Serum Immunoglobulin Concentrations in Patients with Alcoholic Liver Disease

GASTROENTEROLOGY

Vol. 57, No. I Printed in U.S.A .

Copyright © 1969 by The Williams & Wilkins Co.

SERUM IMMUNOGLOBULIN CONCENTRATIONS IN PATIENTS WITH ALCOHOLIC LIVER DISEASE I. DoDD WILSON, M .D., GERALD ONSTAD, M.D., AND RALPH C. WILLIAMS, JR., M.D. Department of M edicine, Univ ersity of Minnesota Medical School, Minneapolis, Minnesota

Measurement of the concentrations of lgG, lgA, lgM, and one of the subgroups of lgG, 'YG3, in the serum of 105 patients with alcoholism and liver disease of varying severity was performed by single diffusion against specific antisera in agar gel. The patients were divided into four groups on the basis of severity of liver disease. Mean serum immunoglobulin concentrations all increased progressively and significantly (P < 0.05) as the liver disease became more severe. Marked individual variation in the serum immunoglobulin concentrations was found in each group of patients. Analysis of the results demonstrated that much of the variation in the serum 'YG3 subgroup concentrations within each patient group was related to specific individual genetic differences in the Gm system. These results tend to support a hypothesis which suggests that the stimulus for increased immunoglobulin synthesis in patients with cirrhosis may be related to an alteration in unknown normal regulatory mechanisms influenced by the severity of the hepatic failure. Increased serum globulin concentrations are characteristic of cirrhosis of the liver. This hyperglobulinemia has been shown to be due primarily to an increase in the serum 'Y-globulin fraction, 1 which in turn, correlates significantly with a proliferation of the antibody-synthesizing cells in the bone marrow, 2 lymph nodes, spleen, 3 and liver itselC Both the rate of ')'-globulin synthesis5 • 6 and the serum immunoglobulin concentrations of the three major classes (lgA, lgG, and lgM) 7 ' 8 are

increased in patients with cirrhosis of the liver. Although some differences exist between the patterns of immunoglobulin alterations which are found, 9 hypergammaglobulinemia is characteristic of all types of cirrhosis.10 Thus, cirrhosis of the liver appears to be associated with a potent stimulus to the proliferation of the antibody-synthesizing cells resulting in the hypergammaglobulinemia that is found. Two hypotheses, either together or individually seem to be reasonable explanations for this increased immunoglobulin synthesis. The first suggests that the immunoglobulins, being antibody protein, result from an immunological reaction, presumably directed against antigens released or unmasked by liver injury. 4 Although a large body of evidence is accumulating which bears on this suggestion, no direct experimental confirmation has evolved. A second hypothesis, which we propose, suggests that some aspect of liver failure alters the . normal regulatory mechanisms

Received October 29, 1968. Accepted February 11, 1969. Address requests for reprints to: Dr. I. Dodd Wilson, Box 172, University Hospitals, 412 Union Street SE, Minneapolis, Minnesota 55455. . This study was supported by Research Grant 1N 13-H from the American Cancer Society, Grant 1R01-AM11803-01 from the United States Public Health Service, and grants from the Minnesota Chapter of the Arthritis Foundation and the University of Minnesota Graduate School. Dr. Wilson was supported by a Daland Fellowship from the American Philosophical Society. 59

60

WILSON ET AL.

for immunoglobulin synthesis. It seems to us that the finding of diffuse hypergammaglobulinemia in all major forms of cirrhosis favors this hypothesis. If an altered regulatory mechanism is responsible for these changes, the magnitude of the serum immunoglobulin alterations should be proportional to the severity of the hepatic failure and individual variation should be, at least in part, attributable to factors unrelated to the liver disease. As a preliminary test of this hypothesis, we undertook this study in an attempt to answer questions related to this phenomenon: (a) Are the· increases in serum immunoglobulin concentrations directly related to the severity of the hepatic failure? (b) Can individual variations in serum immunoglobulin concentrations between patients with cirrhosis of comparable severity be attributed to factors unrelated to the cirrhosis? Relevant to the second question, an analysis was made of the quantitative profile of immunoglobulin increase in conjunction with the Gm types or genetically determined lgG immunoglobulin classification of individual patients with varying degrees of liver disease. As a test of the first hypothesis, we studied the incidence and titers of two serum anti--y-globulin factors which would not reasonably be expected to arise from reactions directed against antigens released during liver injury. This report describes studies on sera from 105 patients with alcoholism and, in many instances, cirrhosis of the liver.

