303
Clinica
Chimica
Acta,
68 (1976)
303-307
@ Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
CGA 7730
DIRECT MEASUREMENT IN LIVER DISEASE
OF SERUM NON-CAERULOPLASMIN
COPPER
D.J. FROMMER * Department
of Medicine,
Royal
Free
Hospital,
London
NW3 2QG
(U.K.)
(Received November 26,1975)
Summary The serum non-caeruloplasmin copper concentrations were measured in normal subjects and patients with various types of liver disease by a sensitive direct method involving complexing the copper and measurement by atomic absorption spectrophotometry. In normal subjects the mean concentration (? S.D.) was 10.1 + 1.6 pug/100 ml, males having a slightly higher value (10.7 f 1.3 c(g/lOO ml) than females (9.2 + 1.8 pg/lOO ml). In patients with various non-hepatic diseases concentrations were raised (15.8 + 8.9 pg/lOO ml), as also in hepatitis (14.7 + 4.3 pg/lOO ml), cholestasis (16.1 f 6.4 pg/lOO ml) and cirrhosis (16.3 + 8.7 pg/lOO ml). Heterozygotes for Wilson’s disease and patients treated for Wilson’s disease had concentrations (12.9 f 5.9 and 9.8 + 3.7 c(g/lOO ml, respectively) which did not differ significantly from normal whereas untreated patients had very significantly raised concentrations (22.9 + 4.5 pg/lOO ml). Direct measurement of serum non-caeruloplasmin copper is more accurate than indirect measurement and may help in assessing the effect of treatment but it is concluded that measurement of this fraction of serum copper will not enable Wilson’s disease to be differentiated from other forms of liver disease.
Introduction Caeruloplasmin in an cY,-globulin containing non-labile copper which is produced by the liver. It normally constitutes the major fraction of serum copper, the remaining fraction being loosely bound to albumin [l] and amino acids e.g. histidine [ 21. In Wilson’s disease (hepatolenticular degeneration) this serum non-caeruloplasmin copper concentration is known to be raised [3,4] and this * Present
address:
Department
of Medicine,
St. Vincent’s
Hospital.
Sydney,
N.S.W.
2010,
Australia.
304
is believed to be responsible for the deposition of copper in brain, kidney and other tissues in this disease. Other forms of liver disease are also known to be associated with alterations of total serum copper, and caeruloplasmin concentrations, hepatic copper content and urinary copper excretion. However in the majority of these diseases serum non-caeruloplasmin copper concentrations have not been estimated. Until recently serum non-caeruloplasmin copper was measured indirectly by measuring total serum copper and serum caeruloplasmin concentrations and subtracting the copper content of the caeruloplasmin from the total serum copper. Following the description of a direct method of measuring serum noncaeruloplasmin copper [5] a study of the serum non-caeruloplasmin copper concentration in a variety of patients with liver disease was undertaken. Materials and methods Subjects 87 subjects were studied. They consisted of: (a) 19 healthy normal subjects (M : F 8 : 11, 18-65 years). (b) 5 control patients. (M : F 3 : 2, 23-59 years). They suffered from a variety of non-hepatic conditions and all liver function tests including liver biopsy were normal. (c) 13 patients with cholestatic liver disease (M : F 8 : 7 13-67 years). Six had primary biliary cirrhosis, six had extrahepatic biliary obstruction and one had cholestasis of unknown origin. (d) 19 patients with hepatitis (M : F 9 : 10, 6-62 years) including viral hepatitis, active chronic hepatitis, alcoholic hepatitis and hepatitis of uncertain aetiology. (e) 5 patients with cirrhosis (M : F 3 : 2, 13-52 years). (f) 13 subjects who were heterozygotes for Wilson’s disease (M : F 7 : 6,1240 years). (g) 9 patients with Wilson’s disease (M : F 5 : 4, 7-36 years) who had been treated for between 2 and 21 years. (h) 4 newly diagnosed and untreated patients with Wilson’s disease (M : F 3 : 1, 17-22 years). One was asymptomatic, two had portal hypertension and one had mild neurological symptoms. Methods Serum non-caeruloplasmin copper concentrations were measured by the method of Blomfield and Macmahon [5], modified by using 2 g/l (instead of 1 g/l) solution of ammonium pyrrolidine dithiocarbamate. A Perkin-Elmer 403 double beam atomic absorption spectrophotometer was set at 0.4 mm slit width and X 10 scale expansion. Recorder deflection correlated very closely with concentration of copper in standard solutions. The coefficient of variation for 20 duplicate samples was 5.0% at mean value of 14.1 pg/lOO ml. All blood samples were collected into plastic tubes using plastic syringes. Samples were measured in duplicate and the mean value was taken. Serum non-caeruloplasmin copper was estimated indirectly by measuring serum copper by the method of Cumings [6] and subtracting from it the copper
305
. . l .
l
.
