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Decrease of a hepatic binding protein specific for asialoglycoproteins with accumulation of serum asialoglycoproteins in galactosamine-treated rats
GASTROENTEROLOGY
1981;81:527-33
Decrease of a Hepatic Binding Protein Specific for Asialoglycoproteins with Accumulation of Serum Asialoglycoproteins in GalactosamineTreated Rats T. SAWAMURA, Y. SAMESHIMA,
S. KAWASATO, Y. SHIOZAKI, H. NAKADA, and Y. TASHIRO
Third Department of Medicine and Department University, Moriguchi, Osaka, Japan
Acute liver injury was induced experimentally in rats by a single injection of a large amount of D-g@ lactosamine. Hepatocelluiar damage was apparent from decrease of total serum proteins, marked release of transaminases into the circulation, precipitous decrease of total microsomal proteins, and intracellular enzymes such as cytochrome I’,,, and S-nucieotidase. In parallel with such hepatocellular damage, serum asialoglycoproteins accumulated markedly, reaching a maximum level 4 days after the injection and then decreased to the control level. In contrast to this increase, hepatic binding protein, a receptor in the liver which specifically recognizes asialoglycoproteins, decreased very much. At the same time, survival time of j-?]asialoorosomucoid intravenously administered into rats was much prolonged in inverse proportion to the decrease of the binding protein. From these results it was concluded that the decrease of the hepatic binding protein induced by galactosamine treatment is probably re-
Received December 29,1989. Accepted April 15, 1981. Address requests for reprints to: Dr. T. Sawamura, Third Department of Medicine, Kansai Medical University, Moriguchi, Osaka 570, Japan. This work was partly supported by a Grant-in-Aid for Scientific Research and for Special Project Research from the Ministry of Education, Science and Culture, Japan. This work was presented in part at the 11th International Congress of Gastroenterology, Hamburg (abstract). 1980:June 8-13. Sawamura et al. Hepato-gastroenterol (Suppl) 198&215. We are deeply grateful to Dr. Ichio Honjo, President of Kansai Medical University for his encouragement. We thank Miss K. Miki for her assistance with the manuscript. 0 1981 by the American Gastroenterological Association 0016-5085/81/999527-07$02.50
of Physiology,
Kansai Medical
sponsible for the marked asialoglycoproteins.
accumulation
of serum
The biologic significance of the presence of carbohydrate chains in serum glycoproteins has remained obscure for a long time. Ashwell and Morel1 have shown that exposure of the penultimate galactosyl residue of the carbohydrate chain by removal of the terminal sialic acid residue results in rapid clearance of many serum glycoproteins from the circulation and that the asialoglycoproteins thus cleared are recovered in the liver (1).A receptor protein specific for asialoglycoproteins has been found and subsequently isolated from livers of the rabbit (2,3)and rat (4-6), and it has been reported that this binding protein is localized exclusively in hepatocytes (7-g). There are several reports indicating that marked accumulation of asialoglycoproteins occurs in the serum of patients with hepatic disease such as liver cirrhosis and cancer (10-14). It is strongly suggested, therefore, that some intimate correlation should exist between the binding protein in hepatocytes and accumulation of serum asialoglycoproteins. No direct evidence, however, has been published so far concerning the relationship. In the present paper acute liver cell injury was induced experimentally by administration of D-galactosamine as first described by Keppler et al. (15) and Reutter et al. (16).It was revealed that serum asialoglycoproteins were accumulated markedly and the clearance from circulation of [““I]asialoorosomucoid intravenously injected into rats was delayed in parallel with the decrease in the amount of hepatic binding protein. We conclude, therefore, that the decrease of the hepatic binding protein is probably re-
526
GASTROENTEROLOGY
SAWAMLJRA ET AL.
sponsible proteins.
for the accumulation
of serum
Vol. 81.No.3
asialoglyco-
Materials and Methods Animals
and Galactosamine
Treatment
Male Wistar rats weighing 180-200 g were used throughout our experiments. They were fed standard laboratory chow diets (Oriental Yeast Mfg. LTD., Japan) and drinking water ad libitum. D-Galactosamine (0.9 g/kg body wt) was administered by a single intraperitoneal injection. Rats were anesthetized with ether, and blood samples were taken from a jugular vein. An aliquot of the serum was dialyzed against 0.01 M Tris-HCl, pH 7.8, for the measurement of serum asialogiycoproteins because glucose is a competitive inhibitor of the binding assay.
Preparation
of Microsomes
Rat livers were perfused with cold 1.15% KC1 and homogenized with a Teflon-glass homogenizer in 4 volumes of ice-cold 0.25 M sucrose TKM (0.05 M Tris-HCl, pH 7.4, 0.025 M KCl, and 0.005 M MgCl,). The homogenate was centrifuged at 10,000 g for 10 min, and the supernatant was spun for 90 min at 105,000 g as described by Ehrenreich et al. (17). The resulting microsomal pellets were resuspended in TKM and centrifuged to remove sucrose.
Proteins Human orosomucoid, asialoorosomucoid, and [‘?]asialoorosomucoid were prepared as described previously (8). The rabbit liver binding protein specific for asialoglycoproteins was prepared as described by Pricer and Ashwell (4).
