Clin. exp. Immunol. (1976) 25, 403-409.
Differences between plasma and serum complement in patients with chronic liver disease S. INAI, H. KITAMURA, T. FUJITA, J. KOJIMA & K. NAGAKI Section of Immunology and Liver Diseases, Center for Adult Diseases, Osaka, Japan (Received 3 May 1976)
SUMMARY
Of sixty patients with chronic liver disease, eight with cirrhosis or chronic hepatitis had very low serum CH50 but normal plasma CH50. The complement component profiles of these sera revealed markedly decreased C4 and C2 activities and normal C3T (C3-C9) activities. From these results, it is suggested that the early acting complement components had been nonspecifically activated during blood coagulation in these patients. No difference between plasma and serum CH50 was found in patients with hepatitis B(s) antigen.
INTRODUCTION Recently, Gewurz (1973) reported that the complement system in the blood of some patients was nonspecifically activated during coagulation, and that activation could be effectively prevented by the addition of EDTA or heparin to the blood. The haemolytic activities of complement (CH50) and the early acting complement components in these sera were markedly low while those in the plasma were normal. Glovsky & Alenty (1973) and Glovsky et al. (1974) reported similar cases. In a previous paper, we reported (Inai et al., 1967) that decreased CH50 had occasionally been observed in sera from patients with chronic hepatitis or liver cirrhosis. It was shown that the very low haemolytic activity of the fourth complement component (C4) was responsible for low CH50 in these sera. The present study concerns the difference between plasma and serum CH50 of patients with liver disease. It was found that serum CH50 in some patients with chronic liver diseases was very low although the plasma CH50 was within the normal range. A marked decrease in the fourth (C4) and the second (C2) complement component levels in these sera were confirmed. This was not observed in the patients with hepatitis B(s) antigen.
MATERIALS AND METHODS 1. Subjects. Nineteen healthy subjects were selected from doctors and technicians in our hospital. They had no histories related to liver disease. Their liver function tests were normal and they had no HB(s) antigen. Sixty patients with chronic liver disease were selected from patients admitted to the Hospital of the Center for Adult Diseases from December, 1973 to May, 1974. They consisted of twenty-three patients with chronic hepatitis (ten with chronic active hepatitis and thirteen with chronic inactive hepatitis), thirty with cirrhosis (five with alcoholic liver cirrhosis and twenty-five with nonalcoholic liver cirrhosis) and seven with hepatic cancer (four with primary hepatic cancer and three with metastatic hepatic cancer). Autoimmune hepatitis, haemochromatosis and cryptogenic cirrhosis were not included in these patients. The diagnosis in each case was confirmed by needle biopsy. Chronic hepatitis was classified into inactive and active forms by the histological findings. (i) Chronic inactive hepatitis: sinusoidal or periportal fibrosis is low grade and mononuclear cell infiltration is limited to portal area. (ii) Chronic active hepatitis: porto-to-porto or porto-to-central fibrosis is occasionally observed with the low grade of sinusoidal or periportal fibrosis. The limiting plates are invaded and sometimes destroyed by the infiltration of mononuclear cells and/or fibroblasts. Single hepatic cell necrosis and/or focal necrotic changes are also observed.
Correspondence: Dr S. Inai, Section of Immunology, The Center for Adult Diseases, Osaka, Higashinari-Ku, Osaka 537, Japan. D
403
S. Inai et al.
404
2. Separation of plasma and serum. Venous blood was obtained using a siliconized needle (0-8 x 38 mm) and a disposable plastic syringe. Two millilitres of freshly-drawn blood was put immediately into a plastic tube containing trisodium ethylenediamine tetra-acetic acid (EDTA 3Na) to a final concentration of 10 u/ml. After thorough mixing the tube was allowed to stand for 3 hr at room temperature (22 + 20C). EDTA-plasma or heparinized plasma was then separated by centrifugation at 2100 g for 10 min and divided into several aliquots, stored in plastic tubes at - 70OC until use. Another 2 ml of freshlydrawn blood was put immediately into a glass tube. The tube was kept for 3 hr at room temperature (22+ 20C) and centrifuged at 2100 g for 10 min. Serum obtained in this manner was stored at - 70'C until use. 3. Assay of the haemolytic activities of complement and its components and the determination of their protein concentrations. Haemolytic activity of complement was measured according to the method of Mayer (Mayer, 1961). The haemolytic activities of the complement components were estimated by the tube method. Cl, C4, C2 and C3-C9 (C3T) were measured using EAC4gp, EAClgp, EAClhu4hu or EAClhu4huoxy2hu as the intermediate cell, respectively. The details of the methods of these titrations and preparations of intermediate cells or complement reagents have been described previously (Borsos & Rapp, 1963; Inai et al., 1964; Nagaki, Tida & Inai, 1974a). The haemolytic activities of the complement components in serum or plasma were expressed as a percentage of those in the pooled normal human serum titrated simultaneously. The estimation of fIlC/lA and ,f1E levels in the serum or plasma was performed using immunodiffusion plates (Partigen, Behring Institute). The activity of the inactivator of the first complement component (ClINA) was assayed by the method of Gigli, Ruddy & Austen (1968). The protein concentration of CUINA was estimated by the single radial immunodiffusion method using monospecific anti-ClINA, and highly purified CIINA (Nagaki, Iida & Inai, 1974b) as a standard.
