in the particulate fraction where it is ass”ciated with MT (11). In the developing guinea pig, copper concentrations are highest at birth and at this time point, the particulate superoatant contains the greatest portion of copper which can be solubilised. I” addition to the MT copper peak, gel filtration of the particulate supernatent from fetal and young gumea pig liver reveals a low molec..
ment there are marked changes in both the compartmentalisation of hepatic copper end its association with copper binding proteins. The cause of hepatic copper accomulation in fetal liver remains unknown. It is possible that physiological cholestasis in the fetus is important, although the profoundly subnormal levels of caeruloplasmln in the neonate are quite unlike adult cholestasis where worn copper and cseruloplasmin levels are markedly increased (6). E+ study emphasises the dramatic changes in copper metabolism occurring at and around the time of birth. The switch from the fetal to adult mode of metabolism is asso ciated with significant changes in the expression of copper proteins. Genetic failure of this pwioatal adaptation might perpetuate the fetal mode of copper and caeroloplasmi” metabolism into later life. Liver mpper retentio” would persist after birth leading to unphysiological liver copper levels end liver damage with the clinical manifestations of Wilson’s disease presenting from the age of 6 years onwards. In addition to the similarities in copper
alar weight copper peak Mr
changes in hepatic copper proteins in the guinea pig
We have suggested that Wilson’s disease is caused by failure to adapt from the fetal to adult mode of copper metabolism and that the study of liver copper ontogeny might provide clues to the pathogen& of Wilson’s disease. This study traces the developmental chauges in hepatic copper biding proteins in the guinea pig. During the last trimester, as fetal hepatic copper increases, metallothionein is the major copper binding peak in both the soluble and particulate supematant fixtions. After birth this peak decreasesin parallel with the fall in liver copper. Metallothionein is absent from adult soluble supematant and copper is associated with superoxide dismutase and a high molecular weight protein. A novel low molecular weight copper binding component is present in the particulate supematant of neonatal liver, but is absent from the adult. Unlike many other animals, but similar to men, the switch from the fetal to adult mode of copper metabolism occurs at birth. Comparison of the copper protein profiles of the fetus and neonate with Wilson’s disease are required to test the hypothesis tha: Wilson’s disease is caused by developmental arrest of copper ontogeny.
In all mammals, fetal liver copper concentrations increase in the last trimester and decline at varying intervals after birth (l-4). Paradoxicatty, serum copper and caeruloplasmin levels are substantially lower than adult levels at birth, but increase thereafter (2,5-6). The healthy human neonate has a copper profile indistinguishable from Wilson’s disease and it is possible that the disease is caused by perpetuation tabolism into childhwd ny may provide
of the fetal mode of copper me(7). The study of copper ontoge-
insight into both the control of hepatic
(10) and may act as a major store for copper and zinc in normal tissue (11). CuZn-SOD is involved in defence against toxic free radicals (12,13). The nature and funcdon of the high molecular weight fraction is unknown. There have been few attempts to study the ontogeny of all three hepatic copper-binding proteins simultaneously, although this is essential for a fuller understanding of cop. per homeostasis (9). In a previous study of neonatal and adult guinea pigs, we reported capper
binding
proteins
a difference
of newborn
in the hepatic
and adult animals
copper homeostasis and the pathogenesis of Wilson’s disease. Changes in hepatic copper concentration are associated with changes in the expression of hepatic and serum copper binding proteins. Hepatic copper is associated with
(14). The present study traces the qualitative and quantitative changes in the association of copper with hepatic bindingpmteins in the developing guinea pig.
three protein peaks following gel filtration; metallothionein (MT; M, M)oO), CuZn-superoxide dismutw (CuZn
Methods
SOD: M, 32 Ooa) and a poorly characterized hi molec. ular weight fraction (M, 150 MJO) (8,9). Cu-MT increases in copper overload states such as primary biliary cirrhosis
Experimental
Comrpondence: Calin D. Bin@. Academic Department NW3Ztx.“.K.
lrnimols
pigs,
Time-mated Dunkin Hardy guinea fed on GPFDl standard solid diet (SDS, Ltd.), mntainirtg 16 @kg Cu.
ofMedicine. Royal Free Hospital School othledicine. Rowland Hill Street, ,..,ndon,
139 were USed throughout. Normal gestation is 69 days and day 0 is defined as the first 24 hours after birth. All litters suckled normally. Fetal litters were obtained by cesarian section performed under halolhane anaesthesia.
All prmedms
gene perfontted
using copper-free
in-
Animals ww sacrificed and IWfs wereremovedand stored on ice or fnxen at -20 T, .ssIsquired. Bile was collected from the gall bladder and urine from the bladder. Blood was cokcted into hepaMt=d tubes and the plasma was femoved fo&x%tg sennifugalion. Red cells were washed three timer with icecold 6.9% NaCl and, following removal of the huffy coat, =*M=~P
and plsrtiwtrc.
the cells were lysed with deionized stored at -2B “C until required. Fractionation
waler. The Iysale was
in 2.5 vol.