Binding
Assay
The binding assay was carried out according to the “Assay A” procedure of Hudgin et al. (2). The binding ability of total microsomes was expressed by specific binding activity which is defined as nanograms of asialoorosomucoid bound per microgram of protein as used by Pricer and Ashwell (4).
of the Amount Determination Asialoglycoproteins
of Serum
The amount of serum asialoglycoproteins was measured indirectly by the inhibition assay as follows: Rabbit binding protein (4 pg) was preincubated for 15 min at 25’C with various amounts of asialoorosomucoid or dialyzed rat serum, and then [‘251]asialoorosomucoid (1 pg) was added. Subsequently the binding assay was carried out as described above. The amount of serum asialoglycoproteins was estimated from the standard inhibition curve (Figure 1) and was expressed in terms of
I
100 Asialoorosomucoid
1
300
200 added
(ngl0.5ml)
Figure 1. Inhibition of the binding of [‘251]asialoorosomucoid to rabbit binding protein by the addition of cold asialoorosomucoid. Rabbit binding protein (4 pg) was preincubated for 15 min at 25” C with various amounts of cold asialoorosomucoid and then incubated with [‘Z51]asialoorosomucoid, and inhibition of the binding of [1251]asialoorosomucoid by the cold asialoorosomucoid was measured. For the determination of asialoglycoproteins in rat serum, rabbit binding protein was preincubated with dialyzed rat serum (usually 50~1) and then incubated with [‘Z51]asialoorosomucoid. From and degree of the inhibition of the binding of [1251]asialoorosomucoid, the amount of serum asialoglycoproteins was estimated. The amount of the dialyzed serum was adjusted so as to use the optimal range of SO?&-50%inhibition of the standard curve.
nanogram asialoorosomucoid equivalents, i.e. nanograms of asialoorosomucoid required to cause equivalent inhibition of the binding reaction. The amount of serum asialoglycoproteins thus determined could be a minimum estimate, because asialoorosomucoid is one of the most potent ligands for the binding protein (1). As shown in Figure 1, the binding activity of rabbit binding protein (4 pg) decreased in proportion to the amount of asialoorosomucoid added to the incubated solutions, and asialoorosomucoid of 214 ng/ml in the incubation solution was enough to inhibit 50% of the binding activity. Conventionally, 50 ~1 of dialyzed serum was used for the preincubation. If the inhibition of the binding was at the optimal range of 30-50%, the data obtained were used. If not, the volume of the serum was adjusted so as to bring the degree of inhibition to the above optimal range.
Other Assays The activities of serum glutamic-pyruvic trans(SGPT) (EC 2.6.1.2) and glutamic-oxaloacetic transaminase (SGOT) (EC 2.6.1.1.) were determined by the aminase
procedure of Reitman and Frankel (18), and were expressed in Karmen units (19). Cytochrome P,, and 5’-nucleotidase were measured as described by Omura and Sato (20), and Heppel and Hilmoe (21), respectively. Pro-
September
BINDING PROTEIN AND GALACTOSAMINE
1981
Changes in Serum Components Galactosamine
i ,
529
induced by
Figure 2 shows changes in serum transaminase activities and the amount of serum proteins. The SGOT activity increased rapidly and reached a maximum value of about 10 times the control value 3 days after galactosamine administration. Then the activity decreased and returned to the original level after 7 days. The SGPT activity showed a similar response except that the maximum activity was about 30 times higher than that of the control. Serum protein decreased gradually to a minimum value of 66.6% of the control after 3 days, and then it increased to the control level.
10 -
h
-0 . 2
HEPATITIS
8-
Liver Injury by Galactosamine
I4 j
I
I
I
1
2
3
Days
after
I
I
4
5
galactosamine
6
7 injection
Figure 2. Galactosamine-induced changes of rat SGOT, SGPT activities, and serum protein concentration. Male Wistar rats weighing between ~30 and 200 g received single intraperitoneal injection of galactoeamine (0.9 g/kg body wt). The blood samples were taken from the jugular vein to determine the concentration of total serum proteins in g/d1 0, glutamic-oxaloacetic (O), and glutamic-pyruvic transaminase (A) activities in Karmen units (19). The vertical bars on each point of Figures 24 show standard deviation. The values in Figures 2-4 are an average of five or six experiments.
tein was determined (22) with crystalline
by the micro-method of Lowry et al. bovine albumin as the standard.
Hepatic Clearance of ‘251-Asialoorosomucoid from the Circulation [‘26I]Asialoorosomucoid (50 &lOO g body wt) was injected intravenously via the jugular vein of normal and galactosamine-treated rats under ether anesthesia. Blood samples (0.2 ml) were taken from another jugular vein with heparinized syringes and centrifuged. The radioactivity of each plasma specimen (50 pl) was counted in a Packard 5320 autogamma scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.).
Results First we examined the sequential changes in rat serum and liver after a single intraperitoneal injection of D-galactosamine. For this purpose the biochemical changes in serum components and in the liver itself were investigated daily for 7 days after the injection.
The changes of microsomal protein and cytoactivity were also chrome P4M, and 5’-nucleotidase measured after the administration of galactosamine (Figure 3). In agreement with the results of Gallenkamp et al. (23) cytochrome P, content in liver decreased to a minimum value of 7.6% of the control value (0.059 f 0.011 nmol/mg protein) after 3 days and then increased gradually toward the control value after 7 days. 5’-Nucleotidase activity and the amount of microsomal protein also showed similar responses. The minimum value of the former was about 30% of the control value (0.047 f 0.004 pmol/ min/mg protein), and the latter was about 55% of the control (7.48 mg/g wet wt of liver). Changes in the Amounts of Serum Asialoglycoproteins and Hepatic Binding Protein We next examined the amount of serum asialoglycoproteins and hepatic binding protein after a single injection of D-galactosamine. As shown in Figure 4, the amount of serum asialoglycoproteins increased markedly after administration of galactosamine, and it reached a maximum value of about 20 times the control value after 4 days (42.46 f 8.93 ng/ 10 ~1). Then it decreased rapidly to the control value. By contrast the amount of the hepatic binding protein specific for asialoglycoproteins decreased precipitously to a minimum value of about 10% of the control value (0.011 U) after 3 days. Then it increased toward the control value in parallel to the increase in 5’-nucleotidase activity and cytochrome P, content. From Figure 4 it is apparent that the decrease in the amount of the binding protein precedes the increase in the amount of serum asialoglycoproteins, the former reaching its minimum level 1 day before the latter reached its maximum level.
530
SAWAMURA
GASTROENTEROLOGY
ET AL.
