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Acid Mucopolysaccharides and Cirrhosis of the Liver
Vol. 51, No.1 Printed in U.S.A .
GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co.
ACID MUCOPOL YSACCHARIDES AND CIRRHOSIS OF THE LIVER JOHN
T.
GALAMBOS,
M.D.
Section of Gastroente1·ology, Department of Medicine, Em01·y University School of Medicine, and the Medical and Pathology Services, Gmdy JVfem01·ial Ho spital, Atlanta, Ge01·gia
The causes of active progressive chronic liver disease in man are not known . A possible mechanism may be the marked alterations of the hepatic connective tissue ground substance which were observed in this type of human liver disease. Acid mucopolysaccharides are a part of the connective tissue ground substance. Therefore, changes of acid mucopolysaccharides in human liver disease were studied in consecutive liver biopsies and selected autopsies. This report stresses the importance of hepatic acid mucopolysaccharide response to injury in the development of chronic liver disease. The observations are compatible with the suggestion that the physi- . cochemical character of the hepatic mesenchymal response to human liver injury could be responsible for the chronic progressive n ature of the lesion. Material and Methods
This report is based on the study of the relationship between the clinical course and the morphological examination of the livers of patients who were seen by the author between July 1, 1959, and December 31, 1964, and from whom an adequate amount of liver tissue was available for examination. A total of 666 cases was included in this study: 109 patients had normal liver, 64 had ordinary viral hepatitis, 8 had viral hepatitis with subacute hepatic necrosis, 76 had fatty liver (14 of whom also had severe acute parenchymal necrosis), 24 had parReceived July 14, 1965. Accepted March 7, 1966. Address requests for reprints to: Dr. John T. Galambos, Emory University School of Medicine, 69 Butler Street, S.E., Atlanta, Georgia 30303. This study was supported in part by Grant AM 08545 and Training Grant 2A-5151 from the United States Public Health Service.
tal fibrosis, 160 had cirrhosis, 61 had granulomas, 44 had small areas of focal, nonspecific necrosis, 49 had malignancy, 19 had intrahepatic cholestasis only, and 8 had extrahepatic biliary obstruction. Miscellaneous lesions were found in the remaining 44 livers. Tissues were fixed in 10% calcium formalin which contained 0.5 g of cetyl pyridinium chloride per 100 ml. Paraffin sections, 4 JL thick, were stained with hematoxylin and eosin, by silver impregnation for reticulum, and by Mallory's aniline blue stain. The acid mucopolysaccharides were studied histochemically by their reaction with (a) alcian blue, (b) colloidal iron, and (c) toluidine blue. The alcian blue reaction was also observed following incubation with hyaluronidase. The sections were therefore stained by the following methods: 1. Alcian blue, 1.0 g, in 100 ml of 3% acetic acid (pH 3.0) and periodic acid-Schiff (PAS) method1 with hematoxylin nuclear stain. 2. Alcian blue at pH 1.0 for sulfate groups.' 3. Mowry's modification1 of the Hale colloidal iron stain which was modified by a 2.5fold increase of the acetic acid concentration to insure a pH value of 1.1. These sections were counterstained by van Gieson's picrofuchsin for collagen fibers." 4. Toluidine blue, 0.1 o/o in 30% ethanol for metachromasia.1 Testicular hyaluronidase (1 mg per ml) digestion was performed at 37 C for 3 hr in saline-0.1 N acetate buffer (1:1 v/v) at pH 5.0. Control sections were simultaneously incubated in buffer alone. The activity of the enzyme was demonstrated by complete hydrolysis of hyaluronic acid in simultaneously incubated sections of umbilical cord. Histochemically demonstrable acid mucopolysaccharides were graded by a single observer on a 0 to 4+ scale. The sections were reviewed in groups of 25 to 35 specimens. To exclude any bias, only a code number was available at the time of histological grading. To explore the analytical reliability of the histochemical methods used in this study, 30 65
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D
D
D
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X DO XX X
DO
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oorn o 000 0
Jl-9
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00
HEXOSAMINE I 100 mg
LYOPHILIZED DEFATTED LIVER
Fw. 1. There was good correlation between the histological grading of acid mucopolysaccharides (ordinate) and the amount of hexosamine (abscissa) in the acid mucopolysaccharide fraction of 30 human livers at postmortem examination.
additional livers were examined postmortem. A representative sample of liver was fixed and processed as described above . The histochemical grading of the acid mucopolysaccharideswhich was recorded prior to the biochemical determinations-was correlated with the total nondialyzable hexosamine of liver homogenates and also with the acid mucopolysaccharide hexosamine. The tissues were homogenized, extracted three times with ether acetone (1: 1 v/v), and dialyzed and lyophilized. The fatextracted, dried livers (FEDL) were hydrolyzed in 1 N HCl for 16 hr at 110 C. After adsorption on a previously prepared column of Dowex 50 resin, the total hexosamine per gram of FEDL was determined on the HCl eluate according to Boas' modification of the Elson-Morgan method.' Other weighed aliquots of the FEDL were digested by activated papain during continuous dialysis against 0.1 N phosphate buffer of pH 6.5 for 96 hr at 65 C. Freshly activated papain was added to each specimen twice daily to insure complete protein hydrolysis. The remaining protein was precipitated by trichloroacetic acid. The supernatant fluid was dialyzed and the acid mucopolysaccharides were precipitated by cetyl pyridinium chloride. The hexosamine was determined in the acid mucopolysaccharide fraction in the same manner as described above. Acid mucopolysaccharides were considered to be present if the samples reacted with colloidal iron at pH 1.1, were metachromatic with toluidine blue staining, and reacted with alcian blue at pH 3.0. Sulfomucopolysaccharides also reacted with alcian blue at pH 1.0.
Results
Histological grading of acid mucopolysaccharides was related to acid mucopolysaccharide hexosamine (fig. 1) . However, no relationship was readily apparent between histological grading of acid mucopolysaccharides and the total nondialyzable hexosamine in FEDL. In the normal liver, acid mucopolysaccharides were detectable only in the epithelial mucin of bile ducts and in the walls of larger arteries. The amounts of histochemically demonstrable acid mucopolysaccharides varied widely in liver injury. In all these specimens, the reaction with alcian blue, colloidal iron, and metachromasia gave comparable results. Although the hepatic acid mucopolysaccharides were completely hydrolyzable by bovine testicular hyaluronidase in most specimens, in some sections of subacute hepatic necrosis and of "active" cirrhosis of alcoholics a small fraction remained unhydrolyzed under conditions which resulted in complete hydrolysis of the acid mucopolysaccharides in the matrix of umbilical cords. The nonhydrolyzable acid mucopolysaccharides reacted with alcian blue at pH 1.0, indicating that sulfate groups were present. Because this enzyme is known to hydrolyze hyaluronic acid and chondroitin sulfates A and C, these studies
July 1966
ACID MUCOPOLY SACCHARIDES AND CIRRHOSIS
67
Fw. 2. Fatty liver of a chronic alcoholic with focal necrosis. This illustration shows a small midzonal area of parenchymal necrosis which is surrounded with fat-filled hepatocytes. Collagen fibers (C, open arrow) appear gray and acid mucopolysaccharides (solid arrows) appear black. There are small clumps of acid mucopolysaccharides along the collagen fibers. Several cells around this area of fibrosis are surrounded by acid mucopolysaccharides which are interposed between the sinusoids and the hepatocytes (colloidal iron and van Gieson stain; original magnification, X 200) .
