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112663 BID-HEDICAL. RESEARCH DOCUMENT DESCRIPTION FORM 63 68 69 76 Duplicate .in. all cards: --> I| 00003^4 year as-1961- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA, preceeded by one blank space KuA/rs/mA, / /hr\ , <TV 77 78 I-------------------1 Sub-Index Code 3J O) 60 61 62 11 12 13 Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with comma-space. Avoid other punctuation; do not abbreviate. g^ g2 21 22 23 24 Source (Journal, Vol., Number, Pages, Date) 12 61 62 31 A/ /WAvZC/nu- Ai/rr/L 1/cl. /ss^/r^-fU______ !_____ /*/& A'f /f.7f 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 U>c.f 7Sr-fpty R&S 112664 URINARY AND TISSUE GLYCQSAMINQGLYCAN . . PATTERNS IN HEPATIC ANGIOSARCOMA Charles E. Kupchella and Carlo H. Tamburro Cancer Center and Department of Medicine University of Louisville School of Medicine Louisville, Kentucky 40201 0000384 I. INTRODUCTION The recent discovery of a relationship between vinyl chloride and angiosarcoma of the liver has received much attention (1-3). Although there are now systematic detection programs for vinyl chloride workers (3,4), there is as yet no specific chemical abnormality that serves as a good Indicator.of early, vinyl-chloride-induced liver injury and angio sarcoma. Alpha feto-proteln has been a relatively valuable serological, mrfcer for hepatocellular carcinoma (5). but Is has not as yet proven use ful in the detection of angiosarcoma (6). New leads are needed If more specific tests are to be developed for angiosarcoma. The literature suggests that the glycosamlnoglycans In the urine and/or blood should be evaluated as a possible aid In early detection. .The production of sulfated glycosamlnoglycans Is characteristic of malig nant vascular tumors of the skin and some pathologists use this feature as a diagnostic.aid (7). Barr and Bonin (B) observed a strong positive alclan-blue, glycosaminoglycan staining reaction In human angiosarcoma tissue and suggested than an attempt be made to quailtate and quantitate the production of glycosamlnoglycans In the neoplasms, serum, and urine Of those at risk. They pointed out that the urinary glycosamlnoglycans Bay have diagnostic significance In angiosarcoma and. If so, a glycosarainoglycan spot test might easily be employed as a gross screening test of vinyl chloride production workers. A number of other observations place the glycosamlnoglycans in a relevant position with regard to angiosarcoma. Angiosarcoma Is accom panied by connective tissue abnormalities (2,9) and changes in tissue, urinary, and blood glycosamlnoglycans have been found to occur In many connective-tissue disorders -- including connective tissue disorders of the liver (10-14) -- as well as In hepatic cancer (15-17). Supported In part by grants from the B. F. Goodrich Company and the American Cancer Society (IN-111) and a contract with the National Cancer.Institute (N01-CN-55212). 915 v *: BSSvaiS wm . A' V .;. } J. vf - i " * j: A- * zL=s~>.-::'.~- The purpose of this study was to make a preliminary determine Jo* of the glycosaminoglycan patterns In tissue and urine associated with angiosarcoma of the liver and with vlnyl-chlorlde-lnduced liver injury other than angiosarcoma and to compare these patterns with those In normal controls and those associated with other liver disease. Our goal was to evaluate the use of glycosaminoglycan patterns In the early detec tion of vlnyl-chlorlde-lnduced liver Injury and angiosarcoma and to explore the role of the glycosamlnoglycans In the etiology of vinyl chloride Injury. II- PROCEDURES AND MATERIALS USED Urine specimens were collected as occasional samples from: 9 normal controls; 9 Individuals with histories of occupational exposure to vinyl chloride and having abnormal, liver, biochemical studies; 6 with "other" cancers prior to surgery; 3 with angiosarcoma; 8 with active viral hepatitis; 6 with cirrhosis; 2 with lung-liver metastases; and 4 with metabolic disorders of the liver (congenital and indirect hyperbiliru binemia). In one case of angiosarcoma- 24-hr urines were collected on