75 _(b)
75 (a)
50-0
U
0I 25-
25
0 Serum
Plasma Normal
0~~~~~~~~~~~~~~~~~ Serum
Plasma Patients (Group E)
Subjects FIG. 1. Plasma and serum CH50 values in normal subjects (a) and twelve patients in Group III (b). (E . Liver cirrhosis, (El. ) chronic hepatitis.
)
4. Hepatitis B(s) antigen and antibody. Hepatitis B(s) antigen (HB(s)Ag) was tested for by the immune adherence haemagglutination method (Mayumi, Okochi & Nishioka, 1971). Monospecific antiserum against HB(s)Ag was kindly given by Dr Kusuya Nishioka, National Cancer Institute, Tokyo, Japan. Hepatitis B(s) antibody (HB(s)Ab) was assayed by the passive haemagglutination method (Vyas & Shulman, 1970). HB(s)Ag-coated cells, prepared by the method of Imai et al. (1974), were kindly supplied by the Section of Immunological Research, the Division of Research and Development, Eisai Company Ltd. The specificity of HB(s)Ab was established by the haemagglutination-inhibition test using known HB(s)Agpositive serum.
RESULTS (1) Difference between plasma and serum CH5O of the patients with chronic liver diseases As a preliminary study, plasma and serum CH50 of nineteen healthy subjects were assayed. As shown in Fig. 1, the plasma and serum CH50 values were ranged from 37 to 56 units (u); the serum CH50 of each sample was equal to or slightly higher than CH50 of the corresponding plasma. Student's t-test for paired experiment (Snedecor & Cochran, 1967) revealed that serum showed a tendency to have higher CH50 values than corresponding plasma (t= 5 845, with 18 d.f., P< 0.001). Sixty patients with chronic liver diseases were classified into three groups according to the serum
Plasma and serum CH50 in liver disease
405
TABLE 1. Classification of patients according to the serum CH50 and to the difference between plasma CH50 and serum CH50*
No. of patients Group I Group II Group III
Chronic hepatitis Liver cirrhosis Liver cancer Total
23
20
0
3
30
17
4
9
7 60
6 43
1 5
0 12
*
See text.
CH50 and to the difference between the plasma and serum CH50 (Table 1). Group I; serum CH50> 30 u; plasma CH50 minus serum CH50 20 u. Plasma and serum CH50 of the twelve cases in Group III are shown in Fig. 1. As shown, three had chronic hepatitis and nine had liver cirrhosis. Remarkable differences between plasma and serum CH50 (> 20 u) were observed in eight patients in this group. The serum CH50 value of seven of these eight patients were very low although the plasma CH50 values were normal or slightly low. Plasma CH50 in one patient was very high while the serum CH50 was slightly low. As shown in Table 1, twenty out of twenty-three with chronic hepatitis and six out of seven with liver cancer were in Group I. Marked differences between plasma CH50 and serum CH50 were most frequently observed in the patients with cirrhosis. CH50 values of heparinized plasma of the patients in Group III were equal to those of EDTA-plasma.
(2) Complement component profiles in plasma and serum ofpatients in Group III Table 2 shows the complement component profiles of plasma and serum in eight patients whose serum CH50 values were lower than those of plasma CH50. The haemolytic activity of Cl in the sera of these patients was usually higher than that in plasma. The most characteristic findings in complement component profiles were in the C4 and C2 activities. The haemolytic activities of C4 and C2 in the plasma were normal or low, while these activities in serum were very low. In contrast to the markedly decreased levels of C4 activity in these sera, the protein concentrations of C4 (/IlE-globulin) in the sera were normal and nearly equal to that in plasma. Both serum and plasma C3T were within the normal range. No significant difference was found between C3 protein concentrations (f3lC/1A-globulin) in serum and in plasma. The protein concentrations of ClINA in plasma and sera of these patients were within the normal range. Plasma and serum CIINA activities of these patients were also within the normal range.
(3) Relationship between hepatitis B(s) antigen and complement patterns Twenty-seven patients were HB(s)Ag-positive and thirty-three were negative. Twenty-five of twentyseven with HB(s)Ag were in Group I. Only two patients with HB(s)Ag had decreased CH50 values in both plasma and serum (Group II). HB(s)Ag-positive patients were not found in Group III. The most striking finding was that abnormal complement patterns were observed in more than half of the patients with HB(s)Ag-negative cirrhosis (Table 3). HB(s)Ab was present in four of eight patients in Group III (Table 2).
406
S. Inai et al.
-4) 4 0
*;L,
Lf.-
.. -
:.-
.Z N
6 O
If
If)
If
I)
0
I0
e
00
-
e
e
e
m
I"
£q
c 0
en
e
-
-
N
-
-
N
N
+-
,0
-
-4
-N L)e
4
I~ ~~~~ I
E-
0-
.
0
._
0 N 0 N 1O - N-
0
0. 4)0 ._
-
+
C:0
%
+
N If)I
-bo
0
.
0
Q se 0
4._ 0
"0 l -
0s
>
>
L
-
4-
Ln
lb 0 lo,
4..
00
*
%.m--e %O _ ~-
bliC
-
0
44
cd
Z44
W
.
el
.t 0
-
"0 f
ut
-o 0 10 d 0.
N
N 00 l.-r- m
'.0t xc0%
0
-_
4)
N
-0 -00 N- If
-
N
00
O
N IO 0 N
ON
N- o N
0 N
4)
00
If
D 0
0 0
N
en%
Q)
=
*
--
d
en -4
u
4
0
)= . -
4)i 0
z
11.