Vol. 81. No. 3
-c
Gajactosamine-induced changes in the amounts of rat liver cytochrome Paso, 5’nucleotidase, and total microsomal proteins. The livers of galactosamine-treated rats were homogenized and microsomes were prepared according to Ehrenreich et al. (17). The amount of total microsomal proteins in mg per g liver 0, cytochrome P,,, in nmol/mg protein (a), and Y-nucleotidase activity in pmol of inorganic phosphate liberated/min/mg protein (A) were determined as described by Lowry et al. (ZZ), Omura and Sato (ZO), and Heppel and Hilmoe (Zl), respectively.
-0
-0
-0
Days
after
galactosamine
injection
Clearance of [‘Z51]Asialoorosomucoid Circulation
from
The above result suggests that accumulation of serum asialoglycoproteins is exclusively due to the decrease in the binding protein of rat liver. So we studied the clearance of [‘“SI]asialoorosomucoid which was injected intravenously into the control and galactosamine treated rats. As shown in Figure 5, [““I]asialoorosomucoid disappeared rapidly from the circulation in control rats. In galactosaminetreated rats, however, the clearance of [“‘I]asialoorosomucoid was much delayed, and 3 days after injection, when the amount of the binding protein decreased to a minimum level, the half-life of [“SIlasialoorosomucoid injected into the galactosamine-treated rats was prolonged to more than 10 times that of the control rats. Then the rate of disappearance gradually increased in parallel with the recovery of the binding protein in liver and returned to an almost normal rate after 7 days. If the hepatic binding protein is exclusively responsible for the removal of serum asialoglycoproteins from the circulation, the clearance of [‘““I]asialoorosomucoid as expressed by the reciprocal of the half-life should be proportional to the total amount of the hepatic binding protein. Figure 6 does show that the clearance is approximately proportional to the specific activity or to the total binding activity of the microsome fraction (specific binding activity multiplied by the total amount of micro-
somal protein). Since the clearance of asialoorosomucoid by the liver is mediated by cell-surface hepatic binding protein, which constitutes only about 5% of the total amount of hepatic binding protein in hepatocytes (24) and the microsomal fraction contains approximately 80% of the total binding activity (4), the binding activity of this fraction could be used as representative of the total.
Discussion When a large amount of D-galactosamine was injected into rats, the livers were severely injured as has been reported by Keppler et al. (15,16,25) and others (28,27). Hepatocellular injury is clearly indicated by a marked release of transaminases into the circulation and by the decrease of serum proteins. Marked decreases of total microsomal protein and several marker enzymes such as cytochrome P,,, and 5’-nucleotidase were also clear indications of the hepatocellular damage. In addition, an extensive accumulation of asialoglycoproteins in rat serum was observed (Figure 4). This finding is consistent with the clinical observations that serum asialoglycoproteins markedly accumulate in the serum of patients with liver disease such as cirrhosis and cancer (10-14). Since it has been reported that the binding protein specific for asialoglycoproteins is localized exclusively in hepatocytes (7-g), the increase in serum
September
BINDING PROTEIN AND GALACTOSAMINE
1983
1 i
,” I A s lr’ ,
._
-
‘3
I
HEPATITIS
531
protein is synthesized on membrane-bound ribosomes, rapidly inserted into the endoplasmic membranes, glycosylated, and then transported via the Golgi apparatus to the plasma membrane (26,29). A decrease in the amount of any intracellular protein could be due to either a decrease in the rate of biosynthesis or increase in the rate of degradation of the protein or both. Morphologic observations have shown that marked and diffuse hepatocellular damage appears rapidly in the livers of galactosaminetreated rats (15,25-27), and biochemical observations have shown that a marked decrease in the amount of polyribosomes in rat liver is already apparent 4 h after galactosamine treatment (27). Since the half-life of rat liver binding protein has been reported to be -68 h (5) inhibition of the biosynthesis of the binding protein alone could not explain the rapid decrease in the amount of the hepatic binding protein after galactosamine treatment. The precipitous decrease of the binding protein is, therefore, probably due not only to marked inhibition of the biosynthesis of the hepatic binding protein but also accelerated degradation of the protein, and this decrease
Q)
,”
01234567 Days
after
galactosamine
injection
amounts of serum asialoglycoprotein and hepatic binding protein. The amount of serum asialoglycoproteins in ng/lO ~1 serum 0 was determined by the binding inhibition assay as shown in Figure 1. The amount of binding protein (0) was determined by the “Assay A” procedure of Hudgin et al (2). The unit of specific binding activity is ng of asialoorosomucoid bound/pg of protein as defined by Pricer and Ashwell (4).
Figure 4. Galactosamine-induced
changes in the
asialoglycoproteins could be due to either a decrease in the clearance by the hepatic binding protein and/ or increased formation of asialoglycoproteins. The present experiment has clearly shown that the hepatic binding protein markedly decreased in amount after the galactosamine treatment in inverse proportion to the rapid accumulation of asialoglycoproteins in the circulation. It was also revealed that the clearance of [““Ilasialoorosomucoid from the circulation is very much delayed after the treatment. Further, the clearance of asialoglycoprotein as expressed by the reciprocal of the half-life of injected [‘Z”I]asialoglycoprotein was roughly proportional to the amount of the hepatic binding protein (Figure 6). We conclude, therefore, that the decrease in the amount of binding protein in rat liver induced by Dgalactosamine administration is mainly responsible for the accumulation of serum asialoglycoproteins in the treated rats. Recently we have shown that rat liver binding
100 80 60 40
1 5
10
15 Minutes
20 after
25 injection
Figure 5. Disappearance of [‘Z51]asialoorosomucoid from circulation after treatment with n-galactosamine. [1251]Asialoorosomucoid (50 &lOO g body wt) was injected intravenously, and the blood samples were taken to measure the radioactivities. Each point is the average value obtained from two animals. Curve 1, control rat; curve 2.3 days; curve $5 days; and curve 4,7 days after galactosamine treatment.