suggest the presence of sulfomucopolysaccharides other than chondroitin sulfates A and C. The hyaluronidase had no effect on the acid mucopolysaccharides in mast cell granules. A moderate depression of alcian blue reactivity was observed in the livers of alcoholic patients with florid cirrhosis when the pH of the staining solution was lowered from 3.0 to 1.0. This observation suggests that some of the alcian blue reactivity at pH 3.0 was due to carboxyl groups whichunlike sulfate-were not available for reaction with alcian blue at the lower pH. Fatty liver. Lobular and perilobular "piecemeal" necrosis 5 and fibrosis were seen in 6 of the 76 patients with fatty liver.
The lobular pattern was preserved, but at several places the limiting plate was invaded by a periportal inflammatory exudate and by fibrosis. This periportal reaction gave a triangular appearance to the involved portal areas. In such specimens neither frank septum formation nor large zones of confluent necrosis were demonstrable. In the lobule there were scattered, small areas of collapse where an inflammatory exudate was replacing the hepatocytes. Acid mucopolysaccharides were seen within and adjacent to these small areas of lobular or periportal necrosis. In some of these small areas of parenchymal necrosis collagen fibers were demonstrable in addition to collapsed reticulum. In areas of
FIGs. 3
68
AND
4.
July 1966
ACID MUCOPOLYSACCHARIDES AND CIRRHOSIS
focal fibrosis in the lobule, the acid mucopolysaccharides were seen (a) in the ground substance which surrounded the collagen fibers, (b) in small clumps within the liver cell plates between hepatocytes, and (c) in layers of varying thicknesses around the adjacent fatfilled parenchymal cells (fig. 2). Similar accumulations of acid mucopolysaccharides were seen around parenchymal cells adjacent to areas of periportal fibrosis and piecemeal necrosis. Fourteen of 76 specimens with steatosis also showed severe, acute alcoholic hepatitis. Four of the 14 patients with alcoholic hepatitis died during the acute phase of their illness. The autopsy of these livers showed that the biopsy findings were based on representative specimens. Mature, van Gieson-positive collagenous septa and cirrhosis were not found in these livers. The acid mucopolysaccharides were prominent both in the portal areas and in the areas of severe parenchymal necrosis and edema. In the lobule, the acid mucopolysaccharides appeared in large clumps and broad bands in areas of parenchymal necrosis and edema. These bands outlined the periphery of the hepatic acinus and surrounded groups of surviving liver cells, giving the appearance of nodule formation. Van Gieson-positive collagen fibers usually could not be demonstrated in such lesions, but fine fibers could be seen after Mallory's aniline blue stain. Collapsed and fragmented reticulum fibers were also observed in these lesions. The acid mucopolysaccharides were also demonstrable in thin layers along adjacent liver cell plates. These surrounded single cells and small groups of liver cells in areas where polymorphonuclear leukocytic infiltration or parenchymal necrosis was not prominent. Such
69
accumulation of acid mucopolysaccharides around hepatocytes was independent of the presence of Mallory's alcoholic hyalin. Some of these hepatocytes appeared normal while others were vacuolated and enlarged. The acid mucopolysaccharides were interspersed between the sinusoidal lining cells and the parenchymal cells. Follow-up liver biopsies were obtained in 5 of these cases. Each showed the development of fibrous septa and the nodular pattern of a cirrhotic liver. Cirrhosis. Acid mucopolysaccharides were demonstrable in neither the nodular parenchyma nor the collagenous septa in 77 of the 160 cirrhotic livers. No relationship was apparent between the morphological type of cirrhosis and the presence of acid mucopolysaccharides. In the remaining 83 acid mucopolysaccharides were seen: 1. In areas of edema and cell necrosis in the nodular parenchyma. 2. In thin layers around some of those hepatocytes which were next to areas of parenchymal necrosis and inflammation (fig. 3). 3. At the septal-parenchymal junction. Here acid mucopolysaccharides were seen in thin layers around hepatocytes which were adjacent to the fibrous septa, whether acid mucopolysaccharides were demonstrable in these septa or not. The cellularity of the septa was related to the accumulation of acid mucopolysaccharides in the parenchymal-septal junction. The extent and intensity of acid mucopolysaccharide accumulation varied from nodule to nodule and varied even within the same nodule (fig. 4). This pattern was observed in postnecrotic cirrhosis as well as in Laennec's or septal cirrhosis, in both alcoholic and nonalcoholic patients. 4. In areas of piecemeal necrosis and fi-
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FIG. 3. Septal cirrhosis, steatosis, and alcoholic hepatitis. The vicinity of a large area of parenchymal necrosis is shown. Thin layers of acid mucopolysaccharides appear black along single and small groups of parenchymal cells (solid arrows). Several of these cells appear normal while others show evidence of injury. The acid mucopolysaccharides are between the sinusoids and the cell. Reticulum fibers appear as thin gray lines (R, lighter arrows) (alcian blue-PAS method with hematoxylin stain; original magnification, X 200). FIG. 4. Active, progressive liver disease with postnecrotic cirrhosis. The outer half of the thin fibrous septum on the left did not react for acid mucopolysaccharides. The septum adjacent to the nodular parenchyma reacted strongly for acid mucopolysaccharides which appear black (arrows). Individual parenchymal cells and small groups are surrounded by acid mucopolysaccharides. The distribution of acid mucopolysaccharides varies along the edge of this nodule) (alcian blue-PAS method; original magnification, X 100).