alter nate days beginning 2 weeks prior to death. Urine samples were collected without preservative and frozen at -76 until analysis. Specimens were divided Into two 25 ml samples and one 5 ml sample. Urinary creatinine was measured on the 5 ml sample using a Technlcon Autoanalyzer. The degree of urinary glycosaminoglycan polymerization was estimated by dialyzing one 25 ml sample for 24 hours In tap water; the sample was Men treated Identically to an undialyzed sample by the method of DIFerrante (18) using cetylpyridlnlum chloride as a precipitant. After resolubilization of the glycosamlnoglycans In water, duplicate samples were assayed for total uronlc acid by the modi fied carbozole reaction of Bitter and Muir (19). The remaining glyco samlnoglycans were reprecipitated with cetylpyridlnlum chloride and separated Into the wash, hyaluronic acid- ehondroltln sulfate- and heparin fractlons as described by Schiller et. a1.(20). Each of the fractions ms assayed for uronlc acid ( yg per mg of creatinine). Autopsy tissue was obtained In 2 cases of hepatic angiosarcoma (timer tissue and non-tumor tissue adjacent to tunor), 2 cases of cirrhosis, and in 3 control cases (gun-shot wound victims without liver pathology) and analyzed for glycosamlnoglycans by a previously reported modification (21) of the method of Schiller et. al. (20). Uronlc acid was determined In each of the hyaluronic acid- ehondroltln sulfate- and heparin fractions. Pieces of tissue were subjected to a1clan-blue-perlodlc-acldSchiff staining with and without hyaluronidase and diastase pretreatment. These procedures were carried out according to the methods described by Howry (22). Ascitic fluid was also obtained at autopsy in one case of angio sarcoma and analyzed for glycosamlnoglycans. The fluid was centrifuged 916 .^ 7 R&S 112665 Sl- lt!0iii4tc*%:.i\+ .<.*. and the sediment analyzed as tissue above. The supernatant was treated tor the method for urine described above. Ill* RESULTS A summary of the urinary glycosasrlnoglycan measurement Is given in Table I. Normal controls had the least urinary glycosamlnoglycans (measured as uronlc acid) of all groups. All other groups showed some elevation. The levels In angiosarcoma, hepatitis, cirrhosis, and liver metastases were significantly elevated (P < .05) over normal controls. The cirrhotic group exhibited the greatest variance In urinary glycosaml noglycans. No significant differences were found In urinary creatinine levels between groups. There were no significant differences between groups In either the percentage of the total glycosamlnoglycans that was dialyzable (Table I) or In the percentage .of the unfractionated total that appeared In the hyaluronic acid, chondroltln sulfate, and heparin fractions. Seven of 9 vinyl-chloride-exposed Individuals other than those with angiosarcoma had positive chondroltln sulfate fractions with negatlve hyaluronic acid and heparin fractions. This was true In only 3 of 32 other urines evaluated In this same manner. The pattern of dally glycosaorinoglycan excretion prior to death due to angiosarcoma In one Individual Is given In Figure 1. Total tissue glycosasrlnoglycan levels for angiosarcoma tumors, flbrotlc tissue adjacent to twors, cirrhotic liver tissue and normal llver tissue are shown In Figure 2. Fractional hyaluronic acid, chondroltln sulfate, and heparin levels are given In Figure 3. HIstocheartcaTTy, anglosarcomatous tissue exhibited a strong alclanblue positive staining reaction. Alclan-blue staining was only slightly less In "non-tumor* tissue adjacent to tumor masses. The staining reaction In tissue from normal liver was very weak and only slightly stronger In cirrhotic liver tissue. The strong aldan-blue reaction In anglosarcomatous tissue did not occur If sections were pretreated with hyaluronldase. Ascitic fluid sediment was uronlc-add-posltive In only the hyaluronic add fraction -- 11Z pg uronlc add per gram of dry, defatted sediment; ascitic fluid supernatant contained 1.7, 1.2, and 0.2 mg uronlc add per ml In the hyaluronic add, chondroltln sulfate, and heparin fractions, respectively- IV. DISCUSSION The literature indicates that normal male creatinine excretion Is 1.5 g per 24 hrs (23). Thus, our normal mean (Table I) of 3.2 .4 yg cetylpyrldlnlura chloride-precrpltable uronic add per mg creatinine falls in the middle of the normal ranges reported by Varma et. al. (24), i i iittit<*,* il ii it il t! . :j i-; l; 11 i; l .