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Changes of hepatic microtubules and secretory proteins in human alcoholic liver disease
Pharmacoh~,,y Biochemistry & Behavior, Vol. 18, Suppl. 1, pp. 47%482, 1983. ~ Ankho International. Printed in the U.S.A.
Changes of Hepatic Microtubules and Secretory Proteins in Human Alcoholic Liver Disease YOSHIRO MATSUDA, AKIRA TAKADA, I RYUICHI KANAYAMA AND SHUJIRO TAKASE
Division o f Gastroenterology, D e p a r t m e n t o f lnternal Medicine, K a n a z a w a Medical University, Japan
MATSUDA, Y., A. TAKADA, R. KANAYAMA AND S. TAKASE. Changes of hepatic microtubules and secretory proteins in human alcoholic liver disease. PHARMACOL BIOCHEM BEHAV 18: Suppl. 1,47%482, 1983.--It has been shown that alcohol consumption disrupts liver microtubules, impairs protein secretion and leads to ballooning of the hepatocytes in rats. Ethanol-induced hepatomegaly was accounted for by an increase of the hepatocytes volume. To study whether these changes occur in human alcoholic liver disease, hepatic tubular protein and export protein content were measured in 29 cases of alcoholic liver disease and were compared with those of 37 cases of non-alcoholic liver disease and 5 cases of non-hepatobiliary disease. Hepatic polymerized tubulin was significantly decreased in alcoholic liver disease compared to non-alcoholic liver disease (p<0.01), while free tubulin was increased in alcoholic liver disease. Hepatic transferrin (one of the export proteins) content was significantly higher (p<0.01) and serum transferrin level was significantly lower (p<0.05) in alcoholic liver disease than in non-alcoholic liver disease. These findings indicated that even in humans, chronic alcohol consumption decreased hepatic microtubules by impairing polymelization of tubular protein and increased hepatic export protein content. This decrease in hepatic microtubules by chronic alcohol consumption may play an important role in the development of human alcoholic liver disease. Tubular protein Hepatic microtubules Human alcoholic liver disease
Hepatic secretory protein
Transferrin
METHOD
E N L A R G E M E N T of the liver is a prominent feature of the initial damage produced by alcohol intake. Hepatomegaly was shown to be due not only to accumulation of fat but also to accumulation of proteins, including albumin and transferrin, which are primarily destined for export from the liver into the blood [1]. Baraona et al. [2] reported that the retention of soluble proteins and swelling of the hepatocyte in ethanol-fed rats were associated with delayed secretion of these exportable proteins, and they also confirmed that these alterations were associated with a decrease in microtubules, the integrity of which appeared to be a requirement for normal secretion [2]. Recently we have found that a decrease in hepatic microtubules and an increase in hepatic export protein were the most important pathogenetic observations accompanying the development of aicohol-pyrazole hepatitis in rats, the features of which resemble human alcoholic hepatitis [8]. However, there is no report on the changes of hepatic microtubules and exportable proteins in human liver diseases. This study was undertaken to determine whether these alterations of microtubules and protein secretion occur in human alcoholic liver disease.
Patients Twenty-nine male patients with alcoholic liver injury including 20 cases of alcoholic fibrosis, 4 of cirrhosis and 5 of alcoholic hepatitis, and 37 patients with non-alcoholic liver injury (male 21, female 16) including 21 cases of acute hepatitis and 16 of chronic hepatitis were studied. As a control group, 5 patients (male 3, female 2) with nonhepatobiliary disease were also investigated. All patients were admitted to Kanazawa Medical University Hospital between 1981 and 1982, and were diagnosed histologically by liver biopsy. Liver biopsies were performed only for diagnostic or management purposes. Liver samples were obtained percutaneously with a Menghini needle within 2 weeks after alcohol abstinence in the cases of alcoholic liver injury and at various times after hospital admission in nonalcoholic liver diseases. In the cases of non-hepatobiliary disease, liver samples were obtained by wedged biopsy at the time of operation.
~Requests for reprints should be addressed to Akira Takada, M.D., Division of Gastroenterology, Department of Internal Medicine, Kanazawa Medical University, Uchinada, Ishikawa, 920-02, Japan,
479
480
MATSUDA E T A L .
I - -
p
+
200.
p
[--
p/-.O.O5 -
-
I~P
400.