Glossary IgG, IgA, and lgM are the major classes of immunoglobulins. These are composed of heavy (H) and light (L) polypeptide chains. H-chains are heavy polypeptide chains which carry the distinct antigenic differences peculiar to the lgG, lgA, and lgM classes. The H-chains of lgG have been further divided into four distinct subgroups that are detectable with specific antisera. 11 "12 The accepted nomenclature for each, with previous designations in parentheses, is -yG1 <"Yzb or We), -yG2 (-yz. or Ne), -yG3 (-yzc or Vi), and -yG4 (-yzd or Ge).1 3 £-chains are light polypeptide chains. These

Vol. 57, No . I

can be divided into two groups, kappa and lambda, on the basis of antigenic differences.< Gm type refers to a group of genetically determined antigenic differences detectable on the H-chains of lgG. The factors studied were Gm(a) and Gm(f) which are found on the -yGl H-chain, Gm(n) found on -yG2, and Gm(b) found on -yG3. 15-1; In Caucasians, these factors tend to be inherited as groups in a Mendelian codominant fashion with Gm(b), Gm(f) , and Gm(n) forming part of one genetic group while Gm(a) is inherited as part of an allelic gene group. 17-19 M-component is a homogeneous electrophoretically narrow band of immunoglobulin possessing only one type of light chains, usually present in high concentrations, which is detected on serum protein electrophoresis.

Materials and Methods One hundred five patients with alcoholism who were hospitalized at either the University of Minnesota Hospitals or the Hennepin County General Hospital participated in the study. These patients were divided into four groups on the basis of the following criteria : Patient group I: Alcoholism alone. Findings of liver disease were not present on physical examination. Patient group II: Liver disease of mild to moderate severity. Hepatomegaly, other physical stigmata of cirrhosis, or both were found but ascites and hepatic encephalopathy were not present. Patient group III: Advanced alcoholic cirrhosis. In addition to the criteria for group II, either ascites or hepatic precoma had been present within the previous 6 months. Patient group IV: Death from liver failure. Blood samples were obtained from each subject and the sera were separated. The sera were preserved with sodium azide and stored either at 4 C or frozen at -10 C until used. Serum concentrations of lgA, lgG, lgM, and the -yG3 subgroup of lgG were determined by single diffusion in agar gel using the Oudin tube method of Huntley.20 Pooled normal sera were used to standardize immunoglobulin class measurements while myeloma proteins served as standards for -yG3 subgroup quantitations. Normal mean serum concentrations with one standard deviation in this laboratory are: lgG, 1278 ± 361 mg per 100 ml; lgA, 282 ± 128 mg per 100 ml; and lgM, 110 ± 49 mg per 100 ml. 21 Scarcity of the -yG3 subgroup antisera has prevented us from determining these mean values in a normal population.