[: c. ' { \ ,. r ` t. ^u,n -MLiipujiii.i ijw...'lhw l miuM...*** R&S 112666 R&S 112667 2.6 - 4.7 ug/mg; DIFerrante and Rich (25). 2.9 - 4.8 vg/mg; and Kao and Leslie (26), 1.8 - 4.9 ug/mg. Although our study was not controlled for age. Goldberg and Cottier (27) have shown that urinary glycosamlnoglycan excretion Is constant (m ages 20-70. Hanley et. al. (28) have shown that the proportion of glycosamlnoglycans In the chondroltln sulfate fraction Is constant fm ages 20-70. Hanley et. al. also reported that the chondroltln sulfate fraction Is highest at birth and that It gradually drops until age 20. suggesting that urinary chondroltln sulfate reflects tissue growth. The fact that we found no differences between groups In the creatinine concentration Is significant In that It Indicates that occa sional samples do reflect 24-hour excretion when normalized to creatmm*. Precedent for expressing glycosanlnoglycan measurements as a function of creatinine content In occasional urine samples has been established by DIFerrante and Rich (25) and Pennodc (29). Hanley et. al. (28) have shown that the creatinine/uranic acid ratio Is steady from ages 20-70. Our results Indicate that the liver diseases evaluated are accom panied by elevated urinary glycosanlnoglycan excretion. Our tissue data suggests that this reflects liver-tissue glycosanlnoglycan elevation and conforms to the reports by others that both hepatic connective tissue disorders (10-14) and hepatic cancer (15) result In Increased hepatic glycosanlnoglycan levels. It may be significant that the angiosarcoma patients had half the urinary glycosamlnog 1 yean excretion of patients with liver metastases and that our analysis of angiosarcomatous tumor tissue exhibited half the glycosamlnoglycan content reported by Kojtma et. al. (15) for hepatocellular carcinoma. While our data suggest that liver disease results In a decrease in the proportion of highly polymerized glycosanlnoglycans, variance was large within each group and none of the differences between groups were statistically significant. Although we have not completed the characterization of Isolated glycosanlnoglycan fractions, our data Indicate: 1) that the chondroitln sulfates are the primary urinary glycosamlnoglycans In both normal controls and In disease states; 2) that the chondroltln sulfates and `'heparin are the dominant glycosamlnoglycans In normal and cirrhotic livers (Figure 3). Chondroltln sulfate Is elevated in the flbrotic. nontuaor, portions of anglosarcomatous livers while heparin is the predomi nant glycosamlnoglycan In tumor tissue. Hyaluronic acid is also apparent ly elevated relative to chondroltln sulfate In anglosarcomatous tumors (Figure 3); and 3) that hyaluronic acid Is the sole glycosamlnoglycan In ascites fluid sediment. These qualitative data are in general agreement with those reported by others. Goldberg and Cotlier (27), Douglas et. al. (30), ana Varna et. al. (24) have reported that the chondroltln sulfates are the predominant urinary glycosamlnoglycans. Varma et. al. reported that 2/3 of urinary glycosamlnoglycans are chondroitln-4-and chondroltln-6-sulfate and this agrees with our data on normal controls -and on those with liver disease. Kojina et. al. (15) reported that In hepatocellular carcinoma 918 w1J --ut i ii .mujfLy.wp-t".?w ..I i ---- r'iiMV R&S 112668 tissue, chondroltln sulfates and hyaluronic acid were Increased 33 and 10 tines* respectively, over amounts found In healthy livers; the heparin and heparan sulfate proportions dropped. This contrasts with our data on angiosarcoma tissue, l.e. heparin and hyaluronic acid increased 5 and 10 times* respectively, over normal tissue; chondroltln sulfate levels rose but fell In proportion to other glycosaminoglycans. Galambos