NI4 en
4> ) o
0
0
U0
44 0%
N
f '
If) 00
N -
N
lZ
4% f
N 0 . NN N If)o o N £ If
b
oo: -Z
-lcs+ 'l el£ eq en en en
*
.
t 00
N 0% 'IO 0) e
e
N~
61. -
O
00 e
es
(q
e
oo N .-
.
1 -
X O -q -r4
004
-4
L
4-
H0 U
0 N
NT
00 en r- C1I en 00
"0 4
cn 0
0
0 0 0.
0-
--
)t
IU
0. 0
C
*
C4
0
4))
l oo
.
e;. "-
r- 6 o
C1
6 _F
6,
X
_
-
ob IX 0
4 4
0
N 0%
Lf If)
I- 0
1.9I?
"0
4)
g0
4
"0U
0.
"
4)
% 00 00 en O-
r
O4
1%
10
0
4)
Q
I..
0
0 0 ~
4)
".
0 0 c444
0 C) .n) 0) r0 -
4
0
.0
O
bo 4)
c
E0A.
0
0
"
U)4), rA M
0 w c
.0 -A
r.0
c
°4EC
o i,
00
£
C
g
¢*U z *u z *u ¢ *> z *>'U.0U
+
z
4
0
;
0
44 4) cn r. u
¢
z
Z
z
,
407
Plasma and serum CH5O in liver disease TABLE 3. Correlation between HB(s)Ag and the patient groups No. of patients Group I Group II Group III
HB(s)Ag Positive
Chronic hepatitis Liver cirrhosis Liver cancer
Total Negative Chronic hepatitis Liver cirrhosis Liver cancer Total
12
12
0
0
14
12
2
0
1 27
1 25
0 2
0 0
11
8
0
3
16
5
2
9
6 33
5 18
1 3
0 12
DISCUSSION Reduction of serum CH50 in patients with chronic liver disease has been recognized by many investigators including ourselves (Inai etal., 1967). In the present study, we have found markedly decreased serum CH50 in six of thirty patients with liver cirrhosis. However, five of the six patients had normal plasma CH50. Furthermore, the plasma CH50 in two patients with chronic hepatitis was normal though the serum CH50 was very low. Therefore, levels of serum CH50 and complement component levels in a patient with chronic liver disease does not always reflect activity in vivo. It is possible that cases having normal plasma CH50 may have been included among the patients reported to have low serum CH50. It is well known that the liver is the major site of synthesis of some complement components (Stecher & Thorbecke, 1967; Adenolfi, Gardner & Wood, 1968; Alper et al., 1969; Kaboth & Arnold, 1969; Littleton, Kessler & Burkholder, 1970; Rommel et al., 1970; Rubin et al., 1971; Colten; 1972; Torisu, Yokoyama & Kohler, 1972), and it has been postulated that the low serum complement values in some cases of chronic liver disease is due to defective synthesis of components (Fox, Dudley & Sherlock, 1971; Finalyson et al., 1972; Kourilsky, Leroy & Peltier, 1974). However, this does not always seem to be true. The serum CH50 of five patients in Group III was low in spite of the normal levels of fliC/lA, PIlE (Table 2). Therefore, synthesis of complement components may not be impaired in these patients. Consumption of complement during immune processes has been considered as one of the main causes of decreased serum complement levels in some liver diseases. It has been reported that the HB(s)Ag might be related to decreased levels of serum complement in patients with the arthritis of acute viral hepatitis (Alpert, Isselbacher & Schur, 1971; Onion, Crumpacker & Gilliand, 1971), and that patients with HB(s)Ag-positive chronic liver disease more frequently have low C3 protein (flIC/lA) than patients without HB(s)Ag (Grob, Jemelka & Muller, 1971). But Dudley, Fox & Sherlock (1971) considered that low C3 levels in massive hepatic necrosis were due to decreased liver synthesis rather than consumption by immune complexes, since C3 level was uniformly low independent of etioloty. HB(s)Ag and HB(s)Ab could not be found in four of eight patients in Group III (Table 2). Therefore, participation of HB(s)Ag-HB(s)Ab complexes in complement consumption in these patients was unlikely, though we could not absolutely exclude the presence of such complexes in the HB(s)Ab-positive cases. Also the presence of the other immune complexes could not be excluded in all cases in Group III. However, if immune complexes had consumed complement in vivo, both the plasma and the serum CH50 should be low.
408
S. Inai et ac'.
The liver also synthesizes some of the coagulation factors and synthesis is impaired in liver diseases. Nonspecific activation of the complement system can not be solely attributed to impaired coagulation, however, because the degree of impaired coagulation found in the patients in Group III was almost the same as that of the patients in Group I. It has been reported that complement in the serum obtained from a patient was non-specifically activated through the alternative pathway (Day et al., 1973). The activation of the complement system through the alternative pathway probably did not take place in our cases, as complement component profiles suggested that the complement system in these cases was activated through the classical pathway. There is the possibility that Cls in these patients may be activated by unknown mechanism(s) during blood coagulation or by the mechanism(s) independent of coagulation and inhibitable by EDTA. Further investigation to clarify these mechanisms is now in progress.
