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Chin Med J (Taipei) 1997;59:334-40.
Division of Gastroenterology, Department of Medicine, Veterans General Hospital-Taipei and National Yang-Ming University School of Medicine, Taipei, Taiwan, R.O.C.
Background. Primary biliary cirrhosis (PBC), a chronic, progressive, cholestatic liver disease characterized by frequent presence of the anti-mitochondrial antibody (AMA), is rarely seen in the Asian population. Whether or not the presence of AMA alters the clinical, serological and histological features of the disease remains unclear.
Methods. During the 25-year period from 1972 to 1996, 71 Chinese patients with PBC who were diagnosed clinically and/or histologically were reviewed. Seventeen (24%) of them repeatedly tested negative for serum AMA. Clinical, biochemical, immunological, histological data and treatment response to ursodeoxycholic acid were compared between PBC patients with positive and negative serum AMA.
Results. Mean serum immunoglobulin M (IgM) level was significantly lower in AMA-negative PBC patients when compared with AMA-positive PBC patients (422 +/- 163 vs. 651 +/- 412 mg/dL, p=0.042). Serum anti-nuclear antibodies (ANA) were positive in 93% of the AMA-negative PBC patients, a significantly higher percentage when compared with the 66% of AMA-positive patients (p=0.048). There were no significant differences with regard to age, sex, clinical manifestations, liver biochemistries, histological findings, survival and response to l-year ursodeoxycholic acid treatment between the AMA-negative and AMA-positive patients. Univariate logistic regression analysis showed relatively lower serum IgM levels, positive serum ANA and anti-centromere type ANA were significant predictive factors to negative serum AMA in PBC patients (p=0.035, 0.044 and 0.029, respectively). Multi-variate analysis revealed relatively lower serum IgM levels were the only independent significant predictor to negative serum AMA in PBC patients (p=0.015).
Conclusions. Twenty-four percent of Chinese patients with PBC were negative for serum AMA. AMA-negative PBC patients were characterized by relatively lower serum IgM levels and a higher rate of positive serum ANA.
[Chin Med J (Taipei) 1997;59:334-40.]
Keywords: anti-mitochondrial antibody, anti-nuclear antibody, autoimmune cholangitis, immunoglobulin M, primary biliary cirrhosis
Received: November 7, 1996.
Accepted: April 21, 1997.
Address reprint requests to: Shinn-Jang Hwang, M.D., Division of Gastroenterology, Department of Medicine, Veterans General Hospital-Taipei, No. 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan, R.O.C.
Primary biliary cirrhosis (PBC) is a chronic progressive liver disease of unknown cause characterized by inflammatory destruction of the intrahepatic bile ducts [1]. It may lead to cirrhosis and portal hypertension. Women are affected nine times more often than men. The disease is characterized by the presence of serum anti-mitochondrial antibody (AMA) in 68-99% of cases [2-7]. However, AMA-negative PBC, also described as autoimmune cholangiopathy, autoimmune cholangitis or primary autoimmune cholangitis, has been noted recently as a special disease entity from Western countries [7-13]. Whether it is a variant of the AMA-positive PBC or whether it constitutes another specific disease entity remains uncertain.
In Asian countries such as Taiwan, the incidence of PBC is lower than in Western countries, but an increased incidence has been noted in recent years, probably because of clinicians' improved awareness of the disease [14]. No previous information regarding Chinese PBC patients with negative serum AMA has been reported. This study, we reports the clinical and serological features of Chinese PBC patients with negative serum AMA, comparing the findings with those of AMA-positive PBC patients.
During the period from 1972 to 1996, 71 patients diagnosed as PBC were seen at the Gastroenterology Section of Veterans General Hospital-Taipei. All patients were diagnosed as PBC by the presence of the characteristic clinical symptoms of pruritus and hyperpigmentation, biochemical findings of elevated biliary enzymes and/or histological findings. Other causes of cholestasis (such as drug-induced cholestasis, primary sclerosing cholangitis, biliary tract stones, tumors or strictures) were excluded by detailed history taking, ultrasonography and/or endoscopic retrograde cholangiopancreatography (ERCP). ERCP is performed routinely in AMA-negative patients who have typical PBC symptoms and biochemical findings, but refuse receiving liver biopsy. Fifty-four (76%) patients tested positive, and 17 (24%) repeatedly tested negative, for serum AMA. Clinical manifestations, serum biochemistries, immunoglobulin (Ig) levels, positive rate for anti-nuclear antibody (ANA) and anti-smooth-muscle antibody (ASMA), histological findings, survival and response to treatment were compared between AMA-positive and AMA-negative PBC patients.