532
SAWAMURA
ET AL.
GASTROENTEROLOGY
Total
,
0;Z
0:4
binding 0:6
activity
0:8
‘1,;
(
Vol. 81. No. 3
?? ) 1;2
l:‘(
1:6
I
between the rate of disFigure 6. Relationship appearance of [‘*51]asialoorosomucoid in rat serum as expressed by the reciprocal of the half-life and the specific (0) and total 0 binding activities of liver microsomes. Specific binding activity is defined as ng of asialoorosomucoid bound/pg of protein. Total binding activities were calculated by multiplying the specific binding activity with the total amount of microsomal proteins.
Specific
binding
activity
(
?? )
might be responsible for the marked accumulation of serum asialoglycoproteins after administration of D-galactosamine intO rats. The results of the present studies suggest that the marked accumulation of asialoglycoproteins in the serum of patients with liver disease such as cirrhosis and cancer (10-14) is induced by a decrease in the amount of hepatic binding protein in the liver. Asialoglycoproteins, however, could be accumulated by other mechanisms. We have not proved this point, because many phenomena other than changes in hepatic binding protein occur in galactosamine-induced acute hepatocellular injury. Clinical investigations on the asialoglycoproteinemia in hepatic diseases are now in progress in our laboratory.
References 1. Ashwell G, Morel1 AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol 1974;41:99-128. 2. Hudgin RL, Pricer WE, Ashwell G, et al. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol Chem 1974;249:5536-43. 3. Kawasaki T, Ashwell G. Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins. J Biol Chem 1976;251:1296-392. 4. Pricer WE, Ashwell G. Subcellular distribution of a mammalian hepatic binding protein specific for asialoglycoproteins. J Biol Chem 1978;251:7539-44. 5. Tanabe T, Pricer WE, Ashwell G. Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. J Biol Chem 1979;254:1038-43,
6, Sawamura
7.
8.
9.
10.
11.
12.
13.
14. 15.
16. 17.
T, Nakada H, Fujii-Kuriyama Y, Tashiro Y. Some properties of a binding protein specific for asialoglycoproteins and its distribution in rat liver microsomes. Cell Struct Funct 1986;5:133-46. Morel1 AG, Irvine RA, Sternleib I, et al. Physical and chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 1968;243:155-9. Hubbard AL, Wilson G, Ashwell G, Stukenbrok H. An electron microscope autoradiographic study of the carbohydrate recognition system in rat liver. I. Distribution of ‘Z51-ligands among the liver cell type. J Cell Biol 1979:83:47&l. Steer CJ, Clarenburg R. Unique distribution of glycoprotein receptors on parenchymal and sinusoidal cells of rat liver. J Biol Chem 1979;254:4457-61. Marshall JS, Green AM. Studies on human thyroxine-binding globulin. VI. The nature of slow thyroxine-binding globulin. J Clin Invest. 1972;51:3173-81. Marshall JS, Green AM, Pensky J, et al. Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage. J Clin Invest 1977;54:555-62. Marshall JS, Williams ST, Hepner GW. Serum desialylated glycoprotein (dGP) in patients with hepatobiliary dysfunction comparison with other liver function tests (abstr). Gastroenterology 1975:69:844. Marshall JS, Williams ST. Serum inhibitors of desialylated glycoprotein binding to hepatocyte membranes. Biochim Biophys Acta 1978;543:41-52. Sobue G, Kosaka A. Asialoglycoproteinemia in a case of primary hepatic cancer. Hepato-Gastroenterol 198&27:266-3. Keppler D, Lesch R, Reutter W, Decker K. Experimental hepatitis induced by o-galactosamine. Exp Mol Path01 1988;9:27999. Reutter W, Lesch R, Keppler D, Decker K. Galaktosamin Hepatitis. Naturwisschenschaften 1968;55:497-8. Ehrenreich JH, Bergeron JJM, Siekevitz P, Palade GE. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol 1973;59:45-72.
September
1981
18.Reitman S, Frankel S. A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Path01 1957;28:58-63. 19. Karmen A. A note on the spectrophotometric assay of glutamic oxalacetic transaminase in human blood serum. J Clin Invest 1955;34:131-3. 20. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomea. I. Evidence for its hemoprotein nature. J Biol Chem 1984;239:2379-85. 21. Heppel LA, Hilmoe RJ. “5”Nucleotidases. In: Colowick SP, Kaplan N, eds. Methods in enzymology. Vol. 2. New York: Academic Press Inc., 1955546-50. 22. Lowry OH, Rosebrough NJ, Farr AL. Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-75. 23. Gallenkamp H, Brachtel D, Richter E. Influence of D-Galactosamine hydrochloride on microsomal phoepholipid, protein and cytochrome P-450 content in guinea pig liver. Acta Hepato-Gastroenterol 1974;21:101-5.
BINDING PROTEIN AND GALACTOSAMINE
HEPATITIS
533
24. Steer C, Ashwell G. Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. J Biol Chem 1980; 255:3(X%13. 25. Decker K, Keppler D. Galactosamine induced liver injury. In: Popper H, Schaffner F, eds. Progress in liver diseases. Vol. 4. New York: Grune and Stratton, Inc., 1972:183-99 26. Medline A, Schaffner F, Popper H. Ultrastructural features in galactosamine-induced hepatitis. Exp Mol Path01 1970;1220111. 27. Shinozuka H, Farber JL, Koniehi Y, Anukarahanonta T. D-Galactosamlne and acute liver cell injury. Fed Proc 1973; 32151826. 28. Nakada H, Sawamura T, Aoi K, et al. Synthesis and intracellular transport of a binding protein specific for asialoglycoproteins in rat hepatocytes (abstr). Eur J Cell Bioll980;22:155. 29. Nakada H, Sawamura T, Tashiro Y. Biosynthesis and insertion of a hepatic binding protein specific for asialoglycoproteins into rough endoplasmic reticulum membranes. J Biothem 1981;89:135-41.