July 1969

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ALCOHOLIC LIVER DISEASE

Specific antisera (Hyland Laboratories) were used for quantitation of serum lgA, lgG, and lgM. Antisera against rG3 were obtained by immunization of rabbits with preparations of rG3 myeloma proteins in Freund's adjuvant. Antisera made against Gm(b+) and Gm(g+) rG3 myeloma proteins produced similar quantitative results. Serum protein electrophoresis on cellulose acetate was performed on samples from all of the patients in the study. 22 When serum M-components were found, immunoelectrophoresis was used for further characterization. 23 The hereditary factors (a), (b), and (0 of the Gm system were determined by standard inhibition of hemagglutination techniques. 19 Reagents for Gm(a) and Gm(f) typing were kindly supplied by Dr. S. D. Litwin and Dr. H. G. Kunkel. Gm(b) was tested by reagents obtained from Hoechst Pharmaceuticals, Cincinnati, Ohio. Kunkel et al. have described a technique which uses double diffusion in agar 17 gel for Gm(n) typing. A modification of this technique, using inhibition of hemagglutination, was used to determine the Gm(n) type of the sera from our patients (performed with the aid of Dr. William J. Yount, The Rockefeller University, New York, N. Y.). Sera from all patients were studied for antir-globulin factors . Human serum Ripley containing incomplete IgG anti-CD antibodies was used to coat Rh-positive cells. 24 Pepsin-digested incomplete anti-Rh antibody from human serum Ripley was also used to coat Rh positive red blood cells. 21 ' 25 Sera were arbitrarily classified as negative in the hemagglutination reaction with red blood cells coated with whole serum and continued in doubling duced no reaction. Since the concentrations of the anti-r-globulin factor against the site revealed by pepsin digestion in the various sera appear to represent a continuous spectrum, assays for this anti-pepsin site factor were begun with whole serum and continued in doubling dilutions. Uniformity of red cell coating antibodies was monitored by use of dilutions of Coombs serum and known positive and negative sera. The Pearson Product-Moment correlation coefficient (r) was used to measure intercorrelation (see reference 26, p. 78-90). Linear regression coefficients were calculated by the method of least squares (see reference 26, p. 95-99). Differences between the means of more than two groups were tested either by an analysis of variance27 or by x2.28 Since the pa-

tient groups studied may not represent a " normal" population, a nonparametric test, the Kruskal-Wallis one-way analysis of variance by ranks, was used to analyze statistical data rather than a parametric test of the analysis of variance. The 0.05 significance level was selected to evaluate statistical significance.

Results Patient groups. Seven of the 105 patients studied were excluded from the statistical tabulations for one of the following reasons: selective serum IgA deficiency,29 hepatoma, or the presence of well defined M-components on serum protein electrophoresis. The distribution of the remaining 98 patients within the four groups as well as the sex ratio and the mean age of each group are presented in table 1. Serum immunoglobulin concentrations. Mean values, standard deviation, and standard error of the mean of the immunoglobulin concentrations of each patient group are shown in table 2. The individual serum immunoglobulin concentrations of IgA, IgG, IgM, and the ,G3 subgroup of lgG in each group of alcoholic patients are demonstrated in figure 1. By analysis of variance, mean values for all of the classes and subgroups studied increased significantly with increasing severity of liver failure. In all instances, there were increases in absolute mean serum concentrations between groups in relation to increasing severity of liver failure. Gm factors. The results of typing studies for Gm factors in the patient sera are shown in table 3. No differences were found in the relative frequency of any of the Gm factors within the four patient groups. ,G3 subgroup concentration and Gm TABLE

1. Number , sex di stri bution, and m ean age i n each of the patien t grou ps

Patient group

Total

Males

Females

Mean age

I II III IV

27 30 26 15

23 23 15

4

11

11

4

56.4 56 .0 52.0 55 .4

7

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WILSON F;T AL.

type. The serum concentrations of the 'YG3 subgroup of lgG were analyzed in relation to the Gm type of this subgroup heavy chain and the total serum lgG concentration. For each patient the concentration of 'YG3 was converted to a percentage of the total serum lgG. A highly significant relationship was found between the Gm type and the mean percentage that the 'YG3 subgroup represented of the total lgG (table 4). This is further reflected in figure 2 where regression lines for the 'YG3 subgroup concentrations in relation to IgG for each Gm type are plotted with 2. S erum immunoglobulin concentrations in patients with alcoholism and liver disease of varying severity

TABLE

Group Immunoglobulin class I

II

III

-- --IgAa Mean (mg per 100 ml) . .. No . . .... . .