Serum albumin (ALB), globulin (GLO), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GT), total bilirubin (TB) and cholesterol (CHOL) levels were analyzed by a systemic autoanalyzer (Hitachi, model 736-60, Tokyo, Japan). Serum AMA, ASMA and ANA were measured by an indirect immunofluorescence method (Incstar, Stillwater, MN, USA). AMA titers above 1:10, ASMA titers above 1:10 and ANA titers above 1:80 were recorded as positive. Serum IgM, IgA and IgG were analyzed by a nephelometric method (Behring, Marburg, Germany). Hepatitis B surface antigen (HBsAg) was tested by a commercial RIA kit (Abbott Laboratories, North Chicago, IL, USA). Antibody to hepatitis C virus (anti-HCV) was measured by a second-generation enzyme immunoassay (Abbott Laboratories, North Chicago, IL, USA).
Twenty-six (8 AMA-negative, 18 AMA-positive) of the 71 patients were treated with ursodeoxycholic acid (UDCA) 600 mg/day, and the biochemical responses were compared between AMA-positive and AMA-negative PBC patients. One AMA-negative patient received immuno-suppressive agents (prednisone and azathioprine) and UDCA for one year. Two patients received liver transplants. The Mayo clinical risk scores [15], based on a combination of five variables (age, serum albumin and total bilirubin levels, prothrombin time and degree of edema) were also evaluated for comparison of treatment response and for predicting long-term survival.
Data in the text and tables are expressed as mean +/- standard deviation (SD). The intergroup difference was compared by two-samples t test for continuous data. Chi-square test (or Fisher's exact test) was used for intergroup comparison of incontinuous data. Mann Whitney-Wilcoxon rank sum test was used for comparison of data not normally distributed. Univariate and multivariate analyzes using logistic regression (BMDP, 1990 version, CA, USA) were performed to analyze the predictive value of clinical, biochemical and immunological variables to the association of negative AMA in PBC patients. Kaplan-Meier method was used to compare the survival between both groups. Results with a p value < 0.05 were considered statistically significant.
Seventy-one patients were diagnosed as PBC during a 25-year period. There were 13 male and 58 female patients. The mean age of diagnosis was 53 +/- 12 (range 25-78) years. Fifty-four (76%) patients tested positive for serum AMA and 17 (24%) repeatedly tested negative for serum AMA. Fatigue, pruritus and jaundice were the common presenting symptoms. Hyperpigmentation, xanthelasma and/or xanthoma were less commonly manifested. Only five patients were asymptomatic. No difference was found between AMA-positive and AMA-negative PBC patients with respect to sex, age of initial symptoms, diagnosis and the presenting symptoms (Table 1). Seventeen (31%) of AMA-positive PBC patients and four (24%) of AMA-negative PBC patients were associated with autoimmune disease. Seventeen (24%) patients were associated with sicca syndrome. Other autoimmune diseases (including three patients with systemic lupus erythematosus, three patients with autoimmune thyroiditis, one patient with rheumatoid arthritis, scleroderma and mixed connective tissue disease, respectively) were also seen. No difference was seen in the associated autoimmune diseases between both groups.
Comparison of liver biochemistries between AMA-positive and AMA-negative PBC patients are listed in Table 2. There were no significant differences in the serum levels of ALB, GLO, AST, ALT, ALP, gamma-GT, TB and CHOL between the two groups. Table 3 compares the immunological data between AMA-positive and AMA-negative PBC patients. Mean serum IgM level was significantly lower in the AMA-negative patients than in the AMA-positive ones (422 +/- 163 vs. 651 +/- 412 mg/dL, 95% confidence interval for difference 8.7-448.9, p=0.042). The difference in the positive rate of serum ANA between both groups was also statistically significant: 15 of the 17 AMA-negative patients tested for serum ANA and 14 (93%) were positive while 29 (66%) of the 44 patients in the AMA-positive group tested positive for ANA (p= 0.048). The median titer of ANA was also significantly higher in AMA-negative PBC patients compared to AMA-positive patients (1: 1280 vs. 1 :320, p=0.002). The comparison of immunofluorescence staining patterns in ANA-positive patients is listed in Table 3. A speckled pattern was commonly seen in both AMA-positive and AMA-negative PBC patients. An anti-centromere pattern was more frequently seen in the AMA-negative patients than in those AMA-positives (27% vs. 5%, p=0.032).