.u 100" E
I
I
Acute hepatitis (21)
Chronic hepatitis 06)
200. IJ_
Non hepatoI~lioly dis. (5)
Chronic hepatitis (16)
Acute hepotitis
(21)
Alcoholic liver inj.
Non hepatobilioly dis. (5)
(29)
liver inj.
(30)
FIG. 2. Hepatic free tubulin contents.
FIG. 1. Hepatic polymerized tubulin contents.
p
p<0.0l I
Alcoholic
p
1
_ 0.2 260
~240
.c_
o.i
220
c
"~200
i
Non hepotobilkaly dis. (51
Acute hepatitis (21)
Chronic
hepatitis
(16)
Alcoholic
liver inj.
t29l
FIG. 3. Hepatic transferrin contents.
180
T
Non hepoto-
bilkalydis. (51
Acute
hepatitis (21)
Chronic
heoablis
(16)
1
Alcoholic
liver inj.
(291
FIG. 4. Serum transferrin levels. Biochemical Analysis
For the measurement of polymerized and free tubulin content in the liver, liver samples weighing about 10 mg were homogenized with 1 ml of microtubular stable solution at room temperature and measured from the binding of 3Hcolchicine according to the method of Pipeleers et al. [13]. Protein content of the liver was assessed by the method of Lowry et al. [5]. For the measurement of hepatic transferrin and albumin content, other liver samples weighing about 10 mg were homogenized with 0.5 ml of ice-cold 0.25 M sucrose solution and were centrifuged at 750 × g for 10 minutes. An aliquot of the supernatants were used for measurement of the two exportable proteins by the method of Laurel et al. [4]. Serum transferrin and albumin levels were determined by single radial immunodiffusion method [6] using rabbit antihuman transferrin and albumin serum (Hoechst, West Germany). All results were expressed by the mean-+standard deviation of the mean. Student's t-test [14] was used for group comparison of the data. RESULTS Hepatic polymerized tubulin content expressed per g of liver was significantly decreased in alcoholic liver injury
compared to non-alcoholic liver disease and nonhepatobiliary disease (Fig. I). When polymerized tubulin contents were calculated per mg of liver protein, alcoholic liver disease (6.0-+4.0 pmoles per mg of liver protein) also showed a significantly lower value (p<0.01) than those of non-alcoholic and non-hepatobiliary disease (acute hepatitis; 37.9-+30.6; chronic hepatitis, 15.9-+7.3; non-hepatobiliary disease, 22.3-+4.8). On the other hand, the hepatic free tubulin content in alcoholic liver disease was significantly higher than those in non-alcoholic liver disease and nonhepatobiliary disease (Fig. 2). When free tubulin contents expressed as pmoles per mg of liver protein, alcoholic liver disease also showed a significant increase in free tubulin compared to non-alcoholic liver disease or non-hepatobiliary disease (p<0.05). Hepatic transferrin content in alcoholic liver disease was increased significantly compared to those of non-alcoholic liver disease or non-hepatobiliary disease (Fig. 3), while the serum transferrin level in alcoholic liver disease was significantly lower than those in non-alcoholic liver disease or non-hepatobiliary disease (Fig. 4). However, there was no significant difference in hepatic nor serum albumin level between alcoholic and non-alcoholic liver disease or the control group (Figs. 5, 6). In 29 patients with
H E P A T I C C H A N G E S IN H U M A N A L C O H O L I C S
481 n.s,
rl.s.
0.5-
÷
4D 0.4-
3.8
~. 0.3-
.c_
E
E
3.6
"~ 0.2" 3.4!
0.1"
T
i I Non hel:x]tobiiioly dis.
15)
Acute hepo5 Iis
(21)
Chron;c hepat,ti$
(~6)
NonhepoloI~lkaly dis.
Alcoholi liver inj.
Acute hepatitis
(51
(29}
(21)
Chronic hepatitis
(16)
Alcoholic liver in].
(29}
FIG. 6. Serum albumin levels.
FIG. 5. Hepatic albumin contents.
alcoholic liver disease, a significant correlation between hepatic polymerized tubulin and hepatic transferrin content was observed (Fig. 7). However, there was no apparent difference in polymerized tubulin nor transferrin content among the types of alcoholic liver disease.