**
*a* .*
.
:.
.
4-
-!.
.
: l
$
0. t
t
l*t
I
-&-: .
= . . l
_I__--
Ch Fig. 1. Serum non-cae~loplasm~
He Copper
Ci
WH
-WT
wu
eoncentrati0nsin different groupsof patients. N, normal subjects;
C, control subjects; Ch, cholestatic liver disease patients; He, hepatitic liver disease patients; Ci, cirrhotic liver disease patients: WH, heterozygotes for Wilson’s disease; WT. treated Wilson’s disease patients; WU,
untreated Wilson’s disease patients.
90 t
74 f6Q t
l
EXPERIIIENTAL SNCC CClNCENTRATION ( w3ilOO ml
)
Fig. 2. Relationship between calculated and experimentally determined serum non-eaeruloplasmin copper concentrations. The regression line given by Y = 72.60s 6.55s. r = 0.4181, P < 0.001.
306
content of the serum caeruloplasmin. The latter was estimated by an enzymatic method [ 71, the normal range being 20-35 mg/lOO ml [8]. A value of 0.34% for the copper content of caeruloplasmin was used [ 91. Results (Fig. 1) In 22 normal subjects the mean (2 S.D.) serum non-caeruloplasmin copper concentration was 10.1 f 1.6 pg/lOO ml. Females showed a somewhat higher level (10.7 f 1.3 pug/100 ml) than males (9.2 + 1.8 pg/lOO ml) although this difference was not significant. Control patients had significantly (t = 2.7784, P < 0.02) raised concentrations (15.8 f 8.9 pg/lOO ml) compared to the normal mean of 10.1 pg/lOO ml. This was also true of levels in patients with hepatitis (14.7 + 4.3 pug/100 ml; t = 4.3670, P < O.OOl), with cholestasis (16.1 + 6.4 pg/lOO ml, t = 3.7890, P< 0.001) and with cirrhosis (16.3 + 8.7 pug/100 ml; t = 3.1017, P< 0.001). Heterozygotes for Wilson’s disease had mean concentrations (12.9 + 5.9 pg/ 100 ml) which did not differ significantly from normal subjects although eight heterozygotes had values which exceeded the mean f 2 S.D. of the normal subjects. The concentrations in treated patients with Wilson’s disease (9.8 f 3.7 pg/ 100 ml) did not differ significantly from normal subjects, whereas that in untreated patients (22.9 ? 4.5 pg/lOO ml) was very significantly raised (t = 10.1555, P < 0.001). Although there was a very significant correlation (r = 0.4181, P < 0.001) between the observed and calculated values for the whole group, in individual cases the agreement between the two values was relatively poor (Fig. 2). Discussion The normal values of serum non-caeruloplasmin concentrations were within a relatively narrow range (5.1-12.8 /-fug/100 ml) and the mean obtained was close to that of Blomfield and Macmahon viz. 9.1 pg/lOO ml. These normal values are slightly higher than that (4 pg/lOO ml) obtained by Gubler et al. [lo] who measured the slight increase in absorbance produced on adding the copper chelating agent, sodium diethyldithiocarbamate to serum. There have been no previous estimations of serum non-caeruloplasmin concentrations in subjects heterozygous for Wilson’s disease or in patients treated for the disease and this study shows that the concentrations are normal in both groups. In patients with untreated Wilson’s disease the concentrations were raised which confirms previous reports by Cartwright et al. [ 31 and Sass-Kortsak et al. [4]. Mild elevations were found in other forms of liver disease and suggest that estimation of only this fraction of serum copper would not be sufficient to differentiate Wilson’s disease from other causes of liver damage. However, it would probably be of value in assessing the effect of treatment. Raised concentrations have also been found in cirrhosis by Gubler et al. [ll], Leeson and Fourman [12] and Smallwood et al. [13] calculated serum non-caeruloplasmin copper levels in cases of primary bihary cirrhosis and found levels (of up to
307
