75
Clinica Chimica Acta, 95 (1979) 75-82 @ Elsevier/North-Holland Biomedical Press
CCA 1030
VITAMIN
I.R.F.
D BINDING
BROWN
GLOBULIN
*, N.D. CARTER
PHENOTYPES
and ANITA
IN LIVER DISEASE
SOOD
Departments of Chemical Pathology and Child Health, St. George’s Hospital Medical School, Cranmer Terrace, London, SW1 7 (U.K.) (Received
December
19th,
1978)
Summary The binding properties towards vitamin D metabolites of plasma from indiviuals with the three common Gc-globulin phenotypes, Gc-1, Cc-2 and Gc-2-1, have been found to be identical. In patients with liver disease there is a good correlation between the levels of Gc-globulin and albumin in plasma. In addition the Gc-globulin levels correlate well with the ability of plasma to bind 25hydroxycholecalciferol. Patients with the Gc-2-1 phenotype showed a significantly smaller depression in plasma Gc-globulin than those with the Gc-2 and Gc-1 phenotypes. The relation of these findings to the pathogenesis of disorders of calcium metabolism in liver disease is discussed.
Introduction Group specific component protein (Gc-globulin) is an a*-globulin which was first detected in human serum almost 20 years ago [ 11. This protein has a well defined genetic heterogeneity [2]. There are two common codominant autosomal alleles, Gc’ and Gc2 and three phenotypes Gel-1, Gc2-1 and Gc2-2 are usually present in all populations. The recent realisation that Gc-globulin is identical with the vitamin D-binding gloulin of human serum [ 31 has stimulated studies on the role which variations in the circulating levels of this protein might play in the pathogenesis of disorders of calcium metabolism [4]. Gcglobulin is synthesised in the liver [5] and the suggestion that the Gc’ and the Gc2 genes predispose to the development of different types of liver disease [6] prompted us to examine the relationships between Gc-globulin levels, phenotypes and binding properties in patients with liver disease.
* To whom correspondence
should be addressed.
16
Materials and methods
Subjects. Heparinised blood samples were taken from 44 subjects with liver disease viz. 12 with active chronic hepatitis, 11 with alcoholic cirrhosis, 15 with primary biliary cirrhosis, 4 with biliary cirrhosis and large duct obstruction and 2 who were in acute liver failure. Some of these were part of a series published previously [ 71. Control samples were taken from healthy laboratory workers (n = 17, ages 18-45) none of whom were taking drugs (such as oestrogen preparations) which might affect the Gc-globulin levels. Gc-globulin. The levels of Gc-globulin in plasma were measured using M-Partigen immunodiffusion plates (Hoechst Pharmaceuticals) containing specific anti-Gc serum. Samples were diluted 1 : 2 with saline and diffusion allowed to proceed for 2 days at room temperature. The diameters of the precipitin rings formed were measured using a calibrated eye piece. The squares of the diameter values were related to that obtained from a known standard (standard human serum, stabilised, Hoechst Pharmaceuticals) applied under similar conditions. The genetic typing of the plasma samples was carried out by immunoelectrophoresis [ 21. Bindingofuitamin D metabolites. Aliquots of 25-hydroxy[26(27)-methyl-3H]cholecalciferol (Radiochemical Centre, Amersham; specific activity 9.0-11.8 Ci/mmol) dissolved in ethanol (0.04 ml containing 1.8-2.5 pmol) were pipetted out into polypropylene tubes. To each assay tube was added 0.4 ml of plasma diluted usually 1 : 1000 with phosphate buffer (0.05 mol/l, pH 7.6) and the solutions incubated for 2 h at 4°C. Non-specific binding was obtained by incubating buffer instead of diluted plasma. In experiments in which the displacement of the bound label was measured, aliquots of vitamin D metabolites in ethanol were pipetted into the reaction tubes and evaporated to dryness before the addition of the label. The metabolites tested were 25-hydroxycholecalciferol (25(OH)D3, O-10 pmol), 1,25-dihydroxycholecalciferol (1,25(OH)zD3, O-100 pmol), 24R,25dihydroxycholecaIciferol (24R,25(OH)zD3, O-10 pmol), vitamin D3 (O-1000 pmol) and vitamin Dz (O-1000 pmol). At the end of the incubation period, 0.2 ml of dextran-charcoal (1.25 g Norit GSX (Hopkin and Williams Ltd) and 0,125 g dextran (Koch-Light, mol. wt. 60 000-90 000) in 100 ml HzO) was added and after a further 20 min at 4”C, the tubes were centrifuged. 