~_ 0.2 =
2 DISCUSSION
The present study clearly showed that hepatic polymerized tubulin was decreased and hepatic transferrin was increased in human alcoholic liver disease. Total hepatic tubulin content was not significantly different among the liver disease groups and the control group. In nonhepatobiliary disease, about 39% of hepatic tubular protein was polymerized tubulin, the major chemical component of the microtubules, and 61% of tubular protein was free tubulin. On the other hand, hepatic tubular protein in alcoholic liver disease was polymerized only 14%, while 86% was free. These results indicate that polymerization of tubular protein is impaired in alcoholic liver disease. Matsuda e t al. [7] reported that ethanol administration decreased the hepatic microtubules in rats and that this effect was attributed to acetaldehyde metabolized from ethanol. Baraona e t al. [3] have pointed out that acetaldehyde directly inhibited the polymerization of tubular protein purified from rat brain in the same manner as colchicin. In the study of baboons in which ethanol was given chronically for 24 months, a decrease of hepatic microtubules was reported [9]. These results with animals [3, 7, 9] are compatible with those of the present study in human alcoholics. In this study, hepatic transferrin, which is one of the exportable proteins in the liver, was increased and serum transferrin was decreased in alcoholic liver disease, indicating that a retention of transferrin in the liver caused by impairment of its secretion occurs in alcoholic liver injury. Baraona et. al. [2] reported that the retention o f soluble proteins by the hepatocyte in alcohol-fed rats was associated with delayed secretion of these exportable proteins and that these alterations were associated with a decrease in hepatic microtubules. In the animal experiment of Matsuda et al. [7], partial disruption of microtubules of the liver by acetaldehyde lowered the exportable protein secretion from the liver. In the present study, there was a significant negative correlation between hepatic polymerized tubulin and transferrin contents. These results suggested that impairment of polymerization of microtubules by alcohol intake resulted in
• x
=m
O
c_
0.1
x
®
x
.:cirrhosis
~: hepatitis
: fibrosis with or wib"mut fatty liver
20
r:0.562 p<0.01 n:29 40
60
80
polymerized tubulin Ipg/ghverl
FIG. 7. Correlation between hepatic polymerized tubulin and transferrin contents in alcoholic liver disease.
a decrease of the secretion of exportable proteins in human alcoholic liver disease. Albumin is also one of the exportable proteins. However, hepatic and serum albumin levels were not different among groups with liver diseases and the control group. Although the precise mechanism of the discrepancy between the changes of albumin and transferrin could not be clarified from the present study, two possible mechanisms will be considered. One is the difference of pool size, and another is the structural difference of both exportable proteins. In the animal experiment of Baraona et al. [2], albumin accumulated not only in the microsomal fraction but preferentially in the cytosol fraction of the hepatocytes, while transferrin was accumulated mainly in the microsomal fraction, suggesting that the cytosol could serve as a storage site of retained albumin but not transferrin and that a stored pool for albumin in the hepatocyte might be large. The big pool size of albumin may make unclear the effect of alcohol in humans. On the other hand, transferrin but not albumin is a glycoprotein, and glycosilation in or out of the Golgi apparatus may be necessary for its secretion from the liver [1 I1. Mitranic et al. [10] reported that an anti-microtubular agent such as colchicin impaired the glycosilation o f glycoprotein in the Golgi apparatus. As pointed out previously [3], acetaldehyde had the same effect as colchicin on the microtubules, suggesting that alcohol intake may inhibit the glycosilation of transferfin. Tuma et al. [17] reported that ethanol inhibited the in-
482
MATSUDA ET AL.
corporation of glucosamine to secretory glycoprotein in rat liver slices. Stiber et al. [15,16] found abnormal heterogeneity of serum transferrin in alcoholic patient during current abuse, and reported that this abnormality was caused by the reduction of sialic acid content in transferrin. Recently, Nei [12] found that the serum transferrin level decreased in alcoholic patients during the periods of alcohol intake and increased following alcohol abstinence, while serum levels of prealbumin, non-glycosylated exportable protein, was rather high during the periods of alcohol intake and decreased following alcohol abstinence. These studies suggest that alcohol intake may inhibit the glycosilation of transferrin and impair its secretion by disrupting microtubular function, and this possibility may explain the discrepancy of the results concerning both exportable proteins. We recently reported that, in the experimental "alcohol pyrazole hepatitis" of rat, the ballooning and necrosis of the hepatocytes might result from a decrease in hepatic microtubules and an increase in exportable protein which is caused by an increased production of acetaldehyde in the
non-alcohol dehydrogenase pathway [8]. In this study ballooned hepatocytes were prominent in alcoholic liver injury with or without necrosis, indicating that acetaldehyde produced in the non-alcohol dehydrogenase pathway may also play an important role in the development of human alcoholic liver disease. From the results of the present study, it was suggested that an impairment of hepatic microtubular polymerization resulted in the retention of hepatic export proteins in hepatocytes and finally in ballooning or necrosis of the hepatocytes in human alcoholic liver disease. This process may be one of the pathogenetic mechanisms in the development of human alcoholic liver injury.