116 pg/lOO ml) which were very much higher than in the present study. The calculated levels are likely to be less accurate than those obtained by direct estimation since calculated levels will be markedly influenced by small errors in the estimations of total serum copper or serum caeruloplasmin. Another source of error is the value for copper content assigned to caeruloplasmin since estimates have varied from 0.32% to 0.34% depending on the molecular weight assigned to the protein. Acknowledgements The author is grateful for the encouragement of Professor S. Sherlock and the financial support from the Trustees of the Will Edmond Fund Fellowship administered by the Royal College of Physicians of London. References 1 Beam. A. and Kunkel, H. (1954) Proc. Sot. Expt. Biol. Med. 85, 44-48 2 Neumann, P. and SassKortsak, A. (1963) VOX Sang. 8.111-112 3 Cartwright, G.. Hodges, R.. Gubler, C., Mahoney, J., Daum. K., Wintrobe. M. and Bean, W. (1954) J. CIin. Invest. 33.1487-1501 4 Sass-Kortsak, A., Cherniak. M.. Geiger, D. and Slater, R. (1959) J. Clin. Invest. 38. 16’72-1678 5 Blomfield. J. and Macmahon, R. (1969) J. Clin. Pathol. 22, 136-143 6 Cumings. J. (1948) Brain 71. 410-415 7 Houchin. 0. (1958) Clin. Chem. 4, 519-523 8 Cumings, J. (1968) in Handbook of Clinical Neurology (Vmken, P.J. and Bruyn. G.W., eds.) Vol. 6. p. 116. North Holland Publishing Co., Amsterdam 9 Holmberg, C. and Lame& C. (1948) Acta Chem. Scand. 2, 550-556 10 Gubler. C.. Lahey, M. Cartwright, G. and Wintrobe. M. (1953) J. Clin. Invest. 32,405-414 J. CIin. Invest. 11 Gubler, C.. Brown, H., Markowitz, H., Cartwright, G. and Wintrobe. M. (1957) 1208-1216 12 Leeson, P. and Fourman. P. (1967) Am. J. Med. 43,620-635 13 Smallwood, R.. McIlveen. B.. Rosenoer, V. and Sherlock, S. (1971) Gut 12.139-144
36,
Clinica
Chimica
Acta,
68 (1976)
303-307
@ Elsevier Scientific Publishing Company, Amsterdam -
Printed in The Netherlands
CGA 7730
DIRECT MEASUREMENT IN LIVER DISEASE
OF SERUM NON-CAERULOPLASMIN
COPPER
D.J. FROMMER * Department
of Medicine,
Royal
Free
Hospital,
London
NW3 2QG
(U.K.)
(Received November 26,1975)
Summary The serum non-caeruloplasmin copper concentrations were measured in normal subjects and patients with various types of liver disease by a sensitive direct method involving complexing the copper and measurement by atomic absorption spectrophotometry. In normal subjects the mean concentration (? S.D.) was 10.1 + 1.6 pug/100 ml, males having a slightly higher value (10.7 f 1.3 c(g/lOO ml) than females (9.2 + 1.8 pg/lOO ml). In patients with various non-hepatic diseases concentrations were raised (15.8 + 8.9 pg/lOO ml), as also in hepatitis (14.7 + 4.3 pg/lOO ml), cholestasis (16.1 f 6.4 pg/lOO ml) and cirrhosis (16.3 + 8.7 pg/lOO ml). Heterozygotes for Wilson’s disease and patients treated for Wilson’s disease had concentrations (12.9 f 5.9 and 9.8 + 3.7 c(g/lOO ml, respectively) which did not differ significantly from normal whereas untreated patients had very significantly raised concentrations (22.9 + 4.5 pg/lOO ml). Direct measurement of serum non-caeruloplasmin copper is more accurate than indirect measurement and may help in assessing the effect of treatment but it is concluded that measurement of this fraction of serum copper will not enable Wilson’s disease to be differentiated from other forms of liver disease.
Introduction Caeruloplasmin in an cY,-globulin containing non-labile copper which is produced by the liver. It normally constitutes the major fraction of serum copper, the remaining fraction being loosely bound to albumin [l] and amino acids e.g. histidine [ 21. In Wilson’s disease (hepatolenticular degeneration) this serum non-caeruloplasmin copper concentration is known to be raised [3,4] and this * Present
address:
Department
of Medicine,
St. Vincent’s
Hospital.
Sydney,
N.S.W.
2010,
Australia.
