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R&S 112270 BIO-MEDICAL - RESEARCH DOCUMENT DESCRIPTION FORM g3 gg 69 76 Duplicate , in all cards: 1 UUU03W year as-1961 File number [Right justify [Numeric only] 77 78 Sub-Index Code Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 20 21___________ CU(trt/. K.L 40 4160 61 62 C*f .. .......... 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. 61 62 21 22 23 24 Source (Journal, Vol., Number, Pages,Date ) ;/ L 61 62 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 Reprinted from CANCER. Vol. 40. No. 6. Decem ber 1977. Copyright. 1977. by the American Can cer Society. Inc. J. B. Uppincott Company. Printed in U.S.A. 0000363 URINARY GLYCOSAMINOGLYCAN PATTERNS IN ANGIOSARCOMA OF THE LIVER Kevin L. Curran, BA, MS, E.Charles Kupchella, PhD, H. MDand Carlo Tamburro, R&S 112271 Glycosaminoglycans extracted from 24-hour urine specimens from patients with hepatic angiosarcoma and from normal/controls were separated as eetylpyridinium complexes into "hyaluronic acid," "chondroitin sulfate," and "heparin" fractions, then further separated and characterized by anion-cxchange chromatography and hyaluronidase susceptibility. The chromato graphic pattern of the urinary chondroitin sulfate fraction in patients with angiosarcoma of the liver differed from those of controls in that there was a relative increase in the total amount of uronic add in a hyaluronidase-resistant fraction and a decrease in a fraction susceptible to hyaluronidase digestion. These changes appeared to become more pronounced with advancing disease. Chromatographic patterns and determinations of hyaluronidase susceptibility indicated that the resistant fraction was heparan sulfate and that the susceptible fraction was chondroitin-4-sulfate and/or chondroitin-6-sulfrte. Canctr 40:3050-3053, 1977. he emergence of angiosarcoma of the urinary GAG or collagen degradative or syn Tliver and its relationship to vinyl chloride thetic products. Preliminary studies in our labo exposure*'* has prompted a search for methodrsatory, however, demonstrated an increase in to detect this lesion. Although systematic both liver and urinary GAG in patients with screening programs are currently in opera angiosarcoma, chronic active hepatitis* and cir tion, there is still no single chemical in rhosis.11 Most of the increase in urinary GAG dicator which is specific for angiosarcoma or for occurred in the chondroitin sulfate fraction and, changes which may precede this disease. in contrast to what was found for normal and The association of elevated tissue glycosami- other diseases, the urinary chondroitin sulfate nogiycans (GAG) with tumors, including angio fraction was the only uronic acid positive fraction sarcoma, has been established. Glyco- found in the urine of seven of nine cases of vinyl- saminogiycans are also known to be involved in chloride-exposure-associated liver injury other normal connective tissue synthesis and collagen than angiosarcoma. This study was undertaken deposition and are elevated in connective tissue to characterize more completely the urinary disorders.11 Since angiosarcoma of the liver has "chondroitin sulfate" fraction in hepatic angio both neoplasia and fibrogenesis in its etiology" sarcoma. GAG changes could be expected to serve to sig nal the appearance of early lesions and may be useful in evaluating advanced lesions. Gaiambos* suggested that since the liver con Clinical Summaries Case 1--(Hepatic Angiosarcoma--advanced) tributes very little to the overall connective tissue of the body, hepatic fibrogenesis should not be expected to result in significant increases in A 46-year-old white male worked as a chemical helper in a vinyl chloride polymerization plant for thirteen years prior to the diagnosis of angiosarcoma. Twelve years after initial employment, the patient From the University of Louisville, Cancer Center, and the Price Institute for Surgical Research Health Sciences Cen ter, Louisville, Kentucky 40201. This work was supported in pan by an American Cancer Society Institutional Gram, IN-111, a gram from the B. F. Goodrich Company, and contract NOl-CN-55212 with the National Cancer Institute. Address for reprints: C. E. Kupchella, Cancer Center, University of Louisville, Louisville, KY 40201. Accepted for publication April 15, 1977. exhibited a persistent* elevation of lactic dehy drogenase and underwent angiographic studies which demonstrated multiple areas of scattered tumor stain throughout both lobes of the liver with