ACKNOWLEDGMENTS This work was supported by Research Grant No. 54183 administrated by the Educational Ministry of Japan. The expert technical assistance of Ms. K. Sugata and Ms. A. Yamatani is gratefully acknowledged.
REFERENCES 1. Baraona, E., M. A. Leo, S. A. Borowsky and C. S. Lieber. Alcoholic hepatomegaly: accumulation of protein in the liver. Science 190: 794-795, 1975. 2. Baraona, E., M. A. Leo, S. A. Borowsky and C. S. Lieber. Pathogenesis of alcohol-induced accumulation of protein in the liver. J Clin Invest 60: 546--555, 1977. 3. Baraona, E., Y. Matsuda, P. Pikkarainen, F. Finkelman and C. S. Lieber. Exaggeration of the ethanol-induced decrease in liver microtubules after chronic alcohol consumption: Role of acetaldehyde. Gastroenterology 76: 1274, 1979. 4. Laurell, C. B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15: 45-52, 1966. 5. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. Protein measurement with the Folin phenol reagent. J Bh)I Chem 193: 265-275, 1951. 6. Mancini, G., A. O. Carbonara and J. F. Herenans. Immunological quantitation of antigens by single radial immunodiffusion. lmmunohistochemistry 2: 235-254, 1965. 7. Matsuda, Y., E. Baraona, M. Salaspuro and C. S. Lieber. Effects of ethanol on liver microtubules and Golgi apparatus. Possible role in altered hepatic secretion of plasma protein. Lab Invest 41: 455-463, 1979. 8. Matsuda, Y., A. Takada, R. Kanayama and S. Takase. Effects of dietary fat on the development of alcohol pyrazole hepatitis. Presented at World Congress, Stockholm, Sweden, 1982, p. 15. (Abstract)
9. Matsuda, Y., E. Baraona, M. Salaspuro and C. S. Lieber. Pathogenesis and role of microtubular alterations in alcohol induced liver injury. Fed Proc 37: 402, 1978. 10. Mitranic, M. M., J. M. Boggs and M. A. Moscarello. An effect of colchicin on galactosyl- and sialyltransferases of rat liver Golgi membrane. Biochim Biophys Acta 672: 57-64, 1981. 11. Morgan, E. H. and T. Peters. lntracellular aspects oftransferrin synthesis and secretion in the rat. J Biol Chem 246:3508-3511, 1975. 12. Nei, J. Serial changes of hepatic secretory proteins in alcoholic liver injury (in Japanese). Gastroenterologica Jap., in press. 13. Pipeleers, D. G., M. A. Piperleers-Marical, P. Shrline and D. M. Kipnis. A sensitive method for measuring polymerized and deplymerized forms of tubulin in tissues. J Cell Biol 74: 341-350, 1977. 14. Snedecor, G. W. and W. G. Cochran. Statistical Methods, 6th edition. Iowa City, IA: The Iowa State University Press, 1967. 15. Stibler, H., S. Borg and C. Allgulander. Clinical significance of abnormal heterogeneity of transferrin in relation to alcohol consumption. Acta Med Scand 206: 275-281, 1979. 16. Stibler, H. and S. Borg. Evidence of a reduced sialic acid content in serum transferrin in male alcoholics. Alcoholism 5: 545549, 1981. 17. Tuma, D. J., R. B. Jannet and M. F. Sorrell. Effect of ethanol on the synthesis and secretion of hepatic secretory glycoproteins and albumin. Hepatology 1: 590-598, 1981.