557.0 30 283 . 1 51.7

804.8 1260 26 15 306.8 580.4 60.2 150.4

123.7 27 . .. 78.1 . . .... . 15 .0

175 .3 30 117 .5 21.4

221.8 26 88.8 17.4

.. . ...... ..

lgMa Mean (mg per 100 ml). No . . . . . . .. . SD . . SEM . . .

. .

IgGa Mean (mg per 100 ml) .. ..... 1280 No . . 27 SD . . . . . . . . . . . . . . 380 SEM .. . . . . . 73 "YG3 Subgroup of lgGa Mean (mg per 100 ml) . . . No .. .... SD ..

. . . . . ..

SEM ..

IV

--

423.4 27 148.5 28.6

SD. .... ... . . . SEM.

-

.. ..

181 26 101 20

284 .9 15 90 .5 23.4

1644 30 527 96

2162 26 545 107

2601 15 741 192

201 30 108 20

226 26 120 24

316 14 149 40

a The four groups were found to be from more than one population at a P value of <0.001.

Vol. 57, No. 1

their correlation coefficients. Gm(b +) patients had higher serum concentrations than did those who were Gm(b-). Serum concentrations of the 'YG3 subgroup were also higher in patients who were homozygous for the Gm(b) factor than in heterozygotes. This relationship between Gm type and serum 'YG3 concentration was found in each of the four groups of patients. Anti-1'-globulin factors . Serum agglutinator activity for the site on /'-globulin revealed by pepsin digestion at pH 4.1, as expressed both by titer and by the number of positive reactions, increased as liver disease became more severe (P < 0.01, table 5). Neither mean serum agglutinator activity nor the frequency of positive reactions directed against whole incomplete lgG anti-CD antibodies from patient Ripley increased in relation to the severity of hepatic failure (table 5). Serum M-components. Three patients were found to have electrophoretically distinct narrow bands in the beta or gamma areas on serum protein electrophoresis. One patient had two distinct IgG M-components whereas the other 2 had single lgG and lgM M-components, respectively. No clinical evidence for multiple myeloma or Waldenstrom's macroglobulinemia was found. Two of these patients had alcoholic cirrhosis while the third had alcoholism alone.

Discussion All of the mean serum concentrations of the immunoglobulin classes and the lgG subgroups measured increased in patients with alcoholic cirrhosis of the liver. Our data have confirmed previously described increases in mean serum immunoglobulin class and subgroup concentrations in alcoholic cirrhosis and have extended these results to a larger population. A direct relationship between immunoglobulin concentrations and the severity of the hepatic failure was demonstrated. Much of the individual variation in serum concentrations of 'YG3, when related to the severity of liver disease, could be explained by dif-

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FIG . 1. Serum immunoglobulin concentrations in the patient groups: serum lgG (top left) , serum IgA (top represents the value of each individual serum ; right), serum lgM (bottom left), serum -yG3 (bottom right). vertical bar represents the mean ± 2 SE. Mean serum immunoglobulin concentrations rose significantly as

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64

WILSON ET AL. TABLE

3. Gm typing within patient groups

Patient group

Gm(a+)

Grh(a-)

Gm(b+)

I II III IV

14 16 14 6

12 13 12 9

21

Gm(b-)

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4. Effect of Gm type on mean -yG3 subgroup percentage of total I gGa a+, b+

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Vol. 57, No. I

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5. Anti--y-globulin factors in patients with alcoholism and liver disease of varying severity