1981;81:527-33
Decrease of a Hepatic Binding Protein Specific for Asialoglycoproteins with Accumulation of Serum Asialoglycoproteins in GalactosamineTreated Rats T. SAWAMURA, Y. SAMESHIMA,
S. KAWASATO, Y. SHIOZAKI, H. NAKADA, and Y. TASHIRO
Third Department of Medicine and Department University, Moriguchi, Osaka, Japan
Acute liver injury was induced experimentally in rats by a single injection of a large amount of D-g@ lactosamine. Hepatocelluiar damage was apparent from decrease of total serum proteins, marked release of transaminases into the circulation, precipitous decrease of total microsomal proteins, and intracellular enzymes such as cytochrome I’,,, and S-nucieotidase. In parallel with such hepatocellular damage, serum asialoglycoproteins accumulated markedly, reaching a maximum level 4 days after the injection and then decreased to the control level. In contrast to this increase, hepatic binding protein, a receptor in the liver which specifically recognizes asialoglycoproteins, decreased very much. At the same time, survival time of j-?]asialoorosomucoid intravenously administered into rats was much prolonged in inverse proportion to the decrease of the binding protein. From these results it was concluded that the decrease of the hepatic binding protein induced by galactosamine treatment is probably re-
Received December 29,1989. Accepted April 15, 1981. Address requests for reprints to: Dr. T. Sawamura, Third Department of Medicine, Kansai Medical University, Moriguchi, Osaka 570, Japan. This work was partly supported by a Grant-in-Aid for Scientific Research and for Special Project Research from the Ministry of Education, Science and Culture, Japan. This work was presented in part at the 11th International Congress of Gastroenterology, Hamburg (abstract). 1980:June 8-13. Sawamura et al. Hepato-gastroenterol (Suppl) 198&215. We are deeply grateful to Dr. Ichio Honjo, President of Kansai Medical University for his encouragement. We thank Miss K. Miki for her assistance with the manuscript. 0 1981 by the American Gastroenterological Association 0016-5085/81/999527-07$02.50
of Physiology,
Kansai Medical
sponsible for the marked asialoglycoproteins.
accumulation
of serum
The biologic significance of the presence of carbohydrate chains in serum glycoproteins has remained obscure for a long time. Ashwell and Morel1 have shown that exposure of the penultimate galactosyl residue of the carbohydrate chain by removal of the terminal sialic acid residue results in rapid clearance of many serum glycoproteins from the circulation and that the asialoglycoproteins thus cleared are recovered in the liver (1).A receptor protein specific for asialoglycoproteins has been found and subsequently isolated from livers of the rabbit (2,3)and rat (4-6), and it has been reported that this binding protein is localized exclusively in hepatocytes (7-g). There are several reports indicating that marked accumulation of asialoglycoproteins occurs in the serum of patients with hepatic disease such as liver cirrhosis and cancer (10-14). It is strongly suggested, therefore, that some intimate correlation should exist between the binding protein in hepatocytes and accumulation of serum asialoglycoproteins. No direct evidence, however, has been published so far concerning the relationship. In the present paper acute liver cell injury was induced experimentally by administration of D-galactosamine as first described by Keppler et al. (15) and Reutter et al. (16).It was revealed that serum asialoglycoproteins were accumulated markedly and the clearance from circulation of [““I]asialoorosomucoid intravenously injected into rats was delayed in parallel with the decrease in the amount of hepatic binding protein. We conclude, therefore, that the decrease of the hepatic binding protein is probably re-
526
GASTROENTEROLOGY
SAWAMLJRA ET AL.
sponsible proteins.
for the accumulation
of serum
Vol. 81.No.3
asialoglyco-
Materials and Methods Animals
and Galactosamine
Treatment
Male Wistar rats weighing 180-200 g were used throughout our experiments. They were fed standard laboratory chow diets (Oriental Yeast Mfg. LTD., Japan) and drinking water ad libitum. D-Galactosamine (0.9 g/kg body wt) was administered by a single intraperitoneal injection. Rats were anesthetized with ether, and blood samples were taken from a jugular vein. An aliquot of the serum was dialyzed against 0.01 M Tris-HCl, pH 7.8, for the measurement of serum asialogiycoproteins because glucose is a competitive inhibitor of the binding assay.
Preparation
of Microsomes
Rat livers were perfused with cold 1.15% KC1 and homogenized with a Teflon-glass homogenizer in 4 volumes of ice-cold 0.25 M sucrose TKM (0.05 M Tris-HCl, pH 7.4, 0.025 M KCl, and 0.005 M MgCl,). The homogenate was centrifuged at 10,000 g for 10 min, and the supernatant was spun for 90 min at 105,000 g as described by Ehrenreich et al. (17). The resulting microsomal pellets were resuspended in TKM and centrifuged to remove sucrose.
Proteins Human orosomucoid, asialoorosomucoid, and [‘?]asialoorosomucoid were prepared as described previously (8). The rabbit liver binding protein specific for asialoglycoproteins was prepared as described by Pricer and Ashwell (4).