Changes of Hepatic Microtubules and Secretory Proteins in Human Alcoholic Liver Disease YOSHIRO MATSUDA, AKIRA TAKADA, I RYUICHI KANAYAMA AND SHUJIRO TAKASE
Division o f Gastroenterology, D e p a r t m e n t o f lnternal Medicine, K a n a z a w a Medical University, Japan
MATSUDA, Y., A. TAKADA, R. KANAYAMA AND S. TAKASE. Changes of hepatic microtubules and secretory proteins in human alcoholic liver disease. PHARMACOL BIOCHEM BEHAV 18: Suppl. 1,47%482, 1983.--It has been shown that alcohol consumption disrupts liver microtubules, impairs protein secretion and leads to ballooning of the hepatocytes in rats. Ethanol-induced hepatomegaly was accounted for by an increase of the hepatocytes volume. To study whether these changes occur in human alcoholic liver disease, hepatic tubular protein and export protein content were measured in 29 cases of alcoholic liver disease and were compared with those of 37 cases of non-alcoholic liver disease and 5 cases of non-hepatobiliary disease. Hepatic polymerized tubulin was significantly decreased in alcoholic liver disease compared to non-alcoholic liver disease (p<0.01), while free tubulin was increased in alcoholic liver disease. Hepatic transferrin (one of the export proteins) content was significantly higher (p<0.01) and serum transferrin level was significantly lower (p<0.05) in alcoholic liver disease than in non-alcoholic liver disease. These findings indicated that even in humans, chronic alcohol consumption decreased hepatic microtubules by impairing polymelization of tubular protein and increased hepatic export protein content. This decrease in hepatic microtubules by chronic alcohol consumption may play an important role in the development of human alcoholic liver disease. Tubular protein Hepatic microtubules Human alcoholic liver disease
Hepatic secretory protein
Transferrin
METHOD
E N L A R G E M E N T of the liver is a prominent feature of the initial damage produced by alcohol intake. Hepatomegaly was shown to be due not only to accumulation of fat but also to accumulation of proteins, including albumin and transferrin, which are primarily destined for export from the liver into the blood [1]. Baraona et al. [2] reported that the retention of soluble proteins and swelling of the hepatocyte in ethanol-fed rats were associated with delayed secretion of these exportable proteins, and they also confirmed that these alterations were associated with a decrease in microtubules, the integrity of which appeared to be a requirement for normal secretion [2]. Recently we have found that a decrease in hepatic microtubules and an increase in hepatic export protein were the most important pathogenetic observations accompanying the development of aicohol-pyrazole hepatitis in rats, the features of which resemble human alcoholic hepatitis [8]. However, there is no report on the changes of hepatic microtubules and exportable proteins in human liver diseases. This study was undertaken to determine whether these alterations of microtubules and protein secretion occur in human alcoholic liver disease.
Patients Twenty-nine male patients with alcoholic liver injury including 20 cases of alcoholic fibrosis, 4 of cirrhosis and 5 of alcoholic hepatitis, and 37 patients with non-alcoholic liver injury (male 21, female 16) including 21 cases of acute hepatitis and 16 of chronic hepatitis were studied. As a control group, 5 patients (male 3, female 2) with nonhepatobiliary disease were also investigated. All patients were admitted to Kanazawa Medical University Hospital between 1981 and 1982, and were diagnosed histologically by liver biopsy. Liver biopsies were performed only for diagnostic or management purposes. Liver samples were obtained percutaneously with a Menghini needle within 2 weeks after alcohol abstinence in the cases of alcoholic liver injury and at various times after hospital admission in nonalcoholic liver diseases. In the cases of non-hepatobiliary disease, liver samples were obtained by wedged biopsy at the time of operation.
~Requests for reprints should be addressed to Akira Takada, M.D., Division of Gastroenterology, Department of Internal Medicine, Kanazawa Medical University, Uchinada, Ishikawa, 920-02, Japan,
479
480
MATSUDA E T A L .
I - -
p
+
200.
p
[--
p/-.O.O5 -
-
I~P
400.
.u 100" E
I
I
Acute hepatitis (21)
Chronic hepatitis 06)
200. IJ_
Non hepatoI~lioly dis. (5)
Chronic hepatitis (16)
Acute hepotitis
(21)
Alcoholic liver inj.
Non hepatobilioly dis. (5)
(29)
liver inj.
(30)
FIG. 2. Hepatic free tubulin contents.
FIG. 1. Hepatic polymerized tubulin contents.
p
p<0.0l I
Alcoholic
p
1
_ 0.2 260
~240
.c_
o.i
220
c
"~200
i
Non hepotobilkaly dis. (51
Acute hepatitis (21)
Chronic
hepatitis
(16)
Alcoholic
liver inj.
t29l
FIG. 3. Hepatic transferrin contents.