70
GALAMBOS
brosis either in the nodular parenchyma or adjacent to fibrous septa. All those patients whose liver biopsy showed the changes described above had clinical evidence of chronic, active liver disease. 5. In the fibrous septa. When acid mucopolysaccharides were seen in the thick fibrous septa of postnecrotic type cirrhosis they were either along the edge of the septa or around proliferating bile ductules. On sections stained with alcian blue-PAS, the middle of these septa reacted positively to PAS and the areas close to the edge of the septa reacted ·with alcian blue. Plasma cells, histiocytes, mast cells, and round cells with strongly basophilic cytoplasm were prominent in the thick septa of the postnecrotic cirrhotic liver in which acid mucopolysaccharides were demonstrated. However, the converse was not true; i.e., in cellular septa acid mucopolysaccharides were not always detected. Active, chronic liver disease with cirrhosis was characterized by the accumulation of acid mucopolysaccharides both along the edge of some of the fibrous septa and in the nodular parenchyma. Viral hepatitis. Sixty-four liver biopsies showed viral hepatitis without significant increase in acid mucopolysaccharides. Spotty areas of collapse of the hepatic cell plates were usually free of acid mucopolysaccharides, with the exception of an occasional area of spotty necrosis at the lobular periphery. The biopsies of all 8 cases of viral hepatitis with submassive hepatic necrosis showed prominent accumulations of acid mucopolysaccharides in the acute lesions. There was a close association between areas of necrosis, inflammation, edema, and collapsed reticulum, on one hand, and the accumulation of acid mucopolysaccharides, on the other, as was previously described. 6 Liver biopsies during the early acute phase of the disease showed the usual lesions of viral hepatitis as well as confluent areas of submassive necrosis. In the edematous bands of extensive necrosis, only a few parenchymal cells could be identified within a prominent inflammatory
Vol. 51, .No. 1
exudate in which the lymphocyte was the dominant cell type. Kupffer cell hyperplasia was prominent. The areas of necrosis were extensive enough to give the appearance of bridging adjacent portal areas and central veins to portal tracts. Areas of collapsed reticulum were readily identifiable both in the periportal zones and in the center of the lobule. The ground substance in some of the areas of collapsed reticulum reacted strongly for acid mucopolysaccharicles (fig. 5). Van Gieson-positive collagen was not detectable in these lesions. In 3 of the 4 surviving patients in whom liver tissue was available for examination, postnecrotic type cirrhosis had developed. The fourth patient, a young woman, made a complete clinical recovery. Her liver function tests returned to normal. An adequate biopsy of her liver 1 year later showed normal hepatic architecture. Biliary obstruction. In mechanical biliary obstruction the portal tracts were edematous, enlarged, and cellular. Vilithin a few weeks after obstruction, acid mucopolysaccharides were demonstrable in such enlarged edematous portal areas before an increase of van Gieson-positive collagen bundles was detected. Acid mucopolysaccharides were not found in the lobular parenchyma. Obstruction of longer duration was associated with portal and periportal fibrosis. In 2 patients, during the early stages of primary biliary cirrhosis, acid mucopolysaccharides were found around the portal bile ductules. Only small amounts were seen in the edematous and enlarged portal tracts. Toxic injury. Three patients were studied following ingestion of a single large dose of yellow phosphorus. The edematous portal tracts and the zones of periportal necrosis reacted very strongly for acid mucopolysaccharides. Several large macrophages, distended with PAS-positive granules, were scattered throughout the alcian blue-positive ground substance. In the areas of peripheral necrosis following phosphorus ingestion, there were few inflammatory cells. The extent of periportal necrosis was proportional to the intensity of acid muco-
July 1966
ACID MUCOPOLYSACCHARIDES AND CIRRHOSIS
71
55 Fra. 5. Acute viral hepatitis with submassive hepatic necrosis. This is a less severely injured area of the liver. The ground substance react ed strongly for acid mucopolysaccharides which appear gray (arr ows ) in the areas of necrosis. Collapsed reticulum and thin, wavy aniline blue-positive fib ers were seen but van Gieson-positive collagen bundles were not demonstrable in these acid mucopolysaccharide rich areas (alcian blue-PAS method with ironhematoxylin nuclear stain; original magnification, X 40).
polysaccharide accumulation. Parenchymal vacuolization was least severe toward the center of the lobule. The accumulation of acid mucopolysaccharides was least extensive and the parenchymal vacuolation most severe in the patient who died on the 6th day of her illness. Follow-up biopsy showed normal liver in one and septal cirrhosis in the other surviving patient. 7 Cancer. In metastatic carcinoma, acid mucopolysaccharide accumulation was exuberant in the cancer stroma and extended into the hepatic lobule and engulfed those liver cells which were next to the tumor. As a rule, acid mucopolysaccharides were not seen in the cancer-free hepatic parenchyma outside the junctional area. The relationship between acid mucopolysaccharides and host response to cancer will
be the subject of a separate communication. The following lesions were not associated with the accumulation of acid mucopolysaccharides: (a) fatty livers without parenchymal necrosis or fibrosis; (b) periportal fibrosis or cirrhosis in patients who did not have clinical evidence of hepatocellular failure (burned out cirrhosis); (c) acute viral hepatitis; (d) nonspecific focal areas of necrosis; (e) "hard" granulomas (small amounts of alcian blue-positive material were seen in acute granulomatous lesions) ; (f) massive hepatic necrosis due to either fulminant viral hepatitis or heat exhaustion; and (g) submassive central necrosis due to shock. The patients with such massive hepatic lesions were young adults who survived for only 4 to 8 days.
GALAMBOS
72 Discussion
Histochemically demonstrable acid mucopolysaccharides were detected in increased amounts and at abnormal locations in those types of hepatic injury which were usually-though not invariably-followed or already accompanied by cirrhosis. The livers of patients with either severe alcoholic hepatitis or subacute hepatic necrosis were characterized by confluent, extensive parenchymal necrosis, by the absence of collagenous fibrous septa, and by a pronounced inflammatory exudate. Followup biopsies showed the development of collagenous fibrous septa after clinical recovery from the acute illness in 8 of 9 such cases. The acid mucopolysaccharides appeared to play an important role in the development of fibrosis, and may be an essential component of "florid" cirrhosis. Acid mucopolysaccharides were seen not only in areas of severe parenchymal necrosis, but also in smaller areas of necrosis both in periportal areas and in the nodular or lobular parenchyma. Some of these lesions also appeared to be followed by fibroSI S.