Binding
Assay
The binding assay was carried out according to the “Assay A” procedure of Hudgin et al. (2). The binding ability of total microsomes was expressed by specific binding activity which is defined as nanograms of asialoorosomucoid bound per microgram of protein as used by Pricer and Ashwell (4).
of the Amount Determination Asialoglycoproteins
of Serum
The amount of serum asialoglycoproteins was measured indirectly by the inhibition assay as follows: Rabbit binding protein (4 pg) was preincubated for 15 min at 25’C with various amounts of asialoorosomucoid or dialyzed rat serum, and then [‘251]asialoorosomucoid (1 pg) was added. Subsequently the binding assay was carried out as described above. The amount of serum asialoglycoproteins was estimated from the standard inhibition curve (Figure 1) and was expressed in terms of
I
100 Asialoorosomucoid
1
300
200 added
(ngl0.5ml)
Figure 1. Inhibition of the binding of [‘251]asialoorosomucoid to rabbit binding protein by the addition of cold asialoorosomucoid. Rabbit binding protein (4 pg) was preincubated for 15 min at 25” C with various amounts of cold asialoorosomucoid and then incubated with [‘Z51]asialoorosomucoid, and inhibition of the binding of [1251]asialoorosomucoid by the cold asialoorosomucoid was measured. For the determination of asialoglycoproteins in rat serum, rabbit binding protein was preincubated with dialyzed rat serum (usually 50~1) and then incubated with [‘Z51]asialoorosomucoid. From and degree of the inhibition of the binding of [1251]asialoorosomucoid, the amount of serum asialoglycoproteins was estimated. The amount of the dialyzed serum was adjusted so as to use the optimal range of SO?&-50%inhibition of the standard curve.
nanogram asialoorosomucoid equivalents, i.e. nanograms of asialoorosomucoid required to cause equivalent inhibition of the binding reaction. The amount of serum asialoglycoproteins thus determined could be a minimum estimate, because asialoorosomucoid is one of the most potent ligands for the binding protein (1). As shown in Figure 1, the binding activity of rabbit binding protein (4 pg) decreased in proportion to the amount of asialoorosomucoid added to the incubated solutions, and asialoorosomucoid of 214 ng/ml in the incubation solution was enough to inhibit 50% of the binding activity. Conventionally, 50 ~1 of dialyzed serum was used for the preincubation. If the inhibition of the binding was at the optimal range of 30-50%, the data obtained were used. If not, the volume of the serum was adjusted so as to bring the degree of inhibition to the above optimal range.
Other Assays The activities of serum glutamic-pyruvic trans(SGPT) (EC 2.6.1.2) and glutamic-oxaloacetic transaminase (SGOT) (EC 2.6.1.1.) were determined by the aminase
procedure of Reitman and Frankel (18), and were expressed in Karmen units (19). Cytochrome P,, and 5’-nucleotidase were measured as described by Omura and Sato (20), and Heppel and Hilmoe (21), respectively. Pro-
September
BINDING PROTEIN AND GALACTOSAMINE
1981
Changes in Serum Components Galactosamine
i ,
529
induced by
Figure 2 shows changes in serum transaminase activities and the amount of serum proteins. The SGOT activity increased rapidly and reached a maximum value of about 10 times the control value 3 days after galactosamine administration. Then the activity decreased and returned to the original level after 7 days. The SGPT activity showed a similar response except that the maximum activity was about 30 times higher than that of the control. Serum protein decreased gradually to a minimum value of 66.6% of the control after 3 days, and then it increased to the control level.
10 -
h
-0 . 2
HEPATITIS
8-
Liver Injury by Galactosamine
I4 j
I
I
I
1
2
3
Days
after
I
I
4
5
galactosamine
6
7 injection
Figure 2. Galactosamine-induced changes of rat SGOT, SGPT activities, and serum protein concentration. Male Wistar rats weighing between ~30 and 200 g received single intraperitoneal injection of galactoeamine (0.9 g/kg body wt). The blood samples were taken from the jugular vein to determine the concentration of total serum proteins in g/d1 0, glutamic-oxaloacetic (O), and glutamic-pyruvic transaminase (A) activities in Karmen units (19). The vertical bars on each point of Figures 24 show standard deviation. The values in Figures 2-4 are an average of five or six experiments.
tein was determined (22) with crystalline
by the micro-method of Lowry et al. bovine albumin as the standard.
Hepatic Clearance of ‘251-Asialoorosomucoid from the Circulation [‘26I]Asialoorosomucoid (50 &lOO g body wt) was injected intravenously via the jugular vein of normal and galactosamine-treated rats under ether anesthesia. Blood samples (0.2 ml) were taken from another jugular vein with heparinized syringes and centrifuged. The radioactivity of each plasma specimen (50 pl) was counted in a Packard 5320 autogamma scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.).
Results First we examined the sequential changes in rat serum and liver after a single intraperitoneal injection of D-galactosamine. For this purpose the biochemical changes in serum components and in the liver itself were investigated daily for 7 days after the injection.
The changes of microsomal protein and cytoactivity were also chrome P4M, and 5’-nucleotidase measured after the administration of galactosamine (Figure 3). In agreement with the results of Gallenkamp et al. (23) cytochrome P, content in liver decreased to a minimum value of 7.6% of the control value (0.059 f 0.011 nmol/mg protein) after 3 days and then increased gradually toward the control value after 7 days. 5’-Nucleotidase activity and the amount of microsomal protein also showed similar responses. The minimum value of the former was about 30% of the control value (0.047 f 0.004 pmol/ min/mg protein), and the latter was about 55% of the control (7.48 mg/g wet wt of liver). Changes in the Amounts of Serum Asialoglycoproteins and Hepatic Binding Protein We next examined the amount of serum asialoglycoproteins and hepatic binding protein after a single injection of D-galactosamine. As shown in Figure 4, the amount of serum asialoglycoproteins increased markedly after administration of galactosamine, and it reached a maximum value of about 20 times the control value after 4 days (42.46 f 8.93 ng/ 10 ~1). Then it decreased rapidly to the control value. By contrast the amount of the hepatic binding protein specific for asialoglycoproteins decreased precipitously to a minimum value of about 10% of the control value (0.011 U) after 3 days. Then it increased toward the control value in parallel to the increase in 5’-nucleotidase activity and cytochrome P, content. From Figure 4 it is apparent that the decrease in the amount of the binding protein precedes the increase in the amount of serum asialoglycoproteins, the former reaching its minimum level 1 day before the latter reached its maximum level.
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SAWAMURA
GASTROENTEROLOGY
ET AL.