areas of central translucency consistent with the diagnosis of angio sarcoma of the liver. Exploratory laparotomy and liver biopsy confirmed this diagnosis, and the patient was treated with adriamydn, cyclophosphamide, and methotrexate; an 3050 No. 6 Glycosaminoglycans in Angiosarcoma Curran el al. 3051 initial response was associated with a decrease in the alkaline phosphatase activity, improvement in in docyanine green clearance and an increase in radio isotopic uptake in areas of previously defective up take. After completion of the chemotherapy course, hepatic function deteriorated and the patient under went partial hepatic lobe radiation (total dose of 5,000 rads) over a two-month period. Despite radia tion therapy, the clinical course continued to deterio rate with the development of ascites, peripheral edema, increasing jaundice, hypoalbuminemia, and marked elevations of transaminases and alkaline phosphatase activities. This was followed by progres sive hepatic failure, hepatorenal syndrome and he patic coma. Autopsy findings showed extensive in volvement of the liver with angiosareomatous tissue extending into the diaphragm and metastasis to retro peritoneal and mediastinal lymph nodes, lungs, right adrenal gland and cerebellum. The right lobe of the liver demonstrated near elimination of the angiosar coma, presumably due to the radiation treatment. Urinary GAG assays reported here were made on 24hour urine specimens collected over the two-week period before death (Fig. 1). Case 2 (Hepatic Angiosarcoma--moderately advanced) A 54-year-old vinyl chloride polymerization worker was first employed as a polymerization vat cleaner 28 years prior to the diagnosis of angiosarcoma. Two years prior to diagnosis, the patient had persistent biochemical liver, abnormalities although he was oth erwise-asymptomatic with a normal liver-spleen sun. Angiographic studies showed pcliosis hepatis. A liver biopsy revealed focal sinusoidal dilatation, mild chronic inflammatory reaction with portal fibrosis, Kupffer cell hyperplasia and dysplasia. Subsequent biopsies demonstrated continued sinusoidal dilitation, atypical and dysplastic Kupffer cells with premalignant changes. The peliosis hepatis pattern be came more , pronounced and multiple radioisotopic defects were evident on liver scan. A repeat biopsy one year after initial biochemical abnormality demon strated malignant sinusoidal cells. The patient was treated with a combination of adriamycin, cytoxan, and methotrexate with limited clinical and biochemi cal response. Death was preceded by peripheral edema, ascites, progressive hepatic failure, and coma. The GAG analyses reported here were made 10 and 6 months before death (Fig. 1, [middle]). Materials and Methods Twenty-four hour urine specimens were col lected from two patients with angiosarcoma of the liver, and from two normal controls. Urine specimens were stored at --70C until analysis. Cetylpyridinium chloride (Sigma Chemical Company, St. Louis) was added to the entire 24-hour volume to precipitate the GAGs $ . NORMAL URfNE 10 - s- uli -3 -Z -I - HEPATIC ANGIOSARCOMA 9 10 u 2- JU--U J K3 "I** z t tV- o 3 Fto. 1. Elution Patterns of the Urinary Chondroitin Sul fate Fraction. The glycosaminoglycans (GAG) in a 24-hour urine specimen were precipitated with cetylpyridinium chlo ride (CPC) and separated as 0.4 M NaCl soluble ("hyalu ronic add"), 1.2 M NaCl soluble ("chondroitin sulfate"), and 11 M NaCl soluble ("heparin") fractions. Each fraction was then subjected to anion-exchange chromatography. Shown here are typical 1.2 M (chondroitin.sulfate) fraction elution patterns (Advanced m case 1). according to the method of DiFerrante.5 The hyaluronic arid, chondroitin sulfate, and hepa rin fractions were eluted individually according to the method of Schiller tt al. '* Cetylpyridinium chloride was removed14 and the GAGs were sub jected to anion-exchange chromatography as de scribed by Schiller tt al.1* Glycosaminoglycan fractions were applied to 1.0 X 44 cm AG1-X2 (200-400 mesh, chloride form) columns Bio Rad Laboratories, Richmond, California) and eluted stepwise with 0.0, 0.5, 1.0, 1.25, 1.50, 2.0, and 3.0 M NaCl. At a flow rate of 1.0 ml/min, approximately sixteen 10.3 ml fractions of each molar strength of NaCl were collected and a sample of each fraction was analyzed for uronic acid by the method of Bitter and Muir. * Stan dards of heparin (Nutritional Biochemical Com pany), chondroitin sulfate (Sigma Chemical Company), and hyaluronic acid (Nutritional Biochemical