0.25 M sucrose al 4 “C.
‘Ihe homogenate was centrifuged at 105 Ooo x g for 1 hour and the soluble supematanl was removed and stored al 4 C. The p-&t was suspended in 1% (v/v) 2.mercaplo&anal in water, beeze&awed three limes and incubated at 37 ‘C for 3 hours lo disrupt membranes. The resuiting sample was centrifuged at 105 000 X 8 for 1 hour lo yield the ‘particulate supernalant’. Both soluble and particulate supematant fractions were rubiecled to gel filtration on a Sephadex G-75 column (Pbrrmacia) (2.6 X 95 cm) at 4 ‘C using 0.01 M Trig+ acetate buffer, pH 7.4, containing 0.1% (v/v) 2-mercaptcethanol. Fnctionr of 4.5 ml were collected. The column was calibrated with appropriate protein standards (M,
12.3 @Xl-66CU0).
Liver Cu. wg) 28.91 + 7.78 (7) 46.49 f 4.49 (5) 55.@3+ 19.15 (9) 63.55 + 11.m (8) 1M.Of 4l.40(30) 154.9* 60.3 (1.4) 74.73 + 21.4 (18) 42.87 * 20.24 (20) 38.66 * 8.34 (9)
mtifs/mgprofein. 1 unit is de65ed as ffte amount of enzyme causing hal! the maximum inhibitiott of Nitmbluc tetrazolium reduction. protein was determined u&g the Bio-Rad protein assay reagent (16) with b01 ‘e gmtma globulia as standard. Caerttloplasmin was measured in plasma by determining the rate of oxidation ofp-phenylendiamine at 37 “C and pH 6 (17). Rcsul~sme expressed ds absorbanceuniWml. Copper wncentrmions in plasma,
chromatography bations were de-
POrtiOns of fresh liver were homogenised
A8C VV) 49 55 M 67 ob 1 4 12 28
erythrcqte
bile, urine, liver and termined by electrothermal atomic sbsmprion spectmmetry using a Perkin Elmer 3Mo insmmtent equipped with a
of livers
umes of 3 mM Hepes containing
Activity of CuZnSOD was determined in chloroformlethanol extractsof lysates, in soluble supematant and also in column fractions (15). The reaction was performed for 6 tin in a foil-lined box (Xl x 25 x 20 cm) with an 8 wall tight source. Red cell activity is expressed as units@ Hb and soluble supernalant activity as
Plasma cu C@) 0.15 + 0.04 (8) 0.13 f 0.01 (5) Cl.19* 0.03 (9) 0.23 * 0.07(12) 0.25 ? 0.07(24) 0.35 + O.u7(11) 0.41 i 0.14(17) 0.39 * 0.09w) 0.52~o.tottt~
HGA4lO graphite fumrre and autosampler. Liver samples were digested with comxntrated
nitric acid al 90 ‘C
far 1 hour and diluted lo appropriate capper-free deionizedw&r.
concenlratians
with
RSldlS
Copperprojik Developmental changes in liver copper, plasma copper and caeruloplasmin let& are shown in Table 1. Liver copper peaked on the fim day after birth, al which time the con~enwation was tive limes higher than the 28&yold animal. There was tittte change in fetal plasma copper in the last trimester, but a dramatic increase occurred after birth. Before birth, carmloplasmin zctivity was unde-
Bile ti (1(8/101) N.D.1.59 t; 0.M (3) N.D. 2.75 t 0.93 (6) 1.93 f x29(31) 2.18 f 1.71(13) 1.52 f. 0.75 @_I000. The second peak (VJV. = 1.75) had an M, of approximately 30 OW. This peak is assumed to represent CuZn-SOD, in view of its molecular weight and the ohservatirms that pure bovine CuZn-SOD eluted in the same fractions. These fractions were also the only peak to elicit CuZn-SOD activity. The third peak (VJV, = 2.3) had an approximate M, of 10 Ow. This peak is assumed to represent MT, in “iew of its elution pr&le, its lack of abscnption at 280 nm and its cross-reactivity with an anti-rat MT antibody which is known to cuss-react with the guinea pig protein (Dr. Ian Bremner, personal communication).
The developmental changes in the soluble supematant copper binding proteins are shown in Fig. 314.During the last 3 weeks of gestation. copper associated with MT rose from a mean of 1.6 #g/g wet wt. liver on day 45 of gestation to a peak of 23.1 #g/g wet wt. liver on the first day afterhirth,fallingtoameanofZ.l/rglgwetwt.byday4and
35 r
of liver on the 67tb day of gestation. In the 4 days after birth, this peak accounted for 10-11 pg Cu/g wet wt. of liver, thereafter
falling to almost undetectable
levels.