Complete clinical recovery and a return to normal morphological findings were observed in only 2 cases (one with subacute hepatic necrosis with glucocorticoid therapy and the other with phosphorus poisoning) with submassive hepatic necrosis and conspicuous accumulation of acid mucopolysaccharides in areas of injury. The accumulation of acid mucopolysaccharides in the absence of demonstrable collagen bundles appeared to be a reversible lesion. Acid mucopolysaccharides stimulate fibrous organization 8 • 9 and regulate the rate of fiber formation and fiber size. 9 - 11 Hepatic fibrogenesis was shown to be associated with a carbohydrate-rich, intercellular, amorphous ground substance. 12 · 13 Fibroblasts manufacture acid mucopolysaccharides and release them in their environment.14-16 Although acid mucopolysaccharides have been demonstrated in mast cells and basophilic leukocytes, 8 • 17 no evidence is available that these cells or cells of the inflammatory exudate contribute to
Vol. 51, No .1
the extracellular acid mucopolysaccharides. "Fibroblastic" Kupffer cells 18 and fibroblasts (H. Popper, personal communication) have been identified in the hepatic lobule as well as in areas of portal and periductular fibrosis. 13 Although after acute injury we observed extracellular acid mucopolysaccharides before an increase of collagen fibers was seen, usually the acid mucopolysaccharides were associated with active fibrosis. An intimate relationship was observed between acid mucopolysaccharides and collagen fibrils during active connective tissue formation.l 6 The formation of both acid mucopolysaccharides and of collagen seems to be an expression of a similar type of mesenchymal response to injury. \Ve have been unable to document the development of human cirrhosis without the accumulation of acid mucopolysaccharides during the early, active phase of the disease. However, occasionally acid mucopolysaccharides were found after injury which did not progress into cirrhosis. The demonstration of increased amounts of acid mucopolysaccharides in liver biopsies can be of practical diagnostic importance. For example, (a) it is diagnostic of subacute hepatitis (submassive hepatic necrosis) as compared with severe ordinary viral hepatitis; (b) it denotes the early active phase of hepatic fibrosis when the process may be still reversible although it is associated with the development of cirrhosis with a high degree of probability; (c) it is a characteristic feature of chronic, active progressive liver disease; and (d) in cirrhotic livers it denotes current fibroblastic activity in areas where the demonstration of collagen cannot differentiate old fibers from recently formed fibers by light microscopy. Furthermore, the demonstration of these acid mucopolysaccharides could serve as one of the objective criteria for the effectiveness of any therapy to prevent cirrhosis or to suppress fibroblastic activity. In our experience needle biopsy of the liver was representative of the disease when the lesion was diffuse. In these cases the presence or absence of acid mucopolysac-
July 1966
ACID MUCOPOLYSACCHARIDES AND CIRRHOSIS
charides gave valid indications of fibroblastic activity. However, the biopsy may not be representative of the disease because of sampling error if the lesions are discrete and spotty or if the severity of the injury varies throughout the liver. In this case the absence of acid mucopolysaccharides in the biopsy could not preclude current fibroblastic activity in another area. Large accumulations of acid mucopolysaccharides were detectable within 6 to 7 days after hepatocellular injury. Yet in ordinary acute viral hepatitis acid mucopolysaccharides could not be detected in significant amounts, whether the liver biopsies were performed during the early acute phase of the illness or in the later phase of the disease when jaundice reached its peak or was clearing. None of these patients developed cirrhosis. An excessive response of the hepatic mesenchyma to parenchymal injury may induce marked quantitative and qualitative changes in the acid mucopolysaccharides of the ground substance. Acid mucopolysaccharides in the perisinusoidal space could act as ion exchange resins because of the density of their electronegative charges, and may have a significant effect on parenchymal metabolism.19 The role of acid mucopolysaccharides as surface blocking and triggering agents for cell division has been reviewed recently. 20 Summary
The relationship between human liver disease and hepatic acid mucopolysaccharides was studied in 666 patients from whom adequate amounts of liver tissue were obtained for study. The accumulation of histochemically demonstrable acid mucopolysaccharides was associated with active fibrosis. The appearance of acid mucopolysaccharides in human liver lesions emphasizes the responsive role of the hepatic mesenchyma in the pathogenesis of human liver disease. Chronic active liver disease was associated with the accumulation of acid mucopolysaccharides (a) in the perisinusoidal space along parenchymal
73
cells, (b) in areas of necrosis, and (c) at the septal-parenchymal junction in cirrhotic livers. A vicious cycle of mesenchymal r~sponse-parenchymal injury could explain the progressive nature and persistence of chronic active liver disease in some patients. When acid mucopolysaccharides accumulate in areas of injury without infiltration into the lobular parenchyma, and before the development of collagen bundles, the mesenchymal reaction may still be reversible. Those types of hepatic lesions which were not associated with the accumulation of acid mucopolysaccharides were not regularly followed by fibrosis and cirrhosis. REFERENCES 1. Pearse, A. G. E. 1959. Histochemistry, theoretical and applied. Little, Brown and Company, Boston. 2. Lev, R., and S. S. Spicer. 1964. Specific staining of sulfate groups with alcian blue at low pH. J. Histochem. Cytochem.12: 309. 3. Puchtler, H., and F. Sweat. 1964. Histochemical specificity of staining methods for connective tissue fibers; resorcin-fuchsin and Van Giesen's picro-fuchsin. Histochemie 4: 24-34. 4. Boas, N. F. 1953. Method for determination
of hexosamine in tissues. J. Bioi. Chern. 204: 553-563. 5. Popper, H., and F. Schaffner. 1957. Liver:
6. 7.
8.
9. 10.
11.
Structure and function, p. 777. McGrawHill Book Company, Inc., New York. Galambos, J. T. 1964. Chronic persisting hepatitis. Amer. J . Dig. Dis. 9: 817--831. Fletcher, G. F ., and J. T. Galambos. 1963. Phosphorus poisoning in humans. Arch. Intern. Med. (Chicago) 112: 846--852. Asboe-Hansen, G. 1961. Endocrine control of connective tissue, p. 38-43. In L. C. Mills and J. H. Moyer [ed.], Inflammation and disease of connective tissue. W. B. Saunders Company, Philadelphia. Hall, D. A. 1961. The chemistry of connective tissue. Charles C Thomas, Springfield, Ill. Wood, G. C. 1960. The formation of fibrils from collagen solutions. III. Effect of chondroitin sulfate and some other naturally occurring polyanions on the rate of formation. Biochem. J. 75: 605-612. Gross, J. 1964. Discussion of: Metabolism of collagen in mammalian tissues, by W. V. B. Robertson. In Connective tissue : Intra-
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12.
13.
14.
15.
GALAMBOS cellular macromolecules (proceedings of a symposium sponsored by the New York Heart Association), p. 114. Little, Brown and Company, Boston. Popper, H., F. Schaffner, F. Hutterer, F. Paronetto, and T. Barka. 1960. Parenchymal fibrogenesis; the liver. Ann. N. Y. Acad. Sci. 86: 1075-1088. Singer, E. J., F. Hutterer, G. Kent, F. G. Zak, and H. Popper. 1959. Hepatic fibrosis. A. l\1. A. Arch. Path. 68: 103-112. Porter, K R. 1964. Cell fine structure and biosynthesis of intercellular macromolecules. In Connective tissue: Intercellular macromolecules (proceedings of a symposium sponsored by the New York Heart Association). Little, Brown and Company, Boston. Green, D., and D. Hamerman. 1964. Produc-
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tion of hyaluronate and collagen by fibroblast clones in culture. Nature (London) 201: 710. 16. Yardley, J. H ., and G. D. Brown. 1965. Fibroblasts in tissue culture. Lab. Invest. 14: 501. 17. Horn, R. G., and S. S. Spicer. 1964. Sulfated mucopolysaccharide and basic protein in certain granules of rabbit leukocytes. Lab. Invest. 13: 1. 18. Pischinger, A. 1954. Uber das Wesen der Kupfferschen Sternzellen. Z. Zellforsch. 40: 605. 19. Bennett, H. S. 1963. Morphological aspects
of extracellular polysaccharides. chern. Cytochem. 11: 14. 20. Lippman, M . 1965. A proposed role polysaccharides in the initiation trol of cell division. Trans. N. Y. 27: 342-260.