REFERENCES ADENOLFI, R., GARDNER, B. & WOOD, C.B.S. (1968) IMAI, M., GOTO, A., NiSHIOKA, K., KURASHINA, S., MIYAKAWA, & MAYUMI, M. (1974) Antigenicity of reduced Ontogenesis of two components of human complement: and alkylated Australia antigen. J. Immunol. 112, 416. flBE and filC-1A globulins. Nature (Lond.), 219, INAI, S., FUJIKAWA, K., TAKAHASHI, H. & NAGAKI, K. (1964) 189. Studies on the fourth component of complement. I. ALPER, C.A., JOHNSON, A.M., BIRTCH, A.G. & MooRE, F.D. Titration of the fourth component of complement in (1969) Human C'3: evidence for the liver as the primary human serum. Biken j. 6, 237. site of synthesis. Science, 163, 286. ALPERT, E., ISSELBACHER, K.J., & SCHUR, P.H. (1971) The INAI, S., FUJIKAWA, K., NAGAKI, K., TAKAHASHI, H., OZONO, N. & ISHIDA, S. (1967) Serum level of the fourth pathogenesis of arthritis associated with viral hepatitis: component of complement in various diseases. Biken 3. complement-component studies. New Engl. 7. Med. 285, 10, 65. 185. BORSOS, T. & RAPP, H.J. (1963) Chromatographic separation KABOTH, U. & ARNOLD, R. (1969) flIA-globulin bei Leberkranken. Verk. Deutsch. Gess. Inn. Med. 75, of the first component of complement and its assay on a 339. molecular basis. J. Immunol. 91, 851. COLTEN, H.R. (1972) Ontogeny of the human system: in KoURILSKY, O., LEROY, C. & PELTIER, A.P. (1974) Complement and liver cell function in 53 patients with liver vitro biosynthesis of individual complement components diseases. Amer. J. Med. 55, 783. by fetal tissues. ]. clin. Invest. 51, 725. DAY, N.K., MCLEAN, G.R., MICHAEL, J.R.A. & GOOD, R.A. LITTLETON, C., KESSLER, D. & BURKHOLDER, P.M. (1970) Cellular basis for synthesis of the fourth component of (1973) The association of respiratory infection, recurrent guinea-pig complement as determined by a hemolytic hematuria, and focal glomerulonephritis with activation of plaque technique. Immunology, 18, 693. the complement system in the cold. J. clin. Invest. 52, MAYER, M.M. Complement and complement fixation. 1968. (1961) Experimental Immunochemistry, 2nd edn. (ed. DUDLEY, E.F., Fox, R.A. & SHERLOCK, S. (1971) Relationby E.A. Kabat and M.M. Mayer), p. 133. Charles C. ship of hepatitis-associated antigen (H.A.A.) to acute and Thomas, Springfield, Illinois. chronic liver injury. Lancet, ii, 1. FINALYSON, N.D.C., KROHN, K., FAUCONETT, M.H. & MAYUMI, M., OKocHI, K. NISHIOKA, K. (1971) Detection of Australia antigen by means of immune adherence haemagANDERSON, K.E. (1972) Significance of serum complement glutination test. Vox Sang. (Basel), 20, 178. levels in chronic liver diseases. Gastroenterology, 63, NAGAKI, K., IIDA, K. & INAI, S. (1974a) A new method for the 653. preparation of EAC14 cell with human or guinea pig Fox, R.A., DUDLEY, F.J. & SHERLOCK, S. (1971) The serum serum. J. Immunol. Meth. 5, 307. concentration of the third component of complement NAGAKI, K., IIDA, K. & INAI, S. (1974b) The inactivator of the P8lC/lA in liver diseases. Gut, 12, 574. first component of human complement (C1INA). Int. GEWURZ, H. (1973) Nonimmune activation of C: two new Arch. Allergy, 46, 935. phenomena. ]. Immunol. 111, 305. GIGLI, I., RUDDY, S. & AUSTEN, K.F. (1968) The stoichio- ONION, D.K., CRUMPACKER, C.S. & GILLIAND, B.C. (1971) Arthritis of hepatitis associated with Australia antigen. metric measurement of the serum inhibitor of the first Ann. intern. Med. 75, 29. component of complement by inhibition of immune ROMMEL, F.A., GOLDLUST, M.B., BANCROFT, F.C., MAYER, hemolysis. ]. Immunol. 100, 1154. M.M. & TASHJIAN, A.H. (1970) Synthesis of the ninth GLOVSKY, M. & ALENTY, A. (1973) Coagulation related component of complement by a clonal strain of rat hepatdeficiencies of early component of C (Cl, C4, C2) in oma cells. J. Immunol. 105, 396. human serum. ]. Immunol. 111, 305. GLOVSKY, M., INAI, S., NAGAKI, K. & TEJIMA, H. (1974) RuBIN, D., BoRsos, T., RAPP, H.J. & COLTEN, H.R. (1971) Synthesis of the second component of guinea pig Perturbations in early components of complement (C1, complement in vitro. 3. Immunol. 106, 295. C4, C2) in man. Fed. Proc. 33, 647. GROB, P.J., JEMELKA, H.J. & MULLER, J.W. (1971) M1A and SNEDECOR, G.W. & COCHRAN, W.G. (1967) Statistical Methods. p. 91, Iowa State University Press, Ames, Iowa, SH antigen in chronic hepatitis. Gastroenterology, 61, U.S.A. 91.
Plasma and serum CH50 in liver disease STECHIER, V.J. & THORBECKE, G.J. (1967) Sites of synthesis of serum proteins. III. Production of PlC, filE and transferrin by primate and rodent cell lines. J. Immunol. 99, 660. TORISU, M., YOKOYAMA, T. & KOHLER, P.F. (1972) Serum
409
complement after orthoptic transplantation of the human liver. Clin. exp. Immunol. 12, 21. VYAS, G.N. & SHULMAN, N.R. (1970) Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science, 170, 332.