Company) were also evaluated by ion exchange chromatography. The uronic-acid-positive fractions within each individual salt fraction were pooled, dialyzed to remove salt, and concentrated. The fractions eluted by 1.25 or 1.50 M NaCl were tested for 2D fio 03 N> IO 3052 Cancer December 1977 Vol. 40 Table 1. Source Ratio ofTotal Uronic Acid Eluted in 1.25 M/1.5 M NaQ Normal Normal Angiosarcoma, case 2, pre-chemotherapy1 Angiosarcoma, case 2, post-chemotherapy1 Angiosarcoma, case 1, advanced 0.364 0.316 0.843 0.971 5.000 Note: The glycosaminogiycans (GAG) in a 24-hour urine ipedmen were precipitated with cctylpyridinium chloride (CPC) and separated as 0.4 M NaQ soluble ("hyaluronic arid"), 1.2 M NaCl soluble ("chondroitin sulfate"), and 2.1 M NaCl soluble ("heparin") fractions. The CPC was removed from the 1.2 M NaCl-CPCsolubilized fraction and the GAGs further purified by anion-exchange chromatography. The total amount of GAG in the resulting 1.25 M and 1.50 M NaCl columneluted fractions was determined and thfc ratio of the two fractions was calculated. ('One day prior to begin ning of chemotherapy; 'Two days following chemotherapy initiation.) acid and heparin fractions revealed no qualita tive differences between controls and angiosar coma patients. The anion exchange column pat terns of the 1.2 M NaCl solubilized GAGs are shown in Fig. 1. Chromatography of the urinary "chondroitin sulfate" fractions of patients with angiosarcoma yielded a comparatively large, uronic-acid positive peak in 1.25 M NaCl. The ratios of the total amount of uronic acid-positive material eluted with 1.25 M NaCl to the total amount eluted with 1.50 M NaCl are given in Table 1. The susceptibility of the GAGs eluted with 1.25 or 1.50 M NaCl to hyaluronidase degrada tion is given in Table 2. The GAG eluted with 1.25 M NaCl was resistant to hyaluronidase, the enzyme producing only a 43% reduction in tur bidity. The 1.50 M NaCl-eluted GAG fraction was 100% susceptible to hyaluronidase degrada tion. Discussion and Conclusion susceptibility to testicular hyaluronidase (Nutri tional Biochemical Company) using a modifica tion of the cetyltrimethylammonium-bromide, turbidimetric assay described by DiFerrante.* Results The major GAG fraction observed in all urines--both from normal controls or from pa tients with angiosarcoma--was the fraction so lubilized by 1.2 M NaCl/1% cetylpyridinium chloride (the "chondroitin sulfate" fraction). Anion exchange chromatography of hyaluronic Table 2.. Hyaluronidase Susceptibility , Source Dcpoiymerization %1 Heparin, standard Hyaluronic acid, standard Chondroitin sulfate, standard 1.25 M NaCl coiumn-eluate, pooled fractions from angiosarcomatous patients 1.50 M NaCl coiumn-eluate, pooled fractionsJrom angiosarcomatous patients 1.50 M NaCl coiumn-eluate. normal 5.0 93.1 97.6 43.5 100.0 100.0 1 Glycosaminogiycans isolated from urine were tested for hyaluronidase susceptibility by measuring changes in turbidity developed with the addition of cetyltrimethylammonium bromide following incubation with hyaluronidase Normal controls exhibited only minor amounts of 1.25 M NaCl column-eluted GAG and consequently do not ap pear in this table. The chromatographic pattern found here for controls conforms to urinary glycosaminoglycan distributions reported by others. **" These pat terns suggest that there was a relative increase in urinary heparan sulfate and a decrease in chondroitin-4- and/or -6-sulfate in patients with hepatic angiosarcoma. This interpretation agrees with the Dowex 1-X2 chromatographic patterns reported by Kao and Leslie11 and by others. **" Heparan sulfate is reported to be partially susceptible to hyaluronidase digestion,10 and this correlates well with the observed 43% diges tion of the GAG in our 1.25 M NaCl fraction. Since heparan sulfate* has been shown to be associated with blood vessels,10 an increase in the urinary excretion of this GAG is not surpris ing in this vascular lesion. Also, chondroitin-4and -6 sulfates are reportedly eluted from Do wex 1-X2 columns with 1.50 M NaCl1,1* and are susceptible to hyaluronidase10 suggesting that our 1.5 M fraction is chordroitin-4- and/or chondroitin-6-sulfate. Assuming that urinary GAG patterns de scribed here are reflections of hepatic changes, it will be important to determine what processes these changes reflect, i.e., those of neoplastic growth, fibrogenesis, or cell death. In this re gard, it should be noted that 1) the ratio of heparan sulfate to chondroitin sulfate reported here for angiosarcomatous