Document 3e3QE27g9zM3y1GxMggm5mn7y

Reprinted from CANCER. Vol. 40. No. 6, Decem ber 1977. Copyright. 1977. by the American Can cer Society. Inc. J. B. Lipptneou Company. Printed in U.S.A. URINARY GLYCOSAMINOGLYCAN PATTERNS IN ANGIOSARCOMA OF THE LIVER Kevin L. Curran, BA, MS, Charles E. Kupchella, PhD, H. MDand Carlo Tamburro, Glycosaroinogiycans extracted from 24-hour urine specimens from patients with hepatic angiosarcoma and from normal/controls were separated as cetyipyridinium complexes into "hyaluronic acid," "chondroitin sulfate," and "heparin" fractions, then further separated and characterized by anion-exchange 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 acid 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 ehondroitin-4-sulfate and/or chondroitin-6-sulfate. Omar 40:5050-3053. 1977. iie emergence of angiosarcoma of the urinary GAG or collagen degradativc or syn Tliver and its relationship to vinyl chloride thetic products. Preliminary studies in our labo exposure4-' 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. 1#-" there is still no single chemical in rhosis. '* 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 'Ilic association of elevated tissue glycosami- other diseases, the urinary chondroitin sulfate noglycan.s (GAG) with tumors, including angio fraction was the only uronic acid positive fraction sarcoma. has been established.Cllyco- found in the urine of seven of nine cases of vinyl- saminoglycans are also known to be involved in chloride-cxposure-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. '* Since angiosarcoma of the liver has "chondroitin sulfate" fraction in hepatic angio both neoplasia and fibrogenesis in its etiology17 sarcoma. GAG changes could be expected to serve to sig nal the appearance of early lesions and may be useful in evaluating advanced lesions. Galambos* 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 fihrogenesis should not be expected to result in significant increases in A 46-ycar-old white male worked as a chemical helper In a vinyl chloride polymerization plant for thirteen years prior to the diagnosis of angiosarcoma. From ihe University of Louisville. C-inrcr Center, and the I'riLe I n,tunic for Surgical Research Health Sciences CenIrr. Louisville, Kentucky 40201. This work was iiippnnrH in pan liv an Aincnr.in (\inct-r Sim leiv Insimmimal f Irani. IN-IIL a grant from the II I*" (I,hKirn h ( atmpanv, and conirai i ) l*( IN-SS212 with the Nattonal (lancer institute Address for reprints- (I L, Kupi liella. (lamer (lenler. I'niversilv of laiuisville, Louisville, KY 41)201 Twelve years after initial employment, the patieni 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 transluccncy consistent with the diagnosis of angio sarcoma of the liver. Kxploratory laparotomy and liver hiopsy confirmed this diagnosis, and the patient was treated with Ai rrptixl lor pulilic.imm April Is, |')77 adriamycin, cyclophosphamide, and methotrexate; an 3050 6 31 f i 05 ft* CO N> i \ No. ft Glycosaminoglycans in Angiosarcoma * Curran et 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 angiosarcomatous 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. I). 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 scan. Angiographic studies showed peliosis hepatis. A liver biopsy revealed focal sinusoidal' dilatation, mild chronic inflammatory reaction with portal fibrosis, Kuplfer cell hyperplasia and dysplasia. Subsequent biopsies dcmunstraicri continued sinusoidal dilitaliim. atypical and dysplaslic Kuplfer cells with premalignant changes, t he peliosis hepatis pattern hecame 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. I, |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 -7()G until analysts. Cetylpyridinium chloride (Sigma (Ihemical Company, St. Louis) was added to the entire 24-hmtr volume to precipitate the CiACis tsp NORMAL URINE 10 S -1 -2 Fk:. I. Klunon Patterns of the Urinary Chondraitin 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 acid"), 1.2 M NaCl soluble ("chondroitin sulfate"), and 2. t M NaCl soluble ("heparin") fractions. Each fraction was then subjected to anion-exchange chromatography. Shown here are typical 1.2 M (chondroitin suirate) fraction elution patterns (Advanced ease U according to the method of DiFerrante." The hyaluronic acid, chondroitin sulfate, and hepa rin fractions were eluted individually according to the method of Schiller et al. '* Cetylpyridinium chloride wasTemoved14 and the GAGs were sub jected to anion-exchange chromatography as de scribed by Schiller et al.'* 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.1 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 urtmic-acid-pnsiiivc fractions within each individual salt fraction were pooled, dialyzed to rrmove salt, and concentrated. The fractions cluictl by 1.25 or 1.50 M NaCl were tested for 3J 0) 05 CO GO 3052 Cancer December 1977 Vol. 40 Table 1. Source Ratio ofTotal Uranic Acid Eluted in 1.25 M/1.5 M NaCl Normal Normal Angiosarcoma, case 2, pre-chemotherapy1 Angiosarcoma, rase 2. post-chemotherapy* Angiosarcoma, case I, advanced 0.364 0.316 0.843 0.971 3.