Differences between plasma and serum complement in patients with chronic liver disease S. INAI, H. KITAMURA, T. FUJITA, J. KOJIMA & K. NAGAKI Section of Immunology and Liver Diseases, Center for Adult Diseases, Osaka, Japan (Received 3 May 1976)
SUMMARY
Of sixty patients with chronic liver disease, eight with cirrhosis or chronic hepatitis had very low serum CH50 but normal plasma CH50. The complement component profiles of these sera revealed markedly decreased C4 and C2 activities and normal C3T (C3-C9) activities. From these results, it is suggested that the early acting complement components had been nonspecifically activated during blood coagulation in these patients. No difference between plasma and serum CH50 was found in patients with hepatitis B(s) antigen.
INTRODUCTION Recently, Gewurz (1973) reported that the complement system in the blood of some patients was nonspecifically activated during coagulation, and that activation could be effectively prevented by the addition of EDTA or heparin to the blood. The haemolytic activities of complement (CH50) and the early acting complement components in these sera were markedly low while those in the plasma were normal. Glovsky & Alenty (1973) and Glovsky et al. (1974) reported similar cases. In a previous paper, we reported (Inai et al., 1967) that decreased CH50 had occasionally been observed in sera from patients with chronic hepatitis or liver cirrhosis. It was shown that the very low haemolytic activity of the fourth complement component (C4) was responsible for low CH50 in these sera. The present study concerns the difference between plasma and serum CH50 of patients with liver disease. It was found that serum CH50 in some patients with chronic liver diseases was very low although the plasma CH50 was within the normal range. A marked decrease in the fourth (C4) and the second (C2) complement component levels in these sera were confirmed. This was not observed in the patients with hepatitis B(s) antigen.
MATERIALS AND METHODS 1. Subjects. Nineteen healthy subjects were selected from doctors and technicians in our hospital. They had no histories related to liver disease. Their liver function tests were normal and they had no HB(s) antigen. Sixty patients with chronic liver disease were selected from patients admitted to the Hospital of the Center for Adult Diseases from December, 1973 to May, 1974. They consisted of twenty-three patients with chronic hepatitis (ten with chronic active hepatitis and thirteen with chronic inactive hepatitis), thirty with cirrhosis (five with alcoholic liver cirrhosis and twenty-five with nonalcoholic liver cirrhosis) and seven with hepatic cancer (four with primary hepatic cancer and three with metastatic hepatic cancer). Autoimmune hepatitis, haemochromatosis and cryptogenic cirrhosis were not included in these patients. The diagnosis in each case was confirmed by needle biopsy. Chronic hepatitis was classified into inactive and active forms by the histological findings. (i) Chronic inactive hepatitis: sinusoidal or periportal fibrosis is low grade and mononuclear cell infiltration is limited to portal area. (ii) Chronic active hepatitis: porto-to-porto or porto-to-central fibrosis is occasionally observed with the low grade of sinusoidal or periportal fibrosis. The limiting plates are invaded and sometimes destroyed by the infiltration of mononuclear cells and/or fibroblasts. Single hepatic cell necrosis and/or focal necrotic changes are also observed.
Correspondence: Dr S. Inai, Section of Immunology, The Center for Adult Diseases, Osaka, Higashinari-Ku, Osaka 537, Japan. D
403
S. Inai et al.
404
2. Separation of plasma and serum. Venous blood was obtained using a siliconized needle (0-8 x 38 mm) and a disposable plastic syringe. Two millilitres of freshly-drawn blood was put immediately into a plastic tube containing trisodium ethylenediamine tetra-acetic acid (EDTA 3Na) to a final concentration of 10 u/ml. After thorough mixing the tube was allowed to stand for 3 hr at room temperature (22 + 20C). EDTA-plasma or heparinized plasma was then separated by centrifugation at 2100 g for 10 min and divided into several aliquots, stored in plastic tubes at - 70OC until use. Another 2 ml of freshlydrawn blood was put immediately into a glass tube. The tube was kept for 3 hr at room temperature (22+ 20C) and centrifuged at 2100 g for 10 min. Serum obtained in this manner was stored at - 70'C until use. 3. Assay of the haemolytic activities of complement and its components and the determination of their protein concentrations. Haemolytic activity of complement was measured according to the method of Mayer (Mayer, 1961). The haemolytic activities of the complement components were estimated by the tube method. Cl, C4, C2 and C3-C9 (C3T) were measured using EAC4gp, EAClgp, EAClhu4hu or EAClhu4huoxy2hu as the intermediate cell, respectively. The details of the methods of these titrations and preparations of intermediate cells or complement reagents have been described previously (Borsos & Rapp, 1963; Inai et al., 1964; Nagaki, Tida & Inai, 1974a). The haemolytic activities of the complement components in serum or plasma were expressed as a percentage of those in the pooled normal human serum titrated simultaneously. The estimation of fIlC/lA and ,f1E levels in the serum or plasma was performed using immunodiffusion plates (Partigen, Behring Institute). The activity of the inactivator of the first complement component (ClINA) was assayed by the method of Gigli, Ruddy & Austen (1968). The protein concentration of CUINA was estimated by the single radial immunodiffusion method using monospecific anti-ClINA, and highly purified CIINA (Nagaki, Iida & Inai, 1974b) as a standard.