Vol. 81. No. 3
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Gajactosamine-induced changes in the amounts of rat liver cytochrome Paso, 5’nucleotidase, and total microsomal proteins. The livers of galactosamine-treated rats were homogenized and microsomes were prepared according to Ehrenreich et al. (17). The amount of total microsomal proteins in mg per g liver 0, cytochrome P,,, in nmol/mg protein (a), and Y-nucleotidase activity in pmol of inorganic phosphate liberated/min/mg protein (A) were determined as described by Lowry et al. (ZZ), Omura and Sato (ZO), and Heppel and Hilmoe (Zl), respectively.
-0
-0
-0
Days
after
galactosamine
injection
Clearance of [‘Z51]Asialoorosomucoid Circulation
from
The above result suggests that accumulation of serum asialoglycoproteins is exclusively due to the decrease in the binding protein of rat liver. So we studied the clearance of [‘“SI]asialoorosomucoid which was injected intravenously into the control and galactosamine treated rats. As shown in Figure 5, [““I]asialoorosomucoid disappeared rapidly from the circulation in control rats. In galactosaminetreated rats, however, the clearance of [“‘I]asialoorosomucoid was much delayed, and 3 days after injection, when the amount of the binding protein decreased to a minimum level, the half-life of [“SIlasialoorosomucoid injected into the galactosamine-treated rats was prolonged to more than 10 times that of the control rats. Then the rate of disappearance gradually increased in parallel with the recovery of the binding protein in liver and returned to an almost normal rate after 7 days. If the hepatic binding protein is exclusively responsible for the removal of serum asialoglycoproteins from the circulation, the clearance of [‘““I]asialoorosomucoid as expressed by the reciprocal of the half-life should be proportional to the total amount of the hepatic binding protein. Figure 6 does show that the clearance is approximately proportional to the specific activity or to the total binding activity of the microsome fraction (specific binding activity multiplied by the total amount of micro-
somal protein). Since the clearance of asialoorosomucoid by the liver is mediated by cell-surface hepatic binding protein, which constitutes only about 5% of the total amount of hepatic binding protein in hepatocytes (24) and the microsomal fraction contains approximately 80% of the total binding activity (4), the binding activity of this fraction could be used as representative of the total.
Discussion When a large amount of D-galactosamine was injected into rats, the livers were severely injured as has been reported by Keppler et al. (15,16,25) and others (28,27). Hepatocellular injury is clearly indicated by a marked release of transaminases into the circulation and by the decrease of serum proteins. Marked decreases of total microsomal protein and several marker enzymes such as cytochrome P,,, and 5’-nucleotidase were also clear indications of the hepatocellular damage. In addition, an extensive accumulation of asialoglycoproteins in rat serum was observed (Figure 4). This finding is consistent with the clinical observations that serum asialoglycoproteins markedly accumulate in the serum of patients with liver disease such as cirrhosis and cancer (10-14). Since it has been reported that the binding protein specific for asialoglycoproteins is localized exclusively in hepatocytes (7-g), the increase in serum
September
BINDING PROTEIN AND GALACTOSAMINE
1983
1 i
,” I A s lr’ ,
._
-
‘3
I
HEPATITIS
531
protein is synthesized on membrane-bound ribosomes, rapidly inserted into the endoplasmic membranes, glycosylated, and then transported via the Golgi apparatus to the plasma membrane (26,29). A decrease in the amount of any intracellular protein could be due to either a decrease in the rate of biosynthesis or increase in the rate of degradation of the protein or both. Morphologic observations have shown that marked and diffuse hepatocellular damage appears rapidly in the livers of galactosaminetreated rats (15,25-27), and biochemical observations have shown that a marked decrease in the amount of polyribosomes in rat liver is already apparent 4 h after galactosamine treatment (27). Since the half-life of rat liver binding protein has been reported to be -68 h (5) inhibition of the biosynthesis of the binding protein alone could not explain the rapid decrease in the amount of the hepatic binding protein after galactosamine treatment. The precipitous decrease of the binding protein is, therefore, probably due not only to marked inhibition of the biosynthesis of the hepatic binding protein but also accelerated degradation of the protein, and this decrease
Q)
,”
01234567 Days
after
galactosamine
injection
amounts of serum asialoglycoprotein and hepatic binding protein. The amount of serum asialoglycoproteins in ng/lO ~1 serum 0 was determined by the binding inhibition assay as shown in Figure 1. The amount of binding protein (0) was determined by the “Assay A” procedure of Hudgin et al (2). The unit of specific binding activity is ng of asialoorosomucoid bound/pg of protein as defined by Pricer and Ashwell (4).
Figure 4. Galactosamine-induced
changes in the
asialoglycoproteins could be due to either a decrease in the clearance by the hepatic binding protein and/ or increased formation of asialoglycoproteins. The present experiment has clearly shown that the hepatic binding protein markedly decreased in amount after the galactosamine treatment in inverse proportion to the rapid accumulation of asialoglycoproteins in the circulation. It was also revealed that the clearance of [““Ilasialoorosomucoid from the circulation is very much delayed after the treatment. Further, the clearance of asialoglycoprotein as expressed by the reciprocal of the half-life of injected [‘Z”I]asialoglycoprotein was roughly proportional to the amount of the hepatic binding protein (Figure 6). We conclude, therefore, that the decrease in the amount of binding protein in rat liver induced by Dgalactosamine administration is mainly responsible for the accumulation of serum asialoglycoproteins in the treated rats. Recently we have shown that rat liver binding
100 80 60 40
1 5
10
15 Minutes
20 after
25 injection
Figure 5. Disappearance of [‘Z51]asialoorosomucoid from circulation after treatment with n-galactosamine. [1251]Asialoorosomucoid (50 &lOO g body wt) was injected intravenously, and the blood samples were taken to measure the radioactivities. Each point is the average value obtained from two animals. Curve 1, control rat; curve 2.3 days; curve $5 days; and curve 4,7 days after galactosamine treatment.