304
is believed to be responsible for the deposition of copper in brain, kidney and other tissues in this disease. Other forms of liver disease are also known to be associated with alterations of total serum copper, and caeruloplasmin concentrations, hepatic copper content and urinary copper excretion. However in the majority of these diseases serum non-caeruloplasmin copper concentrations have not been estimated. Until recently serum non-caeruloplasmin copper was measured indirectly by measuring total serum copper and serum caeruloplasmin concentrations and subtracting the copper content of the caeruloplasmin from the total serum copper. Following the description of a direct method of measuring serum noncaeruloplasmin copper [5] a study of the serum non-caeruloplasmin copper concentration in a variety of patients with liver disease was undertaken. Materials and methods Subjects 87 subjects were studied. They consisted of: (a) 19 healthy normal subjects (M : F 8 : 11, 18-65 years). (b) 5 control patients. (M : F 3 : 2, 23-59 years). They suffered from a variety of non-hepatic conditions and all liver function tests including liver biopsy were normal. (c) 13 patients with cholestatic liver disease (M : F 8 : 7 13-67 years). Six had primary biliary cirrhosis, six had extrahepatic biliary obstruction and one had cholestasis of unknown origin. (d) 19 patients with hepatitis (M : F 9 : 10, 6-62 years) including viral hepatitis, active chronic hepatitis, alcoholic hepatitis and hepatitis of uncertain aetiology. (e) 5 patients with cirrhosis (M : F 3 : 2, 13-52 years). (f) 13 subjects who were heterozygotes for Wilson’s disease (M : F 7 : 6,1240 years). (g) 9 patients with Wilson’s disease (M : F 5 : 4, 7-36 years) who had been treated for between 2 and 21 years. (h) 4 newly diagnosed and untreated patients with Wilson’s disease (M : F 3 : 1, 17-22 years). One was asymptomatic, two had portal hypertension and one had mild neurological symptoms. Methods Serum non-caeruloplasmin copper concentrations were measured by the method of Blomfield and Macmahon [5], modified by using 2 g/l (instead of 1 g/l) solution of ammonium pyrrolidine dithiocarbamate. A Perkin-Elmer 403 double beam atomic absorption spectrophotometer was set at 0.4 mm slit width and X 10 scale expansion. Recorder deflection correlated very closely with concentration of copper in standard solutions. The coefficient of variation for 20 duplicate samples was 5.0% at mean value of 14.1 pg/lOO ml. All blood samples were collected into plastic tubes using plastic syringes. Samples were measured in duplicate and the mean value was taken. Serum non-caeruloplasmin copper was estimated indirectly by measuring serum copper by the method of Cumings [6] and subtracting from it the copper
305
. . l .
l
.
**
*a* .*
.
:.
.
4-
-!.
.
: l
$
0. t
t
l*t
I
-&-: .
= . . l
_I__--
Ch Fig. 1. Serum non-cae~loplasm~
He Copper
Ci
WH
-WT
wu
eoncentrati0nsin different groupsof patients. N, normal subjects;
C, control subjects; Ch, cholestatic liver disease patients; He, hepatitic liver disease patients; Ci, cirrhotic liver disease patients: WH, heterozygotes for Wilson’s disease; WT. treated Wilson’s disease patients; WU,
untreated Wilson’s disease patients.
90 t
74 f6Q t
l
EXPERIIIENTAL SNCC CClNCENTRATION ( w3ilOO ml
)
Fig. 2. Relationship between calculated and experimentally determined serum non-eaeruloplasmin copper concentrations. The regression line given by Y = 72.60s 6.55s. r = 0.4181, P < 0.001.