75 _(b)
75 (a)
50-0
U
0I 25-
25
0 Serum
Plasma Normal
0~~~~~~~~~~~~~~~~~ Serum
Plasma Patients (Group E)
Subjects FIG. 1. Plasma and serum CH50 values in normal subjects (a) and twelve patients in Group III (b). (E . Liver cirrhosis, (El. ) chronic hepatitis.
)
4. Hepatitis B(s) antigen and antibody. Hepatitis B(s) antigen (HB(s)Ag) was tested for by the immune adherence haemagglutination method (Mayumi, Okochi & Nishioka, 1971). Monospecific antiserum against HB(s)Ag was kindly given by Dr Kusuya Nishioka, National Cancer Institute, Tokyo, Japan. Hepatitis B(s) antibody (HB(s)Ab) was assayed by the passive haemagglutination method (Vyas & Shulman, 1970). HB(s)Ag-coated cells, prepared by the method of Imai et al. (1974), were kindly supplied by the Section of Immunological Research, the Division of Research and Development, Eisai Company Ltd. The specificity of HB(s)Ab was established by the haemagglutination-inhibition test using known HB(s)Agpositive serum.
RESULTS (1) Difference between plasma and serum CH5O of the patients with chronic liver diseases As a preliminary study, plasma and serum CH50 of nineteen healthy subjects were assayed. As shown in Fig. 1, the plasma and serum CH50 values were ranged from 37 to 56 units (u); the serum CH50 of each sample was equal to or slightly higher than CH50 of the corresponding plasma. Student's t-test for paired experiment (Snedecor & Cochran, 1967) revealed that serum showed a tendency to have higher CH50 values than corresponding plasma (t= 5 845, with 18 d.f., P< 0.001). Sixty patients with chronic liver diseases were classified into three groups according to the serum
Plasma and serum CH50 in liver disease
405
TABLE 1. Classification of patients according to the serum CH50 and to the difference between plasma CH50 and serum CH50*
No. of patients Group I Group II Group III
Chronic hepatitis Liver cirrhosis Liver cancer Total
23
20
0
3
30
17
4
9
7 60
6 43
1 5
0 12
*
See text.
CH50 and to the difference between the plasma and serum CH50 (Table 1). Group I; serum CH50> 30 u; plasma CH50 minus serum CH50 20 u. Plasma and serum CH50 of the twelve cases in Group III are shown in Fig. 1. As shown, three had chronic hepatitis and nine had liver cirrhosis. Remarkable differences between plasma and serum CH50 (> 20 u) were observed in eight patients in this group. The serum CH50 value of seven of these eight patients were very low although the plasma CH50 values were normal or slightly low. Plasma CH50 in one patient was very high while the serum CH50 was slightly low. As shown in Table 1, twenty out of twenty-three with chronic hepatitis and six out of seven with liver cancer were in Group I. Marked differences between plasma CH50 and serum CH50 were most frequently observed in the patients with cirrhosis. CH50 values of heparinized plasma of the patients in Group III were equal to those of EDTA-plasma.
(2) Complement component profiles in plasma and serum ofpatients in Group III Table 2 shows the complement component profiles of plasma and serum in eight patients whose serum CH50 values were lower than those of plasma CH50. The haemolytic activity of Cl in the sera of these patients was usually higher than that in plasma. The most characteristic findings in complement component profiles were in the C4 and C2 activities. The haemolytic activities of C4 and C2 in the plasma were normal or low, while these activities in serum were very low. In contrast to the markedly decreased levels of C4 activity in these sera, the protein concentrations of C4 (/IlE-globulin) in the sera were normal and nearly equal to that in plasma. Both serum and plasma C3T were within the normal range. No significant difference was found between C3 protein concentrations (f3lC/1A-globulin) in serum and in plasma. The protein concentrations of ClINA in plasma and sera of these patients were within the normal range. Plasma and serum CIINA activities of these patients were also within the normal range.
(3) Relationship between hepatitis B(s) antigen and complement patterns Twenty-seven patients were HB(s)Ag-positive and thirty-three were negative. Twenty-five of twentyseven with HB(s)Ag were in Group I. Only two patients with HB(s)Ag had decreased CH50 values in both plasma and serum (Group II). HB(s)Ag-positive patients were not found in Group III. The most striking finding was that abnormal complement patterns were observed in more than half of the patients with HB(s)Ag-negative cirrhosis (Table 3). HB(s)Ab was present in four of eight patients in Group III (Table 2).
406
S. Inai et al.
-4) 4 0
*;L,
Lf.-
.. -
:.-
.Z N
6 O
If
If)
If
I)
0
I0
e
00
-
e
e
e
m
I"
£q
c 0
en
e
-
-
N
-
-
N
N
+-
,0
-
-4
-N L)e
4
I~ ~~~~ I
E-
0-
.
0
._
0 N 0 N 1O - N-
0
0. 4)0 ._
-
+
C:0
%
+
N If)I
-bo
0
.
0
Q se 0
4._ 0
"0 l -
0s
>
>
L
-
4-
Ln
lb 0 lo,
4..
00
*
%.m--e %O _ ~-
bliC
-
0
44
cd
Z44
W
.
el
.t 0
-
"0 f
ut
-o 0 10 d 0.
N
N 00 l.-r- m
'.0t xc0%
0
-_
4)
N
-0 -00 N- If
-
N
00
O
N IO 0 N
ON
N- o N
0 N
4)
00
If
D 0
0 0
N
en%
Q)
=
*
--
d
en -4
u
4
0
)= . -
4)i 0
z
11.