180
T
Non hepoto-
bilkalydis. (51
Acute
hepatitis (21)
Chronic
heoablis
(16)
1
Alcoholic
liver inj.
(291
FIG. 4. Serum transferrin levels. Biochemical Analysis
For the measurement of polymerized and free tubulin content in the liver, liver samples weighing about 10 mg were homogenized with 1 ml of microtubular stable solution at room temperature and measured from the binding of 3Hcolchicine according to the method of Pipeleers et al. [13]. Protein content of the liver was assessed by the method of Lowry et al. [5]. For the measurement of hepatic transferrin and albumin content, other liver samples weighing about 10 mg were homogenized with 0.5 ml of ice-cold 0.25 M sucrose solution and were centrifuged at 750 × g for 10 minutes. An aliquot of the supernatants were used for measurement of the two exportable proteins by the method of Laurel et al. [4]. Serum transferrin and albumin levels were determined by single radial immunodiffusion method [6] using rabbit antihuman transferrin and albumin serum (Hoechst, West Germany). All results were expressed by the mean-+standard deviation of the mean. Student's t-test [14] was used for group comparison of the data. RESULTS Hepatic polymerized tubulin content expressed per g of liver was significantly decreased in alcoholic liver injury
compared to non-alcoholic liver disease and nonhepatobiliary disease (Fig. I). When polymerized tubulin contents were calculated per mg of liver protein, alcoholic liver disease (6.0-+4.0 pmoles per mg of liver protein) also showed a significantly lower value (p<0.01) than those of non-alcoholic and non-hepatobiliary disease (acute hepatitis; 37.9-+30.6; chronic hepatitis, 15.9-+7.3; non-hepatobiliary disease, 22.3-+4.8). On the other hand, the hepatic free tubulin content in alcoholic liver disease was significantly higher than those in non-alcoholic liver disease and nonhepatobiliary disease (Fig. 2). When free tubulin contents expressed as pmoles per mg of liver protein, alcoholic liver disease also showed a significant increase in free tubulin compared to non-alcoholic liver disease or non-hepatobiliary disease (p<0.05). Hepatic transferrin content in alcoholic liver disease was increased significantly compared to those of non-alcoholic liver disease or non-hepatobiliary disease (Fig. 3), while the serum transferrin level in alcoholic liver disease was significantly lower than those in non-alcoholic liver disease or non-hepatobiliary disease (Fig. 4). However, there was no significant difference in hepatic nor serum albumin level between alcoholic and non-alcoholic liver disease or the control group (Figs. 5, 6). In 29 patients with
H E P A T I C C H A N G E S IN H U M A N A L C O H O L I C S
481 n.s,
rl.s.
0.5-
÷
4D 0.4-
3.8
~. 0.3-
.c_
E
E
3.6
"~ 0.2" 3.4!
0.1"
T
i I Non hel:x]tobiiioly dis.
15)
Acute hepo5 Iis
(21)
Chron;c hepat,ti$
(~6)
NonhepoloI~lkaly dis.
Alcoholi liver inj.
Acute hepatitis
(51
(29}
(21)
Chronic hepatitis
(16)
Alcoholic liver in].
(29}
FIG. 6. Serum albumin levels.
FIG. 5. Hepatic albumin contents.
alcoholic liver disease, a significant correlation between hepatic polymerized tubulin and hepatic transferrin content was observed (Fig. 7). However, there was no apparent difference in polymerized tubulin nor transferrin content among the types of alcoholic liver disease.