0.4 ml of supernatant was removed and the radioactivity counted in a modified Bray’s scintillation solution [ 81. In some experiments the results were analysed as Scatchard plots [9] and the association constants (KA) and capacities (C,,) calculated [lo]. Albumin. Plasma albumin in all samples was measured on an AutoAnalyser (Technicon Instruments Ltd.) by the bromocresol green (BCG) dye-binding method (Technicon Method AA11 No. 30). In view of the possible misinterpretation of the values obtained by this method in pathological sera [ 111, most of the samples from the patients with liver disease were also assayed by the Laurel1 “rocket” electrophoresis method using antiiuman albumin serum (Dako) [ 121. The immunochemical method gave results which were on average 1.7 g/l less than those from the BCG method. 8 patients had BCG albumins of less than 25 g/l, the level below which the BCG method is reported to become
inaccurate [ 111. However, the results in these patients also were not materially different from those obtained with the rocket method. Thus our indirection of the albumin levels does not appear to be affected by the methodology employed. Results
Binding of 25-(0H,)D3. The binding properties of samples of plasma from normal individu~s of known Gc-phenotype were studied in our standard assay system, The displacement of ‘H-25-(OH)D~ by the addition of increasing amounts of unlabelled 25-(OH)D3 was expressed as a Scatchard plot. In all cases, linear plots were obtained with identical gradients (Fig. 1). Thus the association constants, KA, of the three Gc phenotypes for 25-(OH)D3 are the same and have a value of 1.7 ? 0.2 (S.E.) X 10’ l/mol (n = 7) under the conditions which we used, The capacities of the three phenotypes for 25-(OH)D3 were also the same, with a mean value of 3.0 1 0.2 (S.E.) X 10V6 mol/l or 12.4 + 0.9 (SE.) X 10e6 mol/g Gc-globulin. Birding of other uita~~~ D ~etabo~~tes. The displacement of 3H-25-(OH)D3 from binding to each of the three normal Cc-phenotypes was tested using various vitamin D metabolites (Table I). All three phenotypes behaved similarly towards each compound. 24R,25-(OH)zD3 caused almost the same displacement as 25-(OH)D3 whereas much larger amounts of the other compounds were required for a comparable effect. me-globulin leuels and phenotypes. Fig. 2 shows the levels of Gc-globulin found in 17 normal individuals and in 37 of the patients with chronic liver disease. Although the numbers are small there is no evidence of any difference in the Gc-globuhn levels in normal individuals of different phenotype. The overall mean value in our control population was 0.292 f 0.033 g/l. The patients with
0
1
2
3
4
5
6
BOUND (nM) Fig. 1. Scatchard plots 0.2bindine of ZS_(OH)D~ to the three Gc-globulin phenotypes.
TABLE I DISPLACEMENT
OF LABELLED
Compound
25-(OH)D3
FROM THE 3 Gc-GLOBULIN
Amount added
PHENOTYPES
% displacement
(PrnOl)
25-(OH)D3 24R,25(OH)z 1,25-(OH)zDg Vitamin D3 Vitamin D2
3 3 300 300 300
D3
GC-1
Gc-2
Gc-2-1
49 44 21 57 65
49 44 24 55 60
49 41 25 58 69
chronic liver disease tended to have lower and in some cases markedly lower levels of Gc-globulin. The overall mean level was 0.224 + 0.060 g/l. However, when the results were grouped according to phenotype, the levels of Gc-globulin in patients with the Gc-2-1 phenotype (0.248 + 0.059 g/l) were clearly greater than the levels in both the Gc-1 (0.192 f 0.044 g/l, p < 0.01) and Gc-2 phenotypes (0.182 + 0.049 g/l, p < 0.02). The distribution of the phenotypes in the patients (10 Gc-1, 22 Gc-2-1 and 5 Gc-2) did not differ significantly from that 040
040
. . .
= 0.30
z
A
i
z
5 ii
r?
. .
s 0
0.20 :
0
0.10
0
I
I
1
2 2-1
I
1
I
1
2 2-l
I
LIMRDISEASE CONTROLS (n - 17) (n- 371 Fig. 2. Distribution of plasma Gc-globulin levels according to phenotype Gc-1 ta), Gc-2 (0) and Gc-2-l
W.