NI4 en
4> ) o
0
0
U0
44 0%
N
f '
If) 00
N -
N
lZ
4% f
N 0 . NN N If)o o N £ If
b
oo: -Z
-lcs+ 'l el£ eq en en en
*
.
t 00
N 0% 'IO 0) e
e
N~
61. -
O
00 e
es
(q
e
oo N .-
.
1 -
X O -q -r4
004
-4
L
4-
H0 U
0 N
NT
00 en r- C1I en 00
"0 4
cn 0
0
0 0 0.
0-
--
)t
IU
0. 0
C
*
C4
0
4))
l oo
.
e;. "-
r- 6 o
C1
6 _F
6,
X
_
-
ob IX 0
4 4
0
N 0%
Lf If)
I- 0
1.9I?
"0
4)
g0
4
"0U
0.
"
4)
% 00 00 en O-
r
O4
1%
10
0
4)
Q
I..
0
0 0 ~
4)
".
0 0 c444
0 C) .n) 0) r0 -
4
0
.0
O
bo 4)
c
E0A.
0
0
"
U)4), rA M
0 w c
.0 -A
r.0
c
°4EC
o i,
00
£
C
g
¢*U z *u z *u ¢ *> z *>'U.0U
+
z
4
0
;
0
44 4) cn r. u
¢
z
Z
z
,
407
Plasma and serum CH5O in liver disease TABLE 3. Correlation between HB(s)Ag and the patient groups No. of patients Group I Group II Group III
HB(s)Ag Positive
Chronic hepatitis Liver cirrhosis Liver cancer
Total Negative Chronic hepatitis Liver cirrhosis Liver cancer Total
12
12
0
0
14
12
2
0
1 27
1 25
0 2
0 0
11
8
0
3
16
5
2
9
6 33
5 18
1 3
0 12
DISCUSSION Reduction of serum CH50 in patients with chronic liver disease has been recognized by many investigators including ourselves (Inai etal., 1967). In the present study, we have found markedly decreased serum CH50 in six of thirty patients with liver cirrhosis. However, five of the six patients had normal plasma CH50. Furthermore, the plasma CH50 in two patients with chronic hepatitis was normal though the serum CH50 was very low. Therefore, levels of serum CH50 and complement component levels in a patient with chronic liver disease does not always reflect activity in vivo. It is possible that cases having normal plasma CH50 may have been included among the patients reported to have low serum CH50. It is well known that the liver is the major site of synthesis of some complement components (Stecher & Thorbecke, 1967; Adenolfi, Gardner & Wood, 1968; Alper et al., 1969; Kaboth & Arnold, 1969; Littleton, Kessler & Burkholder, 1970; Rommel et al., 1970; Rubin et al., 1971; Colten; 1972; Torisu, Yokoyama & Kohler, 1972), and it has been postulated that the low serum complement values in some cases of chronic liver disease is due to defective synthesis of components (Fox, Dudley & Sherlock, 1971; Finalyson et al., 1972; Kourilsky, Leroy & Peltier, 1974). However, this does not always seem to be true. The serum CH50 of five patients in Group III was low in spite of the normal levels of fliC/lA, PIlE (Table 2). Therefore, synthesis of complement components may not be impaired in these patients. Consumption of complement during immune processes has been considered as one of the main causes of decreased serum complement levels in some liver diseases. It has been reported that the HB(s)Ag might be related to decreased levels of serum complement in patients with the arthritis of acute viral hepatitis (Alpert, Isselbacher & Schur, 1971; Onion, Crumpacker & Gilliand, 1971), and that patients with HB(s)Ag-positive chronic liver disease more frequently have low C3 protein (flIC/lA) than patients without HB(s)Ag (Grob, Jemelka & Muller, 1971). But Dudley, Fox & Sherlock (1971) considered that low C3 levels in massive hepatic necrosis were due to decreased liver synthesis rather than consumption by immune complexes, since C3 level was uniformly low independent of etioloty. HB(s)Ag and HB(s)Ab could not be found in four of eight patients in Group III (Table 2). Therefore, participation of HB(s)Ag-HB(s)Ab complexes in complement consumption in these patients was unlikely, though we could not absolutely exclude the presence of such complexes in the HB(s)Ab-positive cases. Also the presence of the other immune complexes could not be excluded in all cases in Group III. However, if immune complexes had consumed complement in vivo, both the plasma and the serum CH50 should be low.
408
S. Inai et ac'.
The liver also synthesizes some of the coagulation factors and synthesis is impaired in liver diseases. Nonspecific activation of the complement system can not be solely attributed to impaired coagulation, however, because the degree of impaired coagulation found in the patients in Group III was almost the same as that of the patients in Group I. It has been reported that complement in the serum obtained from a patient was non-specifically activated through the alternative pathway (Day et al., 1973). The activation of the complement system through the alternative pathway probably did not take place in our cases, as complement component profiles suggested that the complement system in these cases was activated through the classical pathway. There is the possibility that Cls in these patients may be activated by unknown mechanism(s) during blood coagulation or by the mechanism(s) independent of coagulation and inhibitable by EDTA. Further investigation to clarify these mechanisms is now in progress.