~_ 0.2 =
2 DISCUSSION
The present study clearly showed that hepatic polymerized tubulin was decreased and hepatic transferrin was increased in human alcoholic liver disease. Total hepatic tubulin content was not significantly different among the liver disease groups and the control group. In nonhepatobiliary disease, about 39% of hepatic tubular protein was polymerized tubulin, the major chemical component of the microtubules, and 61% of tubular protein was free tubulin. On the other hand, hepatic tubular protein in alcoholic liver disease was polymerized only 14%, while 86% was free. These results indicate that polymerization of tubular protein is impaired in alcoholic liver disease. Matsuda e t al. [7] reported that ethanol administration decreased the hepatic microtubules in rats and that this effect was attributed to acetaldehyde metabolized from ethanol. Baraona e t al. [3] have pointed out that acetaldehyde directly inhibited the polymerization of tubular protein purified from rat brain in the same manner as colchicin. In the study of baboons in which ethanol was given chronically for 24 months, a decrease of hepatic microtubules was reported [9]. These results with animals [3, 7, 9] are compatible with those of the present study in human alcoholics. In this study, hepatic transferrin, which is one of the exportable proteins in the liver, was increased and serum transferrin was decreased in alcoholic liver disease, indicating that a retention of transferrin in the liver caused by impairment of its secretion occurs in alcoholic liver injury. Baraona et. al. [2] reported that the retention o f soluble proteins by the hepatocyte in alcohol-fed rats was associated with delayed secretion of these exportable proteins and that these alterations were associated with a decrease in hepatic microtubules. In the animal experiment of Matsuda et al. [7], partial disruption of microtubules of the liver by acetaldehyde lowered the exportable protein secretion from the liver. In the present study, there was a significant negative correlation between hepatic polymerized tubulin and transferrin contents. These results suggested that impairment of polymerization of microtubules by alcohol intake resulted in
• x
=m
O
c_
0.1
x
®
x
.:cirrhosis
~: hepatitis
: fibrosis with or wib"mut fatty liver
20
r:0.562 p<0.01 n:29 40
60
80
polymerized tubulin Ipg/ghverl
FIG. 7. Correlation between hepatic polymerized tubulin and transferrin contents in alcoholic liver disease.
a decrease of the secretion of exportable proteins in human alcoholic liver disease. Albumin is also one of the exportable proteins. However, hepatic and serum albumin levels were not different among groups with liver diseases and the control group. Although the precise mechanism of the discrepancy between the changes of albumin and transferrin could not be clarified from the present study, two possible mechanisms will be considered. One is the difference of pool size, and another is the structural difference of both exportable proteins. In the animal experiment of Baraona et al. [2], albumin accumulated not only in the microsomal fraction but preferentially in the cytosol fraction of the hepatocytes, while transferrin was accumulated mainly in the microsomal fraction, suggesting that the cytosol could serve as a storage site of retained albumin but not transferrin and that a stored pool for albumin in the hepatocyte might be large. The big pool size of albumin may make unclear the effect of alcohol in humans. On the other hand, transferrin but not albumin is a glycoprotein, and glycosilation in or out of the Golgi apparatus may be necessary for its secretion from the liver [1 I1. Mitranic et al. [10] reported that an anti-microtubular agent such as colchicin impaired the glycosilation o f glycoprotein in the Golgi apparatus. As pointed out previously [3], acetaldehyde had the same effect as colchicin on the microtubules, suggesting that alcohol intake may inhibit the glycosilation of transferfin. Tuma et al. [17] reported that ethanol inhibited the in-
482
MATSUDA ET AL.
corporation of glucosamine to secretory glycoprotein in rat liver slices. Stiber et al. [15,16] found abnormal heterogeneity of serum transferrin in alcoholic patient during current abuse, and reported that this abnormality was caused by the reduction of sialic acid content in transferrin. Recently, Nei [12] found that the serum transferrin level decreased in alcoholic patients during the periods of alcohol intake and increased following alcohol abstinence, while serum levels of prealbumin, non-glycosylated exportable protein, was rather high during the periods of alcohol intake and decreased following alcohol abstinence. These studies suggest that alcohol intake may inhibit the glycosilation of transferrin and impair its secretion by disrupting microtubular function, and this possibility may explain the discrepancy of the results concerning both exportable proteins. We recently reported that, in the experimental "alcohol pyrazole hepatitis" of rat, the ballooning and necrosis of the hepatocytes might result from a decrease in hepatic microtubules and an increase in exportable protein which is caused by an increased production of acetaldehyde in the
non-alcohol dehydrogenase pathway [8]. In this study ballooned hepatocytes were prominent in alcoholic liver injury with or without necrosis, indicating that acetaldehyde produced in the non-alcohol dehydrogenase pathway may also play an important role in the development of human alcoholic liver disease. From the results of the present study, it was suggested that an impairment of hepatic microtubular polymerization resulted in the retention of hepatic export proteins in hepatocytes and finally in ballooning or necrosis of the hepatocytes in human alcoholic liver disease. This process may be one of the pathogenetic mechanisms in the development of human alcoholic liver injury.
ACKNOWLEDGMENTS This work was supported by Research Grant No. 54183 administrated by the Educational Ministry of Japan. The expert technical assistance of Ms. K. Sugata and Ms. A. Yamatani is gratefully acknowledged.
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