79
0.40
0.M
f 03 z _J ,’
0.20
2 ” :: 0.10
p
Clinica Chimica Acta, 95 (1979) 75-82 @ Elsevier/North-Holland Biomedical Press
CCA 1030
VITAMIN
I.R.F.
D BINDING
BROWN
GLOBULIN
*, N.D. CARTER
PHENOTYPES
and ANITA
IN LIVER DISEASE
SOOD
Departments of Chemical Pathology and Child Health, St. George’s Hospital Medical School, Cranmer Terrace, London, SW1 7 (U.K.) (Received
December
19th,
1978)
Summary The binding properties towards vitamin D metabolites of plasma from indiviuals with the three common Gc-globulin phenotypes, Gc-1, Cc-2 and Gc-2-1, have been found to be identical. In patients with liver disease there is a good correlation between the levels of Gc-globulin and albumin in plasma. In addition the Gc-globulin levels correlate well with the ability of plasma to bind 25hydroxycholecalciferol. Patients with the Gc-2-1 phenotype showed a significantly smaller depression in plasma Gc-globulin than those with the Gc-2 and Gc-1 phenotypes. The relation of these findings to the pathogenesis of disorders of calcium metabolism in liver disease is discussed.
Introduction Group specific component protein (Gc-globulin) is an a*-globulin which was first detected in human serum almost 20 years ago [ 11. This protein has a well defined genetic heterogeneity [2]. There are two common codominant autosomal alleles, Gc’ and Gc2 and three phenotypes Gel-1, Gc2-1 and Gc2-2 are usually present in all populations. The recent realisation that Gc-globulin is identical with the vitamin D-binding gloulin of human serum [ 31 has stimulated studies on the role which variations in the circulating levels of this protein might play in the pathogenesis of disorders of calcium metabolism [4]. Gcglobulin is synthesised in the liver [5] and the suggestion that the Gc’ and the Gc2 genes predispose to the development of different types of liver disease [6] prompted us to examine the relationships between Gc-globulin levels, phenotypes and binding properties in patients with liver disease.
* To whom correspondence
should be addressed.
16
Materials and methods
Subjects. Heparinised blood samples were taken from 44 subjects with liver disease viz. 12 with active chronic hepatitis, 11 with alcoholic cirrhosis, 15 with primary biliary cirrhosis, 4 with biliary cirrhosis and large duct obstruction and 2 who were in acute liver failure. Some of these were part of a series published previously [ 71. Control samples were taken from healthy laboratory workers (n = 17, ages 18-45) none of whom were taking drugs (such as oestrogen preparations) which might affect the Gc-globulin levels. Gc-globulin. The levels of Gc-globulin in plasma were measured using M-Partigen immunodiffusion plates (Hoechst Pharmaceuticals) containing specific anti-Gc serum. Samples were diluted 1 : 2 with saline and diffusion allowed to proceed for 2 days at room temperature. The diameters of the precipitin rings formed were measured using a calibrated eye piece. The squares of the diameter values were related to that obtained from a known standard (standard human serum, stabilised, Hoechst Pharmaceuticals) applied under similar conditions. The genetic typing of the plasma samples was carried out by immunoelectrophoresis [ 21. Bindingofuitamin D metabolites. Aliquots of 25-hydroxy[26(27)-methyl-3H]cholecalciferol (Radiochemical Centre, Amersham; specific activity 9.0-11.8 Ci/mmol) dissolved in ethanol (0.04 ml containing 1.8-2.5 pmol) were pipetted out into polypropylene tubes. To each assay tube was added 0.4 ml of plasma diluted usually 1 : 1000 with phosphate buffer (0.05 mol/l, pH 7.6) and the solutions incubated for 2 h at 4°C. Non-specific binding was obtained by incubating buffer instead of diluted plasma. In experiments in which the displacement of the bound label was measured, aliquots of vitamin D metabolites in ethanol were pipetted into the reaction tubes and evaporated to dryness before the addition of the label. The metabolites tested were 25-hydroxycholecalciferol (25(OH)D3, O-10 pmol), 1,25-dihydroxycholecalciferol (1,25(OH)zD3, O-100 pmol), 24R,25dihydroxycholecaIciferol (24R,25(OH)zD3, O-10 pmol), vitamin D3 (O-1000 pmol) and vitamin Dz (O-1000 pmol). At the end of the incubation period, 0.2 ml of dextran-charcoal (1.25 g Norit GSX (Hopkin and Williams Ltd) and 0,125 g dextran (Koch-Light, mol. wt. 60 000-90 000) in 100 ml HzO) was added and after a further 20 min at 4”C, the tubes were centrifuged. 