TABLE

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Patient group

< 0.001. I

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Anti-pepsin digested Ripley

15 23 15 7

37 63 77 87

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some unknown way by the cirrhosis. We emphasize that these findings neither ex0 4000 2000 3000 1000 clude other possibilities which might exlgG - mg% plain this hypergammaglobulinemia nor FIG. 2. Correlation coefficients and regression lines imply that the mechanisms are unique for of -yG3 related to lgG in the alcoholic patients divided patients with cirrhosis. Rather they are according to Gm type. taken only as support for the plausibility of our hypothesis. Such altered control ferences in genetic factors as measured in mechanisms might include: (a) failure of the Gm system. Thus, the capacity of the liver to synthesize a sufficient quanthese patients to increase their immuno- tity of a normal inhibitor, such as albuglobulin concentrations in response to min'1 0 :1:1; (b) hepatic release of a trophic their liver failure was at least in part ge- substance, possibly during active liver netically determined. Finally, a highly cell injury 14; (c) failure of the liver to significant increase in the incidence and metabolize an immunotrophic substance titer, related to the severity of liver dis- produced elsewhere in the body. All of ease, was found for agglutinating anti- these possibilities can be tested experibodies against the site on lgG revealed by mentally. Marked individual variation of the digestion with pepsin at pH 4.1. This finding demonstrated that one form of anti- serum immunoglobulin class and lgG subbody, presumably not directed primarily group concentrations was found within the against liver antigen, increased in propor- patient groups. This suggests that either the classification system was inaccurate or tion to the severity of liver failure . These data are compatible with the the response to the cirrhosis was not unihypothesis that one or more regulatory form . An objective, accurate classification mechanisms broadly influencing all serum system for the severity of alcoholic cirimmunoglobulins are directly altered in rhosis does not exist . Liver function tests, 100

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July 1969

65

ALCOHOLIC LIVER DISEASE

although helpful, are not an infallible guide to the presence, severity, or the type of liver disease. 35 ' 3 6 For these reasons, we chose a simple but arbitrary classification based on objective physical and historical findings. The upward progression of the mean class and 'YG3 subgroup serum concentrations in relation to increasing severity of liver disease, as judged by our classification, seems to vindicate our approach. Also, the same relative individual variation is seen in normal populations as well as in our group IV where objectivity was complete. An analysis of the 'YG3 subgroup concentration in relation to the Gm(b) factor was performed to test the possibility that hereditary influences upon serum immunoglobulin concentrations were important in producing the variable results found within each patient group. Gm(b) is found solely on the heavy chain of the 'YG3 subgroups of IgG. Recently, Yount et al.'1' have demonstrated that Gm(b) individuals have higher mean serum 'YG3 concentrations than Gm(b - ) subjects. Since the range of serum IgG concentrations is greater in alcoholic patients, our subjects also represented an ideal group in which to extend and confirm these observations. This greater range of lgG values accentuated the differences found between the Gm groups. Gm(b+) patients were found to have increased 'YG3 concentrations. Serum concentrations of the 'YG3 subgroups were also higher in patients who were homozygous for the Gm(b) factor than in heterozygotes. These data support the hypothesis, at least for the r·G3 subgroup, that much of the variation which occurred within each patient group may be related to genetic factors. In general, these results confirm and extend previous work related to quantitation of IgG subgroups.37 No change in the frequency of positive hemagglutination reactions against 0 positiv~ red blood cells coated with whole Ripley anti-CD IgG was found. This is surprising in view of the reported increased frequency of positive latex fixation tests in cirrhosis38 - 41 and the high correlation