CuZnSOD activity in liver and red cells !kpatic CuZn-SOD activity did not correlate with age and on day 4, activity was markedly decreased (Table 2). There was no con&don between bepstic CzZtwSOD activity and liver copper levels. This contrasts with red cell CuZn-SOD activity which increased sigcdftcantly over the same period (Table 2) and was carrelated positively with plasma copper levels (r = ct.%,’ = 0.84).
0.3 ,I& wet wt. by day 28 (Fig. 38). Over the same period, there was no significant change in the amount of cop per associated with the CuZn-SOD and void volume peaks (Fig. 3a).
The developing guinea pig, like the human neonate, has a copper profile similar to that found in Wilson’s disease (14) and study of copper ontogeny might provide insight into the cause of the disease. Gel filtration of the soluble supematard reveals sign&icant developmental differences in copper binding peaks.
Gel filtration of the Bmercaptoethanol solubilised particulate’ fraction (particulate supentatant) resolved dues peaks (Fig. 2b). The first peak eluted with the void volume, the second with MT and the third was of very low molecular weight (LMW; M, SM)[), VJV,, = 3). There was no peak mnesponding to the CuZo-SOD peak.
During the last 3 weeks of gestation there is a dramatic increast in the amount of copper associated with MT. A sirnils; increase has also been reported in rats and mice (18-U) The amount of copper asswiated with MT falls immediately after birth unlike in the rat where both liver copper actci MT continues to increase for upto 2 weeks
The developmental changes of the copper binding proteins in the particulate fraction are represented in Fig. 3b. By day.5.5of gestation, a meanof i.SflgCulgwet wt. liver was associated with MT, increasing to l8jzg Cd8 wet wt. at birth and then falling after birth. In fetal liver, the LMW peak accounted for a mean of 17.6/lg Cu/g wet M.
(22). Fewi hepatic MT increases in parallel with increasing liver copper cmtcentratians, but it remains unclear whether MT is induced by hepatic copper accumulation (II) oi *b&d the mpper kvc: bicraser as a consequence of MT synthesis (21). Our results suggest that in fetal liver, induction of MT is not simply a function of total
copper
142 liver copper concentration; for whilst liver copper coocentrations BE similar in the &day-old fetus and the Z&dayold adult, MT is the major hepatic copper binding protein in the 45.day-old fetus, whereas it is undetectable in 2& day-old animals. It is possible that liver copper sequestration in the fetus is secondary to induction of MT synthesis by glucocorticoids (21). It is htrtber possible that Zn may have a role in MT metabolism in the developing guinea pig as Z” is a potent inducer of the protein (ZZ), however Zo levels were not determined in the present stody. In adult mammals, hepatic soluble supemataot copper is associated almost exclusively with CoZo-SOD. This protein is believed to protect cells from free radical damage (13), and has been suggested also to wf as a storage
The nature of the void volume peak in both the soluble and particulate supematant remains unresolved. It may represent caeruloplasmin, polymer&d MT or en ““defined protein. As &-MT is not detectable in adult liver. it is unlikely to be derived from polymer&d MT. S&es have shown that in many tissues, including liver, newly absorbed copper appears rapidly in the void volume and subsequently transfers to other copper proteins (8), sug. gesting that the peek is ““t caeruloplasmin. ‘I%e peak might comprise more thas one pmtein and it still remains to be established whether the void volume peaks io the soluble and particulate soperoatents represent the st,me protein. It is dear from these studies that during develop
is not apparent. Despite major changes in liver copper concentration during development, CuZn-SOD activity does not change. Similar observations have been made in chicks and young rats (26,27). In wpper overload states, such as chronic cholestasis and erperimental copper overload, copper accmnulates preferentially in the particulate fraction where it is ass”ciated with MT (11). In the developing guinea pig, copper concentrations are highest at birth and at this time point, the particulate superoatant contains the greatest portion of copper which can be solubilised. I” addition to the MT copper peak, gel filtration of the particulate supernatent from fetal and young gumea pig liver reveals a low molec..
ment there are marked changes in both the compartmentalisation of hepatic copper end its association with copper binding proteins. The cause of hepatic copper accomulation in fetal liver remains unknown. It is possible that physiological cholestasis in the fetus is important, although the profoundly subnormal levels of caeruloplasmln in the neonate are quite unlike adult cholestasis where worn copper and cseruloplasmin levels are markedly increased (6). E+ study emphasises the dramatic changes in copper metabolism occurring at and around the time of birth. The switch from the fetal to adult mode of metabolism is asso ciated with significant changes in the expression of copper proteins. Genetic failure of this pwioatal adaptation might perpetuate the fetal mode of copper and caeroloplasmi” metabolism into later life. Liver mpper retentio” would persist after birth leading to unphysiological liver copper levels end liver damage with the clinical manifestations of Wilson’s disease presenting from the age of 6 years onwards. In addition to the similarities in copper
alar weight copper peak Mr