306
content of the serum caeruloplasmin. The latter was estimated by an enzymatic method [ 71, the normal range being 20-35 mg/lOO ml [8]. A value of 0.34% for the copper content of caeruloplasmin was used [ 91. Results (Fig. 1) In 22 normal subjects the mean (2 S.D.) serum non-caeruloplasmin copper concentration was 10.1 f 1.6 pg/lOO ml. Females showed a somewhat higher level (10.7 f 1.3 pug/100 ml) than males (9.2 + 1.8 pg/lOO ml) although this difference was not significant. Control patients had significantly (t = 2.7784, P < 0.02) raised concentrations (15.8 f 8.9 pg/lOO ml) compared to the normal mean of 10.1 pg/lOO ml. This was also true of levels in patients with hepatitis (14.7 + 4.3 pug/100 ml; t = 4.3670, P < O.OOl), with cholestasis (16.1 + 6.4 pg/lOO ml, t = 3.7890, P< 0.001) and with cirrhosis (16.3 + 8.7 pug/100 ml; t = 3.1017, P< 0.001). Heterozygotes for Wilson’s disease had mean concentrations (12.9 + 5.9 pg/ 100 ml) which did not differ significantly from normal subjects although eight heterozygotes had values which exceeded the mean f 2 S.D. of the normal subjects. The concentrations in treated patients with Wilson’s disease (9.8 f 3.7 pg/ 100 ml) did not differ significantly from normal subjects, whereas that in untreated patients (22.9 ? 4.5 pg/lOO ml) was very significantly raised (t = 10.1555, P < 0.001). Although there was a very significant correlation (r = 0.4181, P < 0.001) between the observed and calculated values for the whole group, in individual cases the agreement between the two values was relatively poor (Fig. 2). Discussion The normal values of serum non-caeruloplasmin concentrations were within a relatively narrow range (5.1-12.8 /-fug/100 ml) and the mean obtained was close to that of Blomfield and Macmahon viz. 9.1 pg/lOO ml. These normal values are slightly higher than that (4 pg/lOO ml) obtained by Gubler et al. [lo] who measured the slight increase in absorbance produced on adding the copper chelating agent, sodium diethyldithiocarbamate to serum. There have been no previous estimations of serum non-caeruloplasmin concentrations in subjects heterozygous for Wilson’s disease or in patients treated for the disease and this study shows that the concentrations are normal in both groups. In patients with untreated Wilson’s disease the concentrations were raised which confirms previous reports by Cartwright et al. [ 31 and Sass-Kortsak et al. [4]. Mild elevations were found in other forms of liver disease and suggest that estimation of only this fraction of serum copper would not be sufficient to differentiate Wilson’s disease from other causes of liver damage. However, it would probably be of value in assessing the effect of treatment. Raised concentrations have also been found in cirrhosis by Gubler et al. [ll], Leeson and Fourman [12] and Smallwood et al. [13] calculated serum non-caeruloplasmin copper levels in cases of primary bihary cirrhosis and found levels (of up to
307
116 pg/lOO ml) which were very much higher than in the present study. The calculated levels are likely to be less accurate than those obtained by direct estimation since calculated levels will be markedly influenced by small errors in the estimations of total serum copper or serum caeruloplasmin. Another source of error is the value for copper content assigned to caeruloplasmin since estimates have varied from 0.32% to 0.34% depending on the molecular weight assigned to the protein. Acknowledgements The author is grateful for the encouragement of Professor S. Sherlock and the financial support from the Trustees of the Will Edmond Fund Fellowship administered by the Royal College of Physicians of London. References 1 Beam. A. and Kunkel, H. (1954) Proc. Sot. Expt. Biol. Med. 85, 44-48 2 Neumann, P. and SassKortsak, A. (1963) VOX Sang. 8.111-112 3 Cartwright, G.. Hodges, R.. Gubler, C., Mahoney, J., Daum. K., Wintrobe. M. and Bean, W. (1954) J. CIin. Invest. 33.1487-1501 4 Sass-Kortsak, A., Cherniak. M.. Geiger, D. and Slater, R. (1959) J. Clin. Invest. 38. 16’72-1678 5 Blomfield. J. and Macmahon, R. (1969) J. Clin. Pathol. 22, 136-143 6 Cumings. J. (1948) Brain 71. 410-415 7 Houchin. 0. (1958) Clin. Chem. 4, 519-523 8 Cumings, J. (1968) in Handbook of Clinical Neurology (Vmken, P.J. and Bruyn. G.W., eds.) Vol. 6. p. 116. North Holland Publishing Co., Amsterdam 9 Holmberg, C. and Lame& C. (1948) Acta Chem. Scand. 2, 550-556 10 Gubler. C.. Lahey, M. Cartwright, G. and Wintrobe. M. (1953) J. Clin. Invest. 32,405-414 J. CIin. Invest. 11 Gubler, C.. Brown, H., Markowitz, H., Cartwright, G. and Wintrobe. M. (1957) 1208-1216 12 Leeson, P. and Fourman. P. (1967) Am. J. Med. 43,620-635 13 Smallwood, R.. McIlveen. B.. Rosenoer, V. and Sherlock, S. (1971) Gut 12.139-144
36,