J. Ristofor mucoand conAcad. Sci.
GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co.
ACID MUCOPOL YSACCHARIDES AND CIRRHOSIS OF THE LIVER JOHN
T.
GALAMBOS,
M.D.
Section of Gastroente1·ology, Department of Medicine, Em01·y University School of Medicine, and the Medical and Pathology Services, Gmdy JVfem01·ial Ho spital, Atlanta, Ge01·gia
The causes of active progressive chronic liver disease in man are not known . A possible mechanism may be the marked alterations of the hepatic connective tissue ground substance which were observed in this type of human liver disease. Acid mucopolysaccharides are a part of the connective tissue ground substance. Therefore, changes of acid mucopolysaccharides in human liver disease were studied in consecutive liver biopsies and selected autopsies. This report stresses the importance of hepatic acid mucopolysaccharide response to injury in the development of chronic liver disease. The observations are compatible with the suggestion that the physi- . cochemical character of the hepatic mesenchymal response to human liver injury could be responsible for the chronic progressive n ature of the lesion. Material and Methods
This report is based on the study of the relationship between the clinical course and the morphological examination of the livers of patients who were seen by the author between July 1, 1959, and December 31, 1964, and from whom an adequate amount of liver tissue was available for examination. A total of 666 cases was included in this study: 109 patients had normal liver, 64 had ordinary viral hepatitis, 8 had viral hepatitis with subacute hepatic necrosis, 76 had fatty liver (14 of whom also had severe acute parenchymal necrosis), 24 had parReceived July 14, 1965. Accepted March 7, 1966. Address requests for reprints to: Dr. John T. Galambos, Emory University School of Medicine, 69 Butler Street, S.E., Atlanta, Georgia 30303. This study was supported in part by Grant AM 08545 and Training Grant 2A-5151 from the United States Public Health Service.
tal fibrosis, 160 had cirrhosis, 61 had granulomas, 44 had small areas of focal, nonspecific necrosis, 49 had malignancy, 19 had intrahepatic cholestasis only, and 8 had extrahepatic biliary obstruction. Miscellaneous lesions were found in the remaining 44 livers. Tissues were fixed in 10% calcium formalin which contained 0.5 g of cetyl pyridinium chloride per 100 ml. Paraffin sections, 4 JL thick, were stained with hematoxylin and eosin, by silver impregnation for reticulum, and by Mallory's aniline blue stain. The acid mucopolysaccharides were studied histochemically by their reaction with (a) alcian blue, (b) colloidal iron, and (c) toluidine blue. The alcian blue reaction was also observed following incubation with hyaluronidase. The sections were therefore stained by the following methods: 1. Alcian blue, 1.0 g, in 100 ml of 3% acetic acid (pH 3.0) and periodic acid-Schiff (PAS) method1 with hematoxylin nuclear stain. 2. Alcian blue at pH 1.0 for sulfate groups.' 3. Mowry's modification1 of the Hale colloidal iron stain which was modified by a 2.5fold increase of the acetic acid concentration to insure a pH value of 1.1. These sections were counterstained by van Gieson's picrofuchsin for collagen fibers." 4. Toluidine blue, 0.1 o/o in 30% ethanol for metachromasia.1 Testicular hyaluronidase (1 mg per ml) digestion was performed at 37 C for 3 hr in saline-0.1 N acetate buffer (1:1 v/v) at pH 5.0. Control sections were simultaneously incubated in buffer alone. The activity of the enzyme was demonstrated by complete hydrolysis of hyaluronic acid in simultaneously incubated sections of umbilical cord. Histochemically demonstrable acid mucopolysaccharides were graded by a single observer on a 0 to 4+ scale. The sections were reviewed in groups of 25 to 35 specimens. To exclude any bias, only a code number was available at the time of histological grading. To explore the analytical reliability of the histochemical methods used in this study, 30 65
66
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GALAMBOS o
NOR MAL CIR RHOSIS X CANCER
D
D
D
0
0
0
X DO XX X
DO
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oorn o 000 0
Jl-9
0
00
HEXOSAMINE I 100 mg
LYOPHILIZED DEFATTED LIVER
Fw. 1. There was good correlation between the histological grading of acid mucopolysaccharides (ordinate) and the amount of hexosamine (abscissa) in the acid mucopolysaccharide fraction of 30 human livers at postmortem examination.
additional livers were examined postmortem. A representative sample of liver was fixed and processed as described above . The histochemical grading of the acid mucopolysaccharideswhich was recorded prior to the biochemical determinations-was correlated with the total nondialyzable hexosamine of liver homogenates and also with the acid mucopolysaccharide hexosamine. The tissues were homogenized, extracted three times with ether acetone (1: 1 v/v), and dialyzed and lyophilized. The fatextracted, dried livers (FEDL) were hydrolyzed in 1 N HCl for 16 hr at 110 C. After adsorption on a previously prepared column of Dowex 50 resin, the total hexosamine per gram of FEDL was determined on the HCl eluate according to Boas' modification of the Elson-Morgan method.' Other weighed aliquots of the FEDL were digested by activated papain during continuous dialysis against 0.1 N phosphate buffer of pH 6.5 for 96 hr at 65 C. Freshly activated papain was added to each specimen twice daily to insure complete protein hydrolysis. The remaining protein was precipitated by trichloroacetic acid. The supernatant fluid was dialyzed and the acid mucopolysaccharides were precipitated by cetyl pyridinium chloride. The hexosamine was determined in the acid mucopolysaccharide fraction in the same manner as described above. Acid mucopolysaccharides were considered to be present if the samples reacted with colloidal iron at pH 1.1, were metachromatic with toluidine blue staining, and reacted with alcian blue at pH 3.0. Sulfomucopolysaccharides also reacted with alcian blue at pH 1.0.
Results
Histological grading of acid mucopolysaccharides was related to acid mucopolysaccharide hexosamine (fig. 1) . However, no relationship was readily apparent between histological grading of acid mucopolysaccharides and the total nondialyzable hexosamine in FEDL. In the normal liver, acid mucopolysaccharides were detectable only in the epithelial mucin of bile ducts and in the walls of larger arteries. The amounts of histochemically demonstrable acid mucopolysaccharides varied widely in liver injury. In all these specimens, the reaction with alcian blue, colloidal iron, and metachromasia gave comparable results. Although the hepatic acid mucopolysaccharides were completely hydrolyzable by bovine testicular hyaluronidase in most specimens, in some sections of subacute hepatic necrosis and of "active" cirrhosis of alcoholics a small fraction remained unhydrolyzed under conditions which resulted in complete hydrolysis of the acid mucopolysaccharides in the matrix of umbilical cords. The nonhydrolyzable acid mucopolysaccharides reacted with alcian blue at pH 1.0, indicating that sulfate groups were present. Because this enzyme is known to hydrolyze hyaluronic acid and chondroitin sulfates A and C, these studies
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ACID MUCOPOLY SACCHARIDES AND CIRRHOSIS
67
Fw. 2. Fatty liver of a chronic alcoholic with focal necrosis. This illustration shows a small midzonal area of parenchymal necrosis which is surrounded with fat-filled hepatocytes. Collagen fibers (C, open arrow) appear gray and acid mucopolysaccharides (solid arrows) appear black. There are small clumps of acid mucopolysaccharides along the collagen fibers. Several cells around this area of fibrosis are surrounded by acid mucopolysaccharides which are interposed between the sinusoids and the hepatocytes (colloidal iron and van Gieson stain; original magnification, X 200) .