and Shaplra (10) reported that the chondroltln sulfates are dominant In normal livers and In hepatic fibrosis, but Kojina et. al. (IS) report that chondroltlnase-reslstant and hyaluronldase-resistant glycosaminogly cans are dominant. Kuroda et. al. (31) also reported that heparan, sulfate Is the dominant glycosamlnoglycan In the normal liver. Our hlstochemlcal observation that nearly all of the Increased alclan-blue positive material In ang1osarcomatous.ilvers was susceptible to hyaluronldase digestion suggests that the observed chondroltln sulfate elevation Is due to chondraltin-4- and/or chondraltln-fi-sulfate. The Increases In liver and urinary glycosamlnoglycans may well reflect an Important role of these substances In the process of flbrogenesls and In tumor growth. Galambos and Shaplra (10) reported that hyaluronic acid was elevated during hepatic flbrogenesls. If a similar flbrotlc process Is operative in angiosarcoma. It may be that the ob served tunor-tissue heparin Increase is reflective of tumor growth. We did observe a four- to six-fold greater heparin level In tumor tissue than In adjacent, non-tumor tissue. The observation that the chondroltln sulfates tend to be the exclusive uronlc-aeld-positive constituents In the urine of Individuals Is paradoxical in that those glycosaminoglycan fractions that are most elevated In englosarcamatous tissue are those that are absent from the urine of Individuals who may well have early, vinyl-chloride-induced liver Injury. This pattern may be due to the selective action of lyso somal, glycolytic enzymes In the liver and/or may reflect the role of the chondroltln sulfates In early flbrotlc changes In the liver. Certainly the potential usefulness of this pattern In early detection warrants the more complete evaluation now ongoing In our laboratory. .Glycosamlnoglycans were measured In urine and tissue of patients with hepatic fibrosis and hepatic cancer Including vinyl-chiorlde exposure-associated liver Injury and angiosarcoma. Angiosarcoma, hepa titis, cirrhosis, and liver metastatic patients exhibited significantly elevated glycosamlnoglycan excretion. Angiosarcoma tissue exhibited elevated glycosamlnoglycan levels with the greatest Increases In the heparin fraction. Hlstochemlcally, anglosarcomatous tissue gave a strong aldan-blue staining reaction which could be prevented by pretreatment with hyaluronldase. Although vinyl-chlorlde-exposure-assoclated liver injury other than angiosarcoma was not accompanied by a significantly elevated glycosaminoglycan excretion, this condition tended to be associated with a urinary glycosamlnoglycan excretion pattern in which the chondroltln sulfate fraction was the only uronlc-acld-posltive fraction. > 919 S . Patient otoud nornl control vinyl chloride exposed other cancer other liver disease angiosarcoma hepatitis cirrhosis. *lSVr metastasis Cases 9 9 S 4 3 8 6 2 ,n " Un,",e *" "* ug uronlc acid par* mg creatinine - (*1S.E.) 3.2 * .4 4.1 * .4 X uronlc acid not dlalyzable ( 1 S.f.1 65 * 5 39 t 5 4.5tl.s 5.1 0.8 39 t 6 37*13 7.6 * 1.6 8.5 * 1.8 12.7 * 3 13.8 .9 41 22 52 * 14 53 * 9 42 30 Co CO IO OO)) CO 920 WPff?W5WP*P,(J(lifJLH !M,J^.!i.!Wi^A:,.'',L - 921 tt ! IJpiHLjM |JHi.W3PPPPB9P,liW JWJ r'. zimvv s*a eoo r s6- tc aoo s *n 400 52 SEiQd !> 300 ** // "H|*lur*nlc Ac>4* . | | * Chanlroltla lutfoti1 "Hiporln* a<MU0 "a z<! 5 200 - 18 100 WO O= dS Non-tumor Adlocint Coi ) Non-tumor Adjactnl Com 2 Goto 3 Cat* 4 NORMAL- Goto 3 Caie 6 PIG. 3. Fractional concentration! of glycosaainoglycans in angiosarcomatoua, cirrhotic and normal human liver tiiaue. ffv. I .* J V .1, . -r''" --:. --!----------- '"H y^WNttw^ytye'iiaaifii'i'u; i <*) rfMni'W'; . i \ k *; V i r`:* <\ .< r?M'' 4 R&S 112673 VI., REFERENCES Creech* J. L. and Johnson, M. N. Angiosarcoma of the Liver In the Manufacture of Polyvinyl Chloride. J. Occup. Med. 16: 150-151, 1974. " Falk, H., Creech, J. L., Heath, 0. U., Johnson, M. N., and Key, M. M. Hepatic Disease Among Workers at a Vinyl Chloride Polymerization Plant. JAMA 230: 59-63, 1974. 