between this hemagglutination reaction using serum Ripley and the latex fixation test. 42 Other attempts of demonstrate increases in specific antibody production in cirrhosis have also provided contradictory results. 43 ' 44 REFERENCES 1. Gray, S. J ., and E. S. G. Barrow. 1943. The electrophoretic analyses of the serum proteins in diseases of the liver. J. Clin. Invest . 22: 191200. 2. Jarrold, T. , and R. W. Vilter. 1949. Hematologic observations in patients with chronic hepatic insufficiency. J . Clin. Invest. 28: 286-292. 3. Glagov, S., G. Kent, and H. Popper. 1959. Relation of splenic and lymph node changes to hypergammaglobulinemia in cirrhosis. Arch. Path. (Chicago) 67: 9- 18. 4. Paronetto, F., E. Rubin, and H . Popper. 1962. Local formation of -y-globulin in the diseased liver, and its relation to hepatic necrosis. Lab. Invest. 11: 150- 158. 5. Havens, W. P. , Jr. , J . Dickensheets, J. N. Bierly, and T . P. Eberhard . 1954. The half-life of !'" labeled normal human gamma globulin in patients with hepatic cirrhosis. J. Immun . 73: 256-258. 6. Cohen, S. 1963. -y-Globulin metabolism . Brit. Med . Bull. 19: 202- 206. 7. Tomasi, T. B., Jr., and W. A. Tisdale. 1964. Serum gamma-globulins in acute and chronic liver diseases. Nature (London) 201: 834-835 . 8. Lee, F. I. 1965. Immunoglobulins in viral hepatitis and active alcoholic liver disease . Lancet 2: 1043- 1046. 9. Bevan, G. , W. P. Baldus, and G. ,J. Gleich. 1968. Serum immunoglobulin (Ig) levels in the differential diagnosis of cholestasis . Gastroenterology 54: 151. 10. Feizi , T. 1968. Immunoglobulins in chronic liver disease. Gut 9: 193-198. 11. Grey, H . M., and H. G. Kunkel. 1964 . H chain subgroups of myeloma proteins and normal 7S -y-globulin . J. Exp. M ed . 120: 253- 266. 12. Terry, W. D. , and J. L. Fa hey. 1964. Subclasses of human "12-globulin based on differences in the heavy peptide chains. Science 146: 400401. 13. Fahey, J . L. , E. C. Franklin, H. G. Kunkel, E. F. Osserman, and W. D. Terry. 1967. Notation for human immunoglobulin subclasses. J. Immun. 99: 465. 14. Mannik, M ., and H. G. Kunkel. 1963. Two major types of normal 7S "!-globulin. J. Exp. M ed. 117: 213-230.

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15. Mll.rtensson, L. , and H . G. Kunkel. 1965. Distribution among the -y-globulin molecules of different genetically determined antigenic specificities in the Gm system. J. Exp. M ed . 122: 799- 811. 16. Terry, W. D., J . L. Fahey, and A. G. Steinberg. 1965. Gm and Inv factor in subclasses of human IgG. J. Exp . Med. 122: 1087-1102. 17. Kunkel, H . G. , W. J. Yount, and S. D. Litwin. 1966. Genetically determined antigen of the Ne subgroup of gamma-globulin : detection by precipitin analysis. Science 154: 1041-1043. 18. Fudenberg, H., and E. C. Franklin . 1963. Human gamma globulin. Genetic control and its relation to disease . Ann. Intern. M ed. 58: 171180. 19. Martensson, L. 1964. On the relationships between the -y-globulin genes of the Gm system . A study of the Gm gene products in sera, myeloma globulins, and specific antib odies with special reference to the gene Gm f. J . Exp. M ed. 120: 1169- 1188. 20. Huntley, C. C. 1963. Simple gel diffusion micromethod for gamma-globulin determinat ion. Peqiatrics 31: 123-129. 21. Wilson, I. D. , R. C. Williams, Jr., and L. Tobian, Jr. 1967. Renal tubular acidosis. Three cases with immunoglobulin abnormalities m the patients and their kindreds. Amer. J. M ed. 43: 356-370. 22. Kohn , J . 1957. A cellulose acetate supporting medium for zone electrophoresis. Clin. Chim. A cta 2: 297-303 . 23. Scheidegger, J. J . 1955. Une micromethode de l'immunoelectrophorese . Int. Arch. Allerg. 7: 103-110. 24. Waller, M . V., and J . M . Vaughn. 1956. Use of anti-Rh sera for demonstrating agglutination activating factor in rheumatoid arthritis. Proc . Soc. Exp. Bioi. Med . 92: 198-200. 25. Lawrence, T . G. , Jr., and R. C. Williams, Jr. 1967. Studies of human anti- -y-globulin factors reacting with pepsin-digested -y-globulins. J. Exp. Med. 125: 233- 248. 26. Downie, N. M. , and R. W . Heath. 1959. Basic statistical methods. Harper and Row, Publishers, Inc., New York. 27. Siegel, S. 1956. Nonparametric statistics for the behavioral sciences, p . 184-193. McGraw-Hill Book Company, Inc., New York. 28. Hill. A. B. 1956. Principles of medical statistics, p. 119- 143. Oxford University Press. New York. 29. Wilson, I. D. , G. R. Onstad, R. C. Williams, Jr., and J . B. Carey, Jr. 1968. Selective immunoglobulin A deficiency in two patients with a!-