suggest the presence of sulfomucopolysaccharides other than chondroitin sulfates A and C. The hyaluronidase had no effect on the acid mucopolysaccharides in mast cell granules. A moderate depression of alcian blue reactivity was observed in the livers of alcoholic patients with florid cirrhosis when the pH of the staining solution was lowered from 3.0 to 1.0. This observation suggests that some of the alcian blue reactivity at pH 3.0 was due to carboxyl groups whichunlike sulfate-were not available for reaction with alcian blue at the lower pH. Fatty liver. Lobular and perilobular "piecemeal" necrosis 5 and fibrosis were seen in 6 of the 76 patients with fatty liver.
The lobular pattern was preserved, but at several places the limiting plate was invaded by a periportal inflammatory exudate and by fibrosis. This periportal reaction gave a triangular appearance to the involved portal areas. In such specimens neither frank septum formation nor large zones of confluent necrosis were demonstrable. In the lobule there were scattered, small areas of collapse where an inflammatory exudate was replacing the hepatocytes. Acid mucopolysaccharides were seen within and adjacent to these small areas of lobular or periportal necrosis. In some of these small areas of parenchymal necrosis collagen fibers were demonstrable in addition to collapsed reticulum. In areas of
FIGs. 3
68
AND
4.
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ACID MUCOPOLYSACCHARIDES AND CIRRHOSIS
focal fibrosis in the lobule, the acid mucopolysaccharides were seen (a) in the ground substance which surrounded the collagen fibers, (b) in small clumps within the liver cell plates between hepatocytes, and (c) in layers of varying thicknesses around the adjacent fatfilled parenchymal cells (fig. 2). Similar accumulations of acid mucopolysaccharides were seen around parenchymal cells adjacent to areas of periportal fibrosis and piecemeal necrosis. Fourteen of 76 specimens with steatosis also showed severe, acute alcoholic hepatitis. Four of the 14 patients with alcoholic hepatitis died during the acute phase of their illness. The autopsy of these livers showed that the biopsy findings were based on representative specimens. Mature, van Gieson-positive collagenous septa and cirrhosis were not found in these livers. The acid mucopolysaccharides were prominent both in the portal areas and in the areas of severe parenchymal necrosis and edema. In the lobule, the acid mucopolysaccharides appeared in large clumps and broad bands in areas of parenchymal necrosis and edema. These bands outlined the periphery of the hepatic acinus and surrounded groups of surviving liver cells, giving the appearance of nodule formation. Van Gieson-positive collagen fibers usually could not be demonstrated in such lesions, but fine fibers could be seen after Mallory's aniline blue stain. Collapsed and fragmented reticulum fibers were also observed in these lesions. The acid mucopolysaccharides were also demonstrable in thin layers along adjacent liver cell plates. These surrounded single cells and small groups of liver cells in areas where polymorphonuclear leukocytic infiltration or parenchymal necrosis was not prominent. Such
69
accumulation of acid mucopolysaccharides around hepatocytes was independent of the presence of Mallory's alcoholic hyalin. Some of these hepatocytes appeared normal while others were vacuolated and enlarged. The acid mucopolysaccharides were interspersed between the sinusoidal lining cells and the parenchymal cells. Follow-up liver biopsies were obtained in 5 of these cases. Each showed the development of fibrous septa and the nodular pattern of a cirrhotic liver. Cirrhosis. Acid mucopolysaccharides were demonstrable in neither the nodular parenchyma nor the collagenous septa in 77 of the 160 cirrhotic livers. No relationship was apparent between the morphological type of cirrhosis and the presence of acid mucopolysaccharides. In the remaining 83 acid mucopolysaccharides were seen: 1. In areas of edema and cell necrosis in the nodular parenchyma. 2. In thin layers around some of those hepatocytes which were next to areas of parenchymal necrosis and inflammation (fig. 3). 3. At the septal-parenchymal junction. Here acid mucopolysaccharides were seen in thin layers around hepatocytes which were adjacent to the fibrous septa, whether acid mucopolysaccharides were demonstrable in these septa or not. The cellularity of the septa was related to the accumulation of acid mucopolysaccharides in the parenchymal-septal junction. The extent and intensity of acid mucopolysaccharide accumulation varied from nodule to nodule and varied even within the same nodule (fig. 4). This pattern was observed in postnecrotic cirrhosis as well as in Laennec's or septal cirrhosis, in both alcoholic and nonalcoholic patients. 4. In areas of piecemeal necrosis and fi-
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FIG. 3. Septal cirrhosis, steatosis, and alcoholic hepatitis. The vicinity of a large area of parenchymal necrosis is shown. Thin layers of acid mucopolysaccharides appear black along single and small groups of parenchymal cells (solid arrows). Several of these cells appear normal while others show evidence of injury. The acid mucopolysaccharides are between the sinusoids and the cell. Reticulum fibers appear as thin gray lines (R, lighter arrows) (alcian blue-PAS method with hematoxylin stain; original magnification, X 200). FIG. 4. Active, progressive liver disease with postnecrotic cirrhosis. The outer half of the thin fibrous septum on the left did not react for acid mucopolysaccharides. The septum adjacent to the nodular parenchyma reacted strongly for acid mucopolysaccharides which appear black (arrows). Individual parenchymal cells and small groups are surrounded by acid mucopolysaccharides. The distribution of acid mucopolysaccharides varies along the edge of this nodule) (alcian blue-PAS method; original magnification, X 100).