REFERENCES ADENOLFI, R., GARDNER, B. & WOOD, C.B.S. (1968) IMAI, M., GOTO, A., NiSHIOKA, K., KURASHINA, S., MIYAKAWA, & MAYUMI, M. (1974) Antigenicity of reduced Ontogenesis of two components of human complement: and alkylated Australia antigen. J. Immunol. 112, 416. flBE and filC-1A globulins. Nature (Lond.), 219, INAI, S., FUJIKAWA, K., TAKAHASHI, H. & NAGAKI, K. (1964) 189. Studies on the fourth component of complement. I. ALPER, C.A., JOHNSON, A.M., BIRTCH, A.G. & MooRE, F.D. Titration of the fourth component of complement in (1969) Human C'3: evidence for the liver as the primary human serum. Biken j. 6, 237. site of synthesis. Science, 163, 286. ALPERT, E., ISSELBACHER, K.J., & SCHUR, P.H. (1971) The INAI, S., FUJIKAWA, K., NAGAKI, K., TAKAHASHI, H., OZONO, N. & ISHIDA, S. (1967) Serum level of the fourth pathogenesis of arthritis associated with viral hepatitis: component of complement in various diseases. Biken 3. complement-component studies. New Engl. 7. Med. 285, 10, 65. 185. BORSOS, T. & RAPP, H.J. (1963) Chromatographic separation KABOTH, U. & ARNOLD, R. (1969) flIA-globulin bei Leberkranken. Verk. Deutsch. Gess. Inn. Med. 75, of the first component of complement and its assay on a 339. molecular basis. J. Immunol. 91, 851. COLTEN, H.R. (1972) Ontogeny of the human system: in KoURILSKY, O., LEROY, C. & PELTIER, A.P. (1974) Complement and liver cell function in 53 patients with liver vitro biosynthesis of individual complement components diseases. Amer. J. Med. 55, 783. by fetal tissues. ]. clin. Invest. 51, 725. DAY, N.K., MCLEAN, G.R., MICHAEL, J.R.A. & GOOD, R.A. LITTLETON, C., KESSLER, D. & BURKHOLDER, P.M. (1970) Cellular basis for synthesis of the fourth component of (1973) The association of respiratory infection, recurrent guinea-pig complement as determined by a hemolytic hematuria, and focal glomerulonephritis with activation of plaque technique. Immunology, 18, 693. the complement system in the cold. J. clin. Invest. 52, MAYER, M.M. Complement and complement fixation. 1968. (1961) Experimental Immunochemistry, 2nd edn. (ed. DUDLEY, E.F., Fox, R.A. & SHERLOCK, S. (1971) Relationby E.A. Kabat and M.M. Mayer), p. 133. Charles C. ship of hepatitis-associated antigen (H.A.A.) to acute and Thomas, Springfield, Illinois. chronic liver injury. Lancet, ii, 1. FINALYSON, N.D.C., KROHN, K., FAUCONETT, M.H. & MAYUMI, M., OKocHI, K. NISHIOKA, K. (1971) Detection of Australia antigen by means of immune adherence haemagANDERSON, K.E. (1972) Significance of serum complement glutination test. Vox Sang. (Basel), 20, 178. levels in chronic liver diseases. Gastroenterology, 63, NAGAKI, K., IIDA, K. & INAI, S. (1974a) A new method for the 653. preparation of EAC14 cell with human or guinea pig Fox, R.A., DUDLEY, F.J. & SHERLOCK, S. (1971) The serum serum. J. Immunol. Meth. 5, 307. concentration of the third component of complement NAGAKI, K., IIDA, K. & INAI, S. (1974b) The inactivator of the P8lC/lA in liver diseases. Gut, 12, 574. first component of human complement (C1INA). Int. GEWURZ, H. (1973) Nonimmune activation of C: two new Arch. Allergy, 46, 935. phenomena. ]. Immunol. 111, 305. GIGLI, I., RUDDY, S. & AUSTEN, K.F. (1968) The stoichio- ONION, D.K., CRUMPACKER, C.S. & GILLIAND, B.C. (1971) Arthritis of hepatitis associated with Australia antigen. metric measurement of the serum inhibitor of the first Ann. intern. Med. 75, 29. component of complement by inhibition of immune ROMMEL, F.A., GOLDLUST, M.B., BANCROFT, F.C., MAYER, hemolysis. ]. Immunol. 100, 1154. M.M. & TASHJIAN, A.H. (1970) Synthesis of the ninth GLOVSKY, M. & ALENTY, A. (1973) Coagulation related component of complement by a clonal strain of rat hepatdeficiencies of early component of C (Cl, C4, C2) in oma cells. J. Immunol. 105, 396. human serum. ]. Immunol. 111, 305. GLOVSKY, M., INAI, S., NAGAKI, K. & TEJIMA, H. (1974) RuBIN, D., BoRsos, T., RAPP, H.J. & COLTEN, H.R. (1971) Synthesis of the second component of guinea pig Perturbations in early components of complement (C1, complement in vitro. 3. Immunol. 106, 295. C4, C2) in man. Fed. Proc. 33, 647. GROB, P.J., JEMELKA, H.J. & MULLER, J.W. (1971) M1A and SNEDECOR, G.W. & COCHRAN, W.G. (1967) Statistical Methods. p. 91, Iowa State University Press, Ames, Iowa, SH antigen in chronic hepatitis. Gastroenterology, 61, U.S.A. 91.
Plasma and serum CH50 in liver disease STECHIER, V.J. & THORBECKE, G.J. (1967) Sites of synthesis of serum proteins. III. Production of PlC, filE and transferrin by primate and rodent cell lines. J. Immunol. 99, 660. TORISU, M., YOKOYAMA, T. & KOHLER, P.F. (1972) Serum
409
complement after orthoptic transplantation of the human liver. Clin. exp. Immunol. 12, 21. VYAS, G.N. & SHULMAN, N.R. (1970) Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science, 170, 332.