0.4 ml of supernatant was removed and the radioactivity counted in a modified Bray’s scintillation solution [ 81. In some experiments the results were analysed as Scatchard plots [9] and the association constants (KA) and capacities (C,,) calculated [lo]. Albumin. Plasma albumin in all samples was measured on an AutoAnalyser (Technicon Instruments Ltd.) by the bromocresol green (BCG) dye-binding method (Technicon Method AA11 No. 30). In view of the possible misinterpretation of the values obtained by this method in pathological sera [ 111, most of the samples from the patients with liver disease were also assayed by the Laurel1 “rocket” electrophoresis method using antiiuman albumin serum (Dako) [ 121. The immunochemical method gave results which were on average 1.7 g/l less than those from the BCG method. 8 patients had BCG albumins of less than 25 g/l, the level below which the BCG method is reported to become
inaccurate [ 111. However, the results in these patients also were not materially different from those obtained with the rocket method. Thus our indirection of the albumin levels does not appear to be affected by the methodology employed. Results
Binding of 25-(0H,)D3. The binding properties of samples of plasma from normal individu~s of known Gc-phenotype were studied in our standard assay system, The displacement of ‘H-25-(OH)D~ by the addition of increasing amounts of unlabelled 25-(OH)D3 was expressed as a Scatchard plot. In all cases, linear plots were obtained with identical gradients (Fig. 1). Thus the association constants, KA, of the three Gc phenotypes for 25-(OH)D3 are the same and have a value of 1.7 ? 0.2 (S.E.) X 10’ l/mol (n = 7) under the conditions which we used, The capacities of the three phenotypes for 25-(OH)D3 were also the same, with a mean value of 3.0 1 0.2 (S.E.) X 10V6 mol/l or 12.4 + 0.9 (SE.) X 10e6 mol/g Gc-globulin. Birding of other uita~~~ D ~etabo~~tes. The displacement of 3H-25-(OH)D3 from binding to each of the three normal Cc-phenotypes was tested using various vitamin D metabolites (Table I). All three phenotypes behaved similarly towards each compound. 24R,25-(OH)zD3 caused almost the same displacement as 25-(OH)D3 whereas much larger amounts of the other compounds were required for a comparable effect. me-globulin leuels and phenotypes. Fig. 2 shows the levels of Gc-globulin found in 17 normal individuals and in 37 of the patients with chronic liver disease. Although the numbers are small there is no evidence of any difference in the Gc-globuhn levels in normal individuals of different phenotype. The overall mean value in our control population was 0.292 f 0.033 g/l. The patients with
0
1
2
3
4
5
6
BOUND (nM) Fig. 1. Scatchard plots 0.2bindine of ZS_(OH)D~ to the three Gc-globulin phenotypes.
TABLE I DISPLACEMENT
OF LABELLED
Compound
25-(OH)D3
FROM THE 3 Gc-GLOBULIN
Amount added
PHENOTYPES
% displacement
(PrnOl)
25-(OH)D3 24R,25(OH)z 1,25-(OH)zDg Vitamin D3 Vitamin D2
3 3 300 300 300
D3
GC-1
Gc-2
Gc-2-1
49 44 21 57 65
49 44 24 55 60
49 41 25 58 69
chronic liver disease tended to have lower and in some cases markedly lower levels of Gc-globulin. The overall mean level was 0.224 + 0.060 g/l. However, when the results were grouped according to phenotype, the levels of Gc-globulin in patients with the Gc-2-1 phenotype (0.248 + 0.059 g/l) were clearly greater than the levels in both the Gc-1 (0.192 f 0.044 g/l, p < 0.01) and Gc-2 phenotypes (0.182 + 0.049 g/l, p < 0.02). The distribution of the phenotypes in the patients (10 Gc-1, 22 Gc-2-1 and 5 Gc-2) did not differ significantly from that 040
040
. . .
= 0.30
z
A
i
z
5 ii
r?
. .
s 0
0.20 :
0
0.10
0
I
I
1
2 2-1
I
1
I
1
2 2-l
I
LIMRDISEASE CONTROLS (n - 17) (n- 371 Fig. 2. Distribution of plasma Gc-globulin levels according to phenotype Gc-1 ta), Gc-2 (0) and Gc-2-l
W.
79
0.40
0.M
f 03 z _J ,’
0.20
2 ” :: 0.10
p