Clinical, biochemical and immunological data of PBC patients were selected as independent variables with the serum AMA marker as a dependent variable in a logistic regression analysis. Serum IgM levels less than 500 mg/dL, a positive ANA and anti-centromere type ANA were significant predictors associated with negative serum AMA in univariate analysis (odds ratios 4.0, 3.0, 2.8, 95% confidence intervals 1.1-14.8, 1.1-8.6, 1.1-6.9, p=0.035, 0.044, 0.029, respectively). Multivariate analysis using the logistic regression showed relatively low IgM levels (<=500 mg/dL) which were the only significant factor associated with negative AMA (odds ratio 6.3, 95% confidence interval 1.3-30.1, p=0.015).
Sixty-six patients tested for HBsAg and 29 patients tested for anti-HCV. Only one (1.5%) AMA-positive patient was positive for serum HBsAg and one (3.4%) AMA-negative patient showed positive serum anti-HCV. The patient with positive serum anti-HCV had previously received a blood transfusion. Hepatocellular carcinoma occurred in two AMA-positive patients during a mean follow-up period of four years.
Thirty-one patients (23 AMA-positive, 8 AMA-negative) received percutaneous needle liver biopsy; Table 4 compares the histological findings between AMA-positive and AMA-negative PBC patients. Granuloma, ductless sign, florid duct sign, degree of fibrosis and inflammation and histologic stage were not significantly different between the two groups.
Eight patients in the AMA-negative group and 18 patients in the AMA-positive group received UDCA treatment. After one year of that treatment, serum levels of TB, ALP, ALT and the Mayo risk scores decreased by a mean of 24, 39, 37 and 3.8% of its pretreatment values, respectively, in the AMA-positive group. In the AMA-negative group, serum levels of TB, ALP, ALT and the Mayo risk scores decreased by a mean of 61, 28, 46 and 13.5% of its pretreatment values, respectively. Serum levels of ALB remained the same in the AMA-positive group and increased by 2.5% in the AMA-negative group. The differences were not statistically significant between the two groups (p > 0.05) (Figure 1). One AMA-negative patients who was treated with immunosuppressive agents and UDCA showed improvement in liver biochemistry. Twenty-five patients in the AMA-positive group and five in the AMA-negative group died of liver failure during the follow-up period (p > 0.05). The comparative survival rate using Kaplan-Meier life table analysis was not statistically significant between the two groups (data not shown).
Diagnosis of PBC usually requires the presence of a combination of an increase of serum biliary enzymes and immunoglobulin levels, histological evidence of bile duct injury and/or portal inflammation, together with the presence of characteristic serum AMA. Ultrasonography of the hepatobiliary system and cholangiography are also helpful to differentiate PBC from primary sclerosing cholangitis and other biliary mechanical obstruction. The 71 Chinese cases fulfilled the diagnostic criteria for PBC with the exception of 17 (24%) cases with negative serum AMA. These results are consistent with previous reports that 1-32% of PBC patients do not have positive serum AMA [2-7]. AMA is a class of autoimmune antibodies which react against different antigens in the mitochondria [1]. A characteristic marker in the diagnosis of PBC, the absence of AMA does not, however, preclude a diagnosis of PBC.
According to results here, the AMA-positive and AMA-negative patients were similar in clinical manifestations, prevalence of autoimmune diseases, serum liver biochemistries, positive rate of ASMA and histological findings. However, a significantly higher rate of positive ANA (93% vs. 66%, p=0.048) and higher median titer of ANA in the AMA-negative patients were found when compared with the AMA-positive patients. These results are consistent with previous reports from Western countries that a higher positive rate of ANA (63-100%) was noted in AMA-negative PBC patients [7-13]. Clinical characteristics of AMA-negative PBC patients reported from Western countries and from the present study are listed in Table 5. A high prevalence of autoantibodies, especially ANA in the AMA-negative PBC patients, can be seen in all series. These findings suggest that, besides AMA, other serologic autoimmune markers indicating different autoimmune processes might exist in AMA-negative PBC patients.