532
SAWAMURA
ET AL.
GASTROENTEROLOGY
Total
,
0;Z
0:4
binding 0:6
activity
0:8
‘1,;
(
Vol. 81. No. 3
?? ) 1;2
l:‘(
1:6
I
between the rate of disFigure 6. Relationship appearance of [‘*51]asialoorosomucoid in rat serum as expressed by the reciprocal of the half-life and the specific (0) and total 0 binding activities of liver microsomes. Specific binding activity is defined as ng of asialoorosomucoid bound/pg of protein. Total binding activities were calculated by multiplying the specific binding activity with the total amount of microsomal proteins.
Specific
binding
activity
(
?? )
might be responsible for the marked accumulation of serum asialoglycoproteins after administration of D-galactosamine intO rats. The results of the present studies suggest that the marked accumulation of asialoglycoproteins in the serum of patients with liver disease such as cirrhosis and cancer (10-14) is induced by a decrease in the amount of hepatic binding protein in the liver. Asialoglycoproteins, however, could be accumulated by other mechanisms. We have not proved this point, because many phenomena other than changes in hepatic binding protein occur in galactosamine-induced acute hepatocellular injury. Clinical investigations on the asialoglycoproteinemia in hepatic diseases are now in progress in our laboratory.
References 1. Ashwell G, Morel1 AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol 1974;41:99-128. 2. Hudgin RL, Pricer WE, Ashwell G, et al. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol Chem 1974;249:5536-43. 3. Kawasaki T, Ashwell G. Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins. J Biol Chem 1976;251:1296-392. 4. Pricer WE, Ashwell G. Subcellular distribution of a mammalian hepatic binding protein specific for asialoglycoproteins. J Biol Chem 1978;251:7539-44. 5. Tanabe T, Pricer WE, Ashwell G. Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. J Biol Chem 1979;254:1038-43,
6, Sawamura
7.
8.
9.
10.
11.
12.
13.
14. 15.
16. 17.
T, Nakada H, Fujii-Kuriyama Y, Tashiro Y. Some properties of a binding protein specific for asialoglycoproteins and its distribution in rat liver microsomes. Cell Struct Funct 1986;5:133-46. Morel1 AG, Irvine RA, Sternleib I, et al. Physical and chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 1968;243:155-9. Hubbard AL, Wilson G, Ashwell G, Stukenbrok H. An electron microscope autoradiographic study of the carbohydrate recognition system in rat liver. I. Distribution of ‘Z51-ligands among the liver cell type. J Cell Biol 1979:83:47&l. Steer CJ, Clarenburg R. Unique distribution of glycoprotein receptors on parenchymal and sinusoidal cells of rat liver. J Biol Chem 1979;254:4457-61. Marshall JS, Green AM. Studies on human thyroxine-binding globulin. VI. The nature of slow thyroxine-binding globulin. J Clin Invest. 1972;51:3173-81. Marshall JS, Green AM, Pensky J, et al. Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage. J Clin Invest 1977;54:555-62. Marshall JS, Williams ST, Hepner GW. Serum desialylated glycoprotein (dGP) in patients with hepatobiliary dysfunction comparison with other liver function tests (abstr). Gastroenterology 1975:69:844. Marshall JS, Williams ST. Serum inhibitors of desialylated glycoprotein binding to hepatocyte membranes. Biochim Biophys Acta 1978;543:41-52. Sobue G, Kosaka A. Asialoglycoproteinemia in a case of primary hepatic cancer. Hepato-Gastroenterol 198&27:266-3. Keppler D, Lesch R, Reutter W, Decker K. Experimental hepatitis induced by o-galactosamine. Exp Mol Path01 1988;9:27999. Reutter W, Lesch R, Keppler D, Decker K. Galaktosamin Hepatitis. Naturwisschenschaften 1968;55:497-8. Ehrenreich JH, Bergeron JJM, Siekevitz P, Palade GE. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol 1973;59:45-72.
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1981
18.Reitman S, Frankel S. A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Path01 1957;28:58-63. 19. Karmen A. A note on the spectrophotometric assay of glutamic oxalacetic transaminase in human blood serum. J Clin Invest 1955;34:131-3. 20. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomea. I. Evidence for its hemoprotein nature. J Biol Chem 1984;239:2379-85. 21. Heppel LA, Hilmoe RJ. “5”Nucleotidases. In: Colowick SP, Kaplan N, eds. Methods in enzymology. Vol. 2. New York: Academic Press Inc., 1955546-50. 22. Lowry OH, Rosebrough NJ, Farr AL. Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-75. 23. Gallenkamp H, Brachtel D, Richter E. Influence of D-Galactosamine hydrochloride on microsomal phoepholipid, protein and cytochrome P-450 content in guinea pig liver. Acta Hepato-Gastroenterol 1974;21:101-5.
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HEPATITIS
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24. Steer C, Ashwell G. Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. J Biol Chem 1980; 255:3(X%13. 25. Decker K, Keppler D. Galactosamine induced liver injury. In: Popper H, Schaffner F, eds. Progress in liver diseases. Vol. 4. New York: Grune and Stratton, Inc., 1972:183-99 26. Medline A, Schaffner F, Popper H. Ultrastructural features in galactosamine-induced hepatitis. Exp Mol Path01 1970;1220111. 27. Shinozuka H, Farber JL, Koniehi Y, Anukarahanonta T. D-Galactosamlne and acute liver cell injury. Fed Proc 1973; 32151826. 28. Nakada H, Sawamura T, Aoi K, et al. Synthesis and intracellular transport of a binding protein specific for asialoglycoproteins in rat hepatocytes (abstr). Eur J Cell Bioll980;22:155. 29. Nakada H, Sawamura T, Tashiro Y. Biosynthesis and insertion of a hepatic binding protein specific for asialoglycoproteins into rough endoplasmic reticulum membranes. J Biothem 1981;89:135-41.