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

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coholic cirrhosis. Gastroenterology 54: 253259. Rothschild, M . A., M . Oratz, J . Mongelli, and S. S. Schreiber. 1965. Albumin metabolism in rabbits during gamma globulin infusions. J . Lab . Clin. Med. 66: 733-740. Waldmann, T. A. , R. S. Gordon, Jr., and W. Rosse. 1964. Studies on the metabolism of the serum proteins and lipids in a patient with analbuminemia. Amer. J . Med . 37: 960- 968. Bj¢rneboe, M . 1946. Studies on the serum proteins in hepatitis. I. The relation between serum albumin and serum globulin. Acta M ed. Scand. 123: 393-401. Rothschild , M. A., M. Oratz , E. C. Franklin, and S. S. Schreiber. 1962. The effect of hypergammaglobulinemia on albumin metabolism in hyperimmunized rabbits studied with albumin-I1 31 . J. Clin. Invest . 41: 1564-1571. Kent, G., H . Popper, A. Dubin, and C. Bruce. 1957. The spleen in ethionine-induced cirrhosis. Its role on -y-globulin elevation. Arch. Path. (Chicago) 64: 398-408. Cardstri:im, E ., Y. Edlund, H . A. Hansen . K. Hugosson, and B. Werdinius . 1963 . Hepatic tests in the differential diagnosis of jaundice. Scand . J. Clin. Lab . Invest . 15: suppl. 73, 1-19. Zieve, L., and E. Hill. 1955. An evaluation of factors influencing the discriminative effectiveness of a group of liver function tests. III. Relative effectiveness of hepatic tests in cirrhosis. Gastroenterology 28: 785-802. Yount, W. J., H. G. Kunkel , and S. D. Litwin, 1967. Studies of the Vi (-y,,) subgroup of -y-globulin. A relationship between concentration and genetic type among normal individuals. J. Exp. M ed . 125: 177-190. Howell, D. S., J . M . Malcolm, and R. Pike. 1960. The FII agglutinating factors is serums of patients with non-rheumatic diseases. Amer. J . M ed. 29: 662- 671. Atwater, E. C., and R. F. Jacox . 1963. The latexfixation test in patients with liver disease. Ann. Intern. Med . 58: 419-425. Bonomo, L. , J. LoSpalluto, and M . Ziff. 1963. Anti-gamma globulin factors in liver disease. Arthritis Rheum. 6: 104- 114. Bouchier, I. A. D., K. Rhodes, and S. Sherlock. 1964. Serological abnormalities in patients with liver disease. Brit. Med . J. 1: 592- 594. Waller, M ., and S. D. Lawler. 1962. A study of the properties of the rhesus antibody (Ri) diagnostic for the rheumatoid factor and its application to Gm grouping. Vox Sang . 7: 591606. Havens, W. P ., Jr. , R. M . Myerson, and J .

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Klatchko. 1957. Production of tetanus antitoxin by patients with hepatic cirrhosis. New Eng. J. Med . 257: 637-643. 44 . Cherrick, G. R., L. Pothier, J. J. Dufour, and

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S. Sherlock. 1959. Immunologic response of tetanus toxoid inoculation in patients with hepatic cirrhosis. New Eng. J . Med . 261: 340342.