Different ANA subtypes have been reported in patients with PBC from different geographic areas [10-12]. Results here revealed a high prevalence of speckled pattern in PBC patients with positive ANA, with no statistical significance found between AMA-negative and AMA-positive PBC patients. The cause of the higher prevalence of anti-centromere type in our AMA-negative PBC patients has yet to be determined.
Elevated serum levels of immunoglobulins have been frequently described in patients with PBC [16]. Serum levels of IgM are more frequently elevated than those of IgG and IgA in PBC patients. The elevation of IgM levels may indicate a defect in the control of immunoglobulin synthesis [17]. Michieletti et al. [11] and Lacerda et al. [13] have described a tendency for AMA-negative patients to have lower levels of serum IgM than the AMA-positives. Results here also showed a significantly lower mean level of IgM in the AMA-negative group when compared with the AMA-positive group. In univariate and multivariate analyses, serum IgM level of less than 500 mg/dL is also a significant predictor of AMA-negative in PBC patients. These findings show the close relationship between negative AMA and relatively low IgM levels in those patients. However, the actual mechanism demands further investigation.
Whether PBC patients have increased risk of HCC remains controversial [18,19]. HCC was found in two patients in this series during the follow-up period, giving 0.7% case of HCC per year. The risk of HCC in PBC patients seems higher than that of the general population in Taiwan and is similar to the risk of those with other chronic liver diseases [20].
UDCA is safe and well tolerated and has been widely used in the treatment of PBC patients. UDCA has provided significant improvement of both symptoms and liver biochemistries in PBC patients [21]. The response to immunosuppressive therapy in the treatment of PBC remains controversial. Corticosteroids are ineffective and may actually worsen the bone disease in AMA-positive PBC cases. Carrougher et al. [8] and Ben-Ari et al. [10] treated five AMA-negative PBC patients, characteristically positive for serum ANA, with immunosuppressive therapy; all showed rapid clinical and biochemical remission. However, Goodman et al. [7] and Taylor et al. [12] later reported the opposite results. Results here showed similar responses to UDCA treatment in AMA-positive and AMA-negative patients. The presence or absence of AMA did not seem to influence the effect of UDCA in the teatment of PBC. Only one AMA-negative PBC patient with positive ANA was treated with immunosuppressive agents and UDCA and the response is good. Further evaluation is needed to clarify the effectiveness of immunosuppressive agents in the treatment of AMA-negative, ANA-positive PBC patients.
The name "primary biliary cirrhosis" seems to be a misnomer and accurately describes only the fibrotic features of late-stage disease in PBC patients. "Chronic nonsuppurative destructive cholangitis" highlights the principal diagnostic features of lymphocytic infiltration of the portal tracts with destruction of the smaller bile ducts but it is a cumbersome term to use. Increased serum IgM levels, circulating autoantibodies and an association with other autoimmune diseases in PBC patients may suggest that abnormalities of the immune regulation are present. The recently advocated name "autoimmune cholangitis" may be the most suitable, since it avoids the problems arising from including the term "cirrhosis" in PBC and its close relationship to autoimmunity, although this nomenclature has not yet been agreed upon. The disease may be further subdivided into AMA-positive and AMA-negative cases. AMA-negative and AMA-positive PBC may be just parts of a spectrum of disorders in which bile duct destruction frequently associates with several serum autoantibodies. Further characterization of the pathogenesis is necessary for the classification. In conclusion, 24% of Chinese patients with PBC tested negative for serum AMA. These patients were characterized by relatively lower serum IgM levels and higher positive rates of serum ANA.
Acknowledgements
The authors gratefully acknowledge Ms. Shwu-Jen Wu and Wen-I Huang for their secretarial assistance and preparation of this manuscript. This work was supported by a grant from Veterans General Hospital-Taipei.
Copyright: 1997, Chinese Medical Association (Taipei)