3. Makk, L., Creech, J. L., Whelan, J. G., and Johnson, M. N. Liver Damage and Angiosarcoma In Vinyl Chloride Workers: A Systematic . Detection Program. JAMA 230: 64-68, 1974. 4* Creech, J. L. Makk, L., Whelan, J. and Tamfaurro, C. H. Hepatotoxi city Among Polyvinyl Chloride Production Workers Ourlng First Year of Surveillance Program. Gastroenterology 67: 786, 1974. . 5. Kohn, J. and Weaver, P. C. Serum Alpha Fetoprotein In Hepatocellular Carcinoma. Lancet 2_: 334-336, 1974. 6. Tamfaurro, C. H., Makk, L. and Creech, J. L. Unpublished observation. 7. Girard, D., Johnston, W. C., and Grahm, J. H. Cutaneous Angiosar coma. Cancer 25: 868-883, 1970. 8. Barr, R. and Bonin, M. "Letters." JAMA 231(9): 914, 1975. 9. Popper, H., and Thomas, L. B. Alterations of Liver and Spleen Among Workers Exposed to Vinyl Chloride. Ann. NY Acad. Scl. 246: 172-194, 1975. 10. 6a1ambos, J. T. and Shaplra, R. Natural History of Hepatitis: IY Glycosanrinoglycuronans and Collagen In the Hepatic Connective -.^Tissue, J. Clin. Invest. 52(11): 2952-2962, 1973. U. Koizumi, T., Nakamura, N., and Abe, H. Changes In Acid Mucopoly saccharide In the Liver In Hepatic Fibrosis. Blochlm. Blophys. Acta. 148: 749-756, 1967. 12. Kajima, J. Studies on the Metabolism of Hepatic Connective Tissue In Fibrosis of the Liver. Med. J. Osaka Univ. .16: 419-429, 1964. 13. Rubin, E. Autoradiographic Characterization of Sulfated Acid Mucopolysaccharides in Experimental Cirrhosis. J. Hlstochem. Cytochem. 14: 688-689, 1966. 14. Patrick. R. S. and Kennedy, J. S. The Synthesis of Sulfated Mucopolysaccharide at Sites of Hepatic Fibrosis Induced by * Carbon Tetrachloride, Amyloidosis, and the Implantation of Catgut. J. Pathol. Bacteriol. 88: 549-555, 1964. 15. Kojima, J., Nakamura, N., Kanatanl, M. and Ohmori, K. The Glycosamlnog 1 yeans In Human Hepatic Cancer. Cancer Res. 35(3)_: 542-547, 1975. 924 I Artghllerf, L. J. Metabolism of Acid Mucopolysaccharides In Hepatoma and In Normal Liver. Oncology 30: 304-317, 1974. Yamamoto, K., and Teryama, H. Comparison of Cell Coat Acid Muco polysaccharides of Normal Liver and Various Ascites Hepatoma Cells. Cancer Res. tt: 2257-2264, 1973. xa. QIFerrante, N. M. The Measurement of Urinary Mucopolysaccharides. Anal. Blochem. 21: 98-106, 1967. 19. Bitter, T., and Muir, H. A Modified Uronlc Acid Carbazole Reaction. Anal. Blochem. 4: 330-334, 1962. 20. 'A 21. .22 Schiller, S., Slover, G. A., and Dorftnan, A. A Method for the Separation of Acid Mucopolysaccharides: Its Application to the Isolation of Heparin from the Skin of Rats. J. Biol. Chem. 236141: 983-987, 1961. Kupchella, C., and Steggerda, F. The Distribution of Acid Muco polysaccharides In the Canine Gastrointestinal Mucosa. Trans. NY Acad. Scl. 34: 351-360, 1971. Howry, R. W. Aldan Blue Techniques for the Hlstochenrical Study of Acidic Carbohydrates. J. Hlstochem. and Cytochem. 4: 407, 1356. ~ 23. Sundennan, F. W. and Boerner, F. Normal Values In Clinical Medldne. W. B. Saunders. Philadelphia, p. 353, 1949. 24. Varma, R. S., Vanna, R., Allen, W. S., and Hardi, A. H. Urinary Excretion of Acid Mucopolysaccharides in Schizophrenia. 81ochem. Med. 11(4): 358-369, 1974. 25. OlFerrante, N. and Rich, C. The Determination of Acid Amlnopolysaccharlde in Urine. J. Lab. Clin. Med. 48: 491-494, 1956. 26. Kao, K.''and Leslie J. Micro Fractionation and Determination of Urinary Glycosanrinoglycans. Blochem. Med. 9(4): 317-326, 1974. 27. Goldberg, J. and Cotller, E. Specific Isolation and Analysis of Mucopolysaccharides (Glycosamlnoglycans) from Human Urine. Clin. Chlm. Acta. 41; 19-27, 1972. 28. Manley, G., Severn, M. and Hawksworth, 0. Excretion Patterns of Glycosanrinoglycans-and Glycoproteins In Normal Hunan Urine. J. Clin. Pathol. 21: 339-345, 1968. 29. Pennock, C. A. A Modified Screening Test for Glycosaminog1yean Excretion. J. Clin. Path. 22: 310, 1969. a 92S R&S 112674 R&S 112675 ; jt,. 30. Douglas, C., Nowak, J. and Danes, B. Mucopolysaccharides In Urine During Normal Hunan Developnent. Pedlatr. Res. 7: 724-727, 1973. 31 Kurgda, J., Salto, S., Seno, N., Nagase, S-, and Anno, K. Isolation and Chemical Characterization of Mucopolysaccharides from Rat Tiaors. Cancer Res. 34(2): 308-312, 1974. n 4