70
GALAMBOS
brosis either in the nodular parenchyma or adjacent to fibrous septa. All those patients whose liver biopsy showed the changes described above had clinical evidence of chronic, active liver disease. 5. In the fibrous septa. When acid mucopolysaccharides were seen in the thick fibrous septa of postnecrotic type cirrhosis they were either along the edge of the septa or around proliferating bile ductules. On sections stained with alcian blue-PAS, the middle of these septa reacted positively to PAS and the areas close to the edge of the septa reacted ·with alcian blue. Plasma cells, histiocytes, mast cells, and round cells with strongly basophilic cytoplasm were prominent in the thick septa of the postnecrotic cirrhotic liver in which acid mucopolysaccharides were demonstrated. However, the converse was not true; i.e., in cellular septa acid mucopolysaccharides were not always detected. Active, chronic liver disease with cirrhosis was characterized by the accumulation of acid mucopolysaccharides both along the edge of some of the fibrous septa and in the nodular parenchyma. Viral hepatitis. Sixty-four liver biopsies showed viral hepatitis without significant increase in acid mucopolysaccharides. Spotty areas of collapse of the hepatic cell plates were usually free of acid mucopolysaccharides, with the exception of an occasional area of spotty necrosis at the lobular periphery. The biopsies of all 8 cases of viral hepatitis with submassive hepatic necrosis showed prominent accumulations of acid mucopolysaccharides in the acute lesions. There was a close association between areas of necrosis, inflammation, edema, and collapsed reticulum, on one hand, and the accumulation of acid mucopolysaccharides, on the other, as was previously described. 6 Liver biopsies during the early acute phase of the disease showed the usual lesions of viral hepatitis as well as confluent areas of submassive necrosis. In the edematous bands of extensive necrosis, only a few parenchymal cells could be identified within a prominent inflammatory
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exudate in which the lymphocyte was the dominant cell type. Kupffer cell hyperplasia was prominent. The areas of necrosis were extensive enough to give the appearance of bridging adjacent portal areas and central veins to portal tracts. Areas of collapsed reticulum were readily identifiable both in the periportal zones and in the center of the lobule. The ground substance in some of the areas of collapsed reticulum reacted strongly for acid mucopolysaccharicles (fig. 5). Van Gieson-positive collagen was not detectable in these lesions. In 3 of the 4 surviving patients in whom liver tissue was available for examination, postnecrotic type cirrhosis had developed. The fourth patient, a young woman, made a complete clinical recovery. Her liver function tests returned to normal. An adequate biopsy of her liver 1 year later showed normal hepatic architecture. Biliary obstruction. In mechanical biliary obstruction the portal tracts were edematous, enlarged, and cellular. Vilithin a few weeks after obstruction, acid mucopolysaccharides were demonstrable in such enlarged edematous portal areas before an increase of van Gieson-positive collagen bundles was detected. Acid mucopolysaccharides were not found in the lobular parenchyma. Obstruction of longer duration was associated with portal and periportal fibrosis. In 2 patients, during the early stages of primary biliary cirrhosis, acid mucopolysaccharides were found around the portal bile ductules. Only small amounts were seen in the edematous and enlarged portal tracts. Toxic injury. Three patients were studied following ingestion of a single large dose of yellow phosphorus. The edematous portal tracts and the zones of periportal necrosis reacted very strongly for acid mucopolysaccharides. Several large macrophages, distended with PAS-positive granules, were scattered throughout the alcian blue-positive ground substance. In the areas of peripheral necrosis following phosphorus ingestion, there were few inflammatory cells. The extent of periportal necrosis was proportional to the intensity of acid muco-
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ACID MUCOPOLYSACCHARIDES AND CIRRHOSIS
71
55 Fra. 5. Acute viral hepatitis with submassive hepatic necrosis. This is a less severely injured area of the liver. The ground substance react ed strongly for acid mucopolysaccharides which appear gray (arr ows ) in the areas of necrosis. Collapsed reticulum and thin, wavy aniline blue-positive fib ers were seen but van Gieson-positive collagen bundles were not demonstrable in these acid mucopolysaccharide rich areas (alcian blue-PAS method with ironhematoxylin nuclear stain; original magnification, X 40).
polysaccharide accumulation. Parenchymal vacuolization was least severe toward the center of the lobule. The accumulation of acid mucopolysaccharides was least extensive and the parenchymal vacuolation most severe in the patient who died on the 6th day of her illness. Follow-up biopsy showed normal liver in one and septal cirrhosis in the other surviving patient. 7 Cancer. In metastatic carcinoma, acid mucopolysaccharide accumulation was exuberant in the cancer stroma and extended into the hepatic lobule and engulfed those liver cells which were next to the tumor. As a rule, acid mucopolysaccharides were not seen in the cancer-free hepatic parenchyma outside the junctional area. The relationship between acid mucopolysaccharides and host response to cancer will
be the subject of a separate communication. The following lesions were not associated with the accumulation of acid mucopolysaccharides: (a) fatty livers without parenchymal necrosis or fibrosis; (b) periportal fibrosis or cirrhosis in patients who did not have clinical evidence of hepatocellular failure (burned out cirrhosis); (c) acute viral hepatitis; (d) nonspecific focal areas of necrosis; (e) "hard" granulomas (small amounts of alcian blue-positive material were seen in acute granulomatous lesions) ; (f) massive hepatic necrosis due to either fulminant viral hepatitis or heat exhaustion; and (g) submassive central necrosis due to shock. The patients with such massive hepatic lesions were young adults who survived for only 4 to 8 days.
GALAMBOS
72 Discussion
Histochemically demonstrable acid mucopolysaccharides were detected in increased amounts and at abnormal locations in those types of hepatic injury which were usually-though not invariably-followed or already accompanied by cirrhosis. The livers of patients with either severe alcoholic hepatitis or subacute hepatic necrosis were characterized by confluent, extensive parenchymal necrosis, by the absence of collagenous fibrous septa, and by a pronounced inflammatory exudate. Followup biopsies showed the development of collagenous fibrous septa after clinical recovery from the acute illness in 8 of 9 such cases. The acid mucopolysaccharides appeared to play an important role in the development of fibrosis, and may be an essential component of "florid" cirrhosis. Acid mucopolysaccharides were seen not only in areas of severe parenchymal necrosis, but also in smaller areas of necrosis both in periportal areas and in the nodular or lobular parenchyma. Some of these lesions also appeared to be followed by fibroSI S.