urine is similar to that reported for cirrhotic human liver tissue by Becker,1 and 2) the shift from a hyaluronidase- No. 6 Glycosaminoglycans in Angiosarcoma Curran et al. 3053 susceptible, to a hyaluronidase-resistant GAG is consistent with the suggestion by Hutterer and Rubin11 that the stabilization of collagen de pends on a shift to a hyaluronidase-resistant GAG envelope surrounding the collagen bundle. Although Hutterer and Rubin attribute this to an augmentation of dermatan sulfate, Becker1 reported that the GAG pattern in human cir rhosis was characterized by the augmentation of dermatan sulfate and heparan sulfate. If the ob served changes in urinary GAG are reflective of vinyl chloride-exposure-associated fibrosis, the fact that fibrosis is a precursor of angiosarcoma n indicates that the observations reported here constitute a promising lead in early detection of vinyl-chloride-induced liver disease. REFERENCES 1. Anghileri, L. J.; Metabolism of acid mucopolysaccha rides in hepatoma and normal liver. Oncology 30:304-317, 1974. 2. Becker, K.: Add mucopolysaccharides in experimental and human cirrhosis. In Collagen Metabolism in the Liver, H. Popper and K. Becker, Eds. New York, Stratton Inter continental Medical Book Corporation, 1973; pp. 45-52, 3. Bitter, T., and Muir, H.: A modified uronic add carbazole reaction. Anal. Biodum. 4:330-334, 1962. 4. Creech, J. L-, and Jphnson, M. N.: Angiosarcoma of the liver in the manufacture of polyvinyl chloride. J. Occupa tional MaL 16:150-151, 1974. 5. DiFerrante, N.: The measurement of urinary muco polysaccharides. AnaL Biodum. 21:98-106, 1967. 6. DiFerrante, N.: Turbidimetric measurement of add mucopolysaccharides and hyaiuronidase activity. J. Biol. Oum. 220:303-306, 1956. 7. Falk, H., Creech. J. L-, Heath, D. W., Johnson, M. N., and Key, M. M.: Hepatic disease among workers at a vinyl chloride polymerization plant. JAMA 230:59-63, 1974. 8. Galambos, J. T.: Connective tissue metabolism and drrhosis. In Collagen Metabolism in the Liver, H. Popper and K. Becker, Eds. New York, Stratton Intercontinental Medical Book Corporation, 1973; pp. 57-61. 9. Gasic, G., and Gasic, T.: Removal of sialic add from the cell coat in tumor cells and vascular endothdium and its effects in metastasis. Pnc. Natl. Acad. ScL USA. 48:1172'-! 177, 1962. 10. Greenberg, R. A., Tamburro, C. H., and Kupcheila, C. E.: A prospective medical surveillance program for the detection and prevention of occupationaily-reiated cancer. In Prevention and Detection of Cancer. H. E. Nieburgs, Editor, Part 1, Volume 2, Mated Dekko-, Inc., NY (In press). 11. Hutterer, F., and Rubin, E.: Mucopolysaccharides in reversible and irreversible experimental hepatic fibrosis. In Collagen Metabolism in the Liver, H. Popper and K. Becker, Eds. New York, Stratton Intercontinental Medical Book Corporation, 1973; pp. 53-56. 12. Kao, K. Y. T., and Leslie, J. G.: Micro fractionation and determination of urinary glycosaminoglycans. Biodum. Med. 9:317-326. 1974. 13. Koizumi, T., Nakamura, N., and Abe, H.: Changes in add mucopolysaccharide in the liver in hepatic fibrosis. Biodum. Biopkyt. Ada. 148:749-756, 1967. 14. Korn, E- D.: Isolation of heparin from mouse mast ceil tumor. J. Biol. Oum. 234:1325-1329, 1959. 15. Kupchdla, C. E., and Tamburro, C. H.: Urinary and tissue glycosaminoglycan patterns in hepatic angiosarcoma. In Prevention and Detection of Cancer, H. E. Nieburgs, Editor, Part 1, Volume 1, Mated Dcfcker, loe^ NY (In press). 16. 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. 17. Popper, H., and Thomas, L. B.: Alterations of liver and spleen among workers exposed to vinyl chloride. Am. NT Acad. ScL 246:172-194, 1975. 18. Rich, C., and Myers, W. P. L.: Excretion of add mucopolysaccharides in the urine ofpatients with malignant neoplastic diseases. J. Lab. and Cti*. Med. 54:223-228, 1959. 19. Schiller, S., Slover, G. A., and Dorfman, A.: A method for the separation of add mucopolysaccharides: Its application to the isolation of heparin from the skin of rats. J. BioL Oum. 236:983-987. 1961. 20. Sharon, N.: Complex Carbohydrates: Their Chem istry, Biosynthesis, and Functions. Reading, Massachusetts, Addison-Wesiey Publishing Company, 1975. 21. Varadi, D. P,, Cifondli, J. A., and Dorfman, A.: The add mucopolysaccharides in normal urine. Biodum. Biopkyt. Ada. 141:103-117, 1967. 22. Yamamoto, K., and Tcrayama, H.: Comparison of cell coat add mucopolysaccharides of normal liver and vari ous ascites hepatoma cells. Cancer Bet. 33:2257-2264, 1973. R&S 112274