Complete clinical recovery and a return to normal morphological findings were observed in only 2 cases (one with subacute hepatic necrosis with glucocorticoid therapy and the other with phosphorus poisoning) with submassive hepatic necrosis and conspicuous accumulation of acid mucopolysaccharides in areas of injury. The accumulation of acid mucopolysaccharides in the absence of demonstrable collagen bundles appeared to be a reversible lesion. Acid mucopolysaccharides stimulate fibrous organization 8 • 9 and regulate the rate of fiber formation and fiber size. 9 - 11 Hepatic fibrogenesis was shown to be associated with a carbohydrate-rich, intercellular, amorphous ground substance. 12 · 13 Fibroblasts manufacture acid mucopolysaccharides and release them in their environment.14-16 Although acid mucopolysaccharides have been demonstrated in mast cells and basophilic leukocytes, 8 • 17 no evidence is available that these cells or cells of the inflammatory exudate contribute to
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the extracellular acid mucopolysaccharides. "Fibroblastic" Kupffer cells 18 and fibroblasts (H. Popper, personal communication) have been identified in the hepatic lobule as well as in areas of portal and periductular fibrosis. 13 Although after acute injury we observed extracellular acid mucopolysaccharides before an increase of collagen fibers was seen, usually the acid mucopolysaccharides were associated with active fibrosis. An intimate relationship was observed between acid mucopolysaccharides and collagen fibrils during active connective tissue formation.l 6 The formation of both acid mucopolysaccharides and of collagen seems to be an expression of a similar type of mesenchymal response to injury. \Ve have been unable to document the development of human cirrhosis without the accumulation of acid mucopolysaccharides during the early, active phase of the disease. However, occasionally acid mucopolysaccharides were found after injury which did not progress into cirrhosis. The demonstration of increased amounts of acid mucopolysaccharides in liver biopsies can be of practical diagnostic importance. For example, (a) it is diagnostic of subacute hepatitis (submassive hepatic necrosis) as compared with severe ordinary viral hepatitis; (b) it denotes the early active phase of hepatic fibrosis when the process may be still reversible although it is associated with the development of cirrhosis with a high degree of probability; (c) it is a characteristic feature of chronic, active progressive liver disease; and (d) in cirrhotic livers it denotes current fibroblastic activity in areas where the demonstration of collagen cannot differentiate old fibers from recently formed fibers by light microscopy. Furthermore, the demonstration of these acid mucopolysaccharides could serve as one of the objective criteria for the effectiveness of any therapy to prevent cirrhosis or to suppress fibroblastic activity. In our experience needle biopsy of the liver was representative of the disease when the lesion was diffuse. In these cases the presence or absence of acid mucopolysac-
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ACID MUCOPOLYSACCHARIDES AND CIRRHOSIS
charides gave valid indications of fibroblastic activity. However, the biopsy may not be representative of the disease because of sampling error if the lesions are discrete and spotty or if the severity of the injury varies throughout the liver. In this case the absence of acid mucopolysaccharides in the biopsy could not preclude current fibroblastic activity in another area. Large accumulations of acid mucopolysaccharides were detectable within 6 to 7 days after hepatocellular injury. Yet in ordinary acute viral hepatitis acid mucopolysaccharides could not be detected in significant amounts, whether the liver biopsies were performed during the early acute phase of the illness or in the later phase of the disease when jaundice reached its peak or was clearing. None of these patients developed cirrhosis. An excessive response of the hepatic mesenchyma to parenchymal injury may induce marked quantitative and qualitative changes in the acid mucopolysaccharides of the ground substance. Acid mucopolysaccharides in the perisinusoidal space could act as ion exchange resins because of the density of their electronegative charges, and may have a significant effect on parenchymal metabolism.19 The role of acid mucopolysaccharides as surface blocking and triggering agents for cell division has been reviewed recently. 20 Summary
The relationship between human liver disease and hepatic acid mucopolysaccharides was studied in 666 patients from whom adequate amounts of liver tissue were obtained for study. The accumulation of histochemically demonstrable acid mucopolysaccharides was associated with active fibrosis. The appearance of acid mucopolysaccharides in human liver lesions emphasizes the responsive role of the hepatic mesenchyma in the pathogenesis of human liver disease. Chronic active liver disease was associated with the accumulation of acid mucopolysaccharides (a) in the perisinusoidal space along parenchymal
73
cells, (b) in areas of necrosis, and (c) at the septal-parenchymal junction in cirrhotic livers. A vicious cycle of mesenchymal r~sponse-parenchymal injury could explain the progressive nature and persistence of chronic active liver disease in some patients. When acid mucopolysaccharides accumulate in areas of injury without infiltration into the lobular parenchyma, and before the development of collagen bundles, the mesenchymal reaction may still be reversible. Those types of hepatic lesions which were not associated with the accumulation of acid mucopolysaccharides were not regularly followed by fibrosis and cirrhosis. REFERENCES 1. Pearse, A. G. E. 1959. Histochemistry, theoretical and applied. Little, Brown and Company, Boston. 2. Lev, R., and S. S. Spicer. 1964. Specific staining of sulfate groups with alcian blue at low pH. J. Histochem. Cytochem.12: 309. 3. Puchtler, H., and F. Sweat. 1964. Histochemical specificity of staining methods for connective tissue fibers; resorcin-fuchsin and Van Giesen's picro-fuchsin. Histochemie 4: 24-34. 4. Boas, N. F. 1953. Method for determination
of hexosamine in tissues. J. Bioi. Chern. 204: 553-563. 5. Popper, H., and F. Schaffner. 1957. Liver:
6. 7.
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Structure and function, p. 777. McGrawHill Book Company, Inc., New York. Galambos, J. T. 1964. Chronic persisting hepatitis. Amer. J . Dig. Dis. 9: 817--831. Fletcher, G. F ., and J. T. Galambos. 1963. Phosphorus poisoning in humans. Arch. Intern. Med. (Chicago) 112: 846--852. Asboe-Hansen, G. 1961. Endocrine control of connective tissue, p. 38-43. In L. C. Mills and J. H. Moyer [ed.], Inflammation and disease of connective tissue. W. B. Saunders Company, Philadelphia. Hall, D. A. 1961. The chemistry of connective tissue. Charles C Thomas, Springfield, Ill. Wood, G. C. 1960. The formation of fibrils from collagen solutions. III. Effect of chondroitin sulfate and some other naturally occurring polyanions on the rate of formation. Biochem. J. 75: 605-612. Gross, J. 1964. Discussion of: Metabolism of collagen in mammalian tissues, by W. V. B. Robertson. In Connective tissue : Intra-
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GALAMBOS cellular macromolecules (proceedings of a symposium sponsored by the New York Heart Association), p. 114. Little, Brown and Company, Boston. Popper, H., F. Schaffner, F. Hutterer, F. Paronetto, and T. Barka. 1960. Parenchymal fibrogenesis; the liver. Ann. N. Y. Acad. Sci. 86: 1075-1088. Singer, E. J., F. Hutterer, G. Kent, F. G. Zak, and H. Popper. 1959. Hepatic fibrosis. A. l\1. A. Arch. Path. 68: 103-112. Porter, K R. 1964. Cell fine structure and biosynthesis of intercellular macromolecules. In Connective tissue: Intercellular macromolecules (proceedings of a symposium sponsored by the New York Heart Association). Little, Brown and Company, Boston. Green, D., and D. Hamerman. 1964. Produc-
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tion of hyaluronate and collagen by fibroblast clones in culture. Nature (London) 201: 710. 16. Yardley, J. H ., and G. D. Brown. 1965. Fibroblasts in tissue culture. Lab. Invest. 14: 501. 17. Horn, R. G., and S. S. Spicer. 1964. Sulfated mucopolysaccharide and basic protein in certain granules of rabbit leukocytes. Lab. Invest. 13: 1. 18. Pischinger, A. 1954. Uber das Wesen der Kupfferschen Sternzellen. Z. Zellforsch. 40: 605. 19. Bennett, H. S. 1963. Morphological aspects
of extracellular polysaccharides. chern. Cytochem. 11: 14. 20. Lippman, M . 1965. A proposed role polysaccharides in the initiation trol of cell division. Trans. N. Y. 27: 342-260.
J. Ristofor mucoand conAcad. Sci.