(88) Note: The giyctnaminoglycans (C IAt) in a 24-hour urine specimen were precipitated with cctylpyridinitim chloride (CPC) and separated as 0.4 M NaCI sololtlc ("hyaluronic acid"), 1.2 N1 NaCI soluble ("chondraitin sulTaie")r and 2.1 M NaCI soluble ("heparin") fractions. The CPC was removed from the 1.2 M NaCl-CPGsolubilized fraction and the GAGs further purified by anion-exchange chromatography. The total amount of GAG in the resulting 1.23 M and 1.30 M Nad columneluted fractions was determined and the 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 NaCI 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 NaCI. The ratios of the total amount of uronic acid-p sitive material eluted with 1.25 M NaCI to the total amount eluted with 1.50 M NaCI are given in Table I. 'the susceptibility of the GAGs eluted with 1.25 or 1.50 M NaCI to hyaiuronidase degrada tion is given in Table 2. The GAG eluted with 1.25 M NaCI was resistant to hyaiuronidase, the enzyme producing only a 43% reduction in tur bidity. The 1.50 M NaCl-eluted GAG fraction was 100% susceptible to hyaiuronidase degrada tion. Discussion and Conclusion susceptibility to testicular hyaluronidasc (Nutri The chromatographic pattern found here for tional Biochemical Company) using a modifica controls conforms to urinary glycosaminogiycan tion of the cetyltrimethylammonium-bromide, distributions reported by others. These pat turbidimetric assay described by DiFerramc.* terns suggest that there was a relative increase in urinary heparan sulfate and a decrease in Results rhondmiiin-4- and/or -6-sulfatc in patients with hepatic angiosarcoma. This interpretation The major GAG fraction observed in all agrees with the Dowex 1-X2 chromatographic urines--both from normal controls or from pa patterns reported by Kao and Leslie11 and by tients with angiosarcoma--was the fraction so others.*-1* lubilized by 1.2 M NaCl/1% cetylpyridinium Heparan sulfate is reported to be partially chloride (the "chondroitin sulfate" fraction). susceptible to hyaiuronidase digestion,10 and Anion exchange chromatography of hyaluronic this correlates well with the observed 43% diges tion of the GAG in our 1.25 M NaCI fraction. Table Z Hyaiuronidase Susceptibility Since heparan sulfate has been shown to be Source associated with blood vessels," an increase in Depolymenzatmn %1 the urinary excretion of this GAG is not surpris Hepann, jtandard 5.0 ing in this vascular lesion. Also, chondroitin-4- Hyaluronic acid, standard 93.1 and -ft sulfates are reportedly eluted from Do Chondroitin sulfate, standard 97.6 wex I-X2 columns with 1.50 M NaCl*-10and are 1.25 M NaCI column-eluate. pooled susceptible to hyaiuronidase" suggesting that 07 Co frmiionN from ;mqimjtrrnmai<MiK piiluniH 4 VS our 1.5 M fraction is chordmitin-4- and/or CO 1.50 M N;i('l Lolumn-cluatr, pooled chondroitin-(i-sulfaie. fraction* from anqiORurromatou* Assuming that urinary GAG patterns de patients 100.0 scribed here are reflections of hepatic changes, it 1.50 M NaCI column-eluatc, normal will be important to determine what processes 1(81.0 these changes reflect, t.e., those of neoplastic 1 (ilycnsaminoiflvcans isolated from urine weft tested for growth, fibrogenesis, or cell death. In this re hyaiuronidase susceptibility by measuring changes in turbidity developed with the addition of cctyltnmethvlammonium bromide fnllowintg incubation with hy;tiun>nid;i*e Normal controls exhibited only minor amounts of 1.25 SI NaCI column-eluted CA(* and winwtpirnfiy do run ap pear in this table. gard, it should be noted that 1) the ratio of heparan sulfate to chondroitin sulfate reported here for angiosnrcomatous urine is similar to that reported for cirrhotic human liver tissue by Becker,2 and 2) the shift from a hyaiuronidase- R&S 116435 No. ft Glycosaminoglycans in Angiosarcoma Curran et ai. 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. Becker' 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 angiosarcoma17 indicates that the observations reported here constitute a promising lead in early detection of vinyl-chloride-induced liver disease. KKFF.RKNCKN 1. Antghileri, L. J-. Metabolism of acid mucojtolvsacrha* rides in hqiatoma and normal liver. Oncology 30:304*317, 1974 2. Becker. K.: Arid mucopolysaccharides in experimental and human cirrhosis. In Collagen Metabolism in the Liver* II. Popper and K. Becker. Eds. New York, Stratton Inter continental Medical Hook Corporation. 1973; pp. 45-52* 3 Uittrr. T.. and Muir, 1L: A modified tinmicaeid carha* /ole reaction. Anal. Ilimktm. 4:33tl-,334. 1962, 4 Crrrc h. J. L-. and Johnson. M, N.: Amfiosarroma of the liver m thr manufacture of polyvinyl chloride, J. (tempo* huwi \hti. If.*1VI |5|, 1974 5, 1 >d n ranie, N.. ilie measurement of urinary tnueo* |Hilv*atchundev Antd. Ittwhem, 21 '9K UK*. I9fi7, f. 1 )i|-'rrTJinle, N.: Tnrbidinietnc measurement of acid mucopolysaccharides and hyalurcmiriase activity J. Itml. Chrm. 220-303-30f, 1956. 7. Talk. II., Crcrrh. |, I,,. Heath. IV W * Inhrwm, M. N.. and Key. M. M-- Mqiauc disease amom* worker* at a vinyl chloride jiolymrrizaiion plant. JAMA 230-59-63, 1974. ti, (talambos, J T.. Connective tissue metabolism and cirrhosis- In Collagen Metabolism tn the Liver, 11. Popper and K Becker. Kris. New York, Stratton intercontinental MetIII ;d Hook Corporation, 1973; pp 57-61, 9 (i.isk. (V, and (lasic. I'-: Removal of sialic acid from the cell i o.ic in tumor cells and vascular rndothelltim and its rlln is m metastasis. /Vw. .-Oar/.; Sn, 48 I [72 1 177, 1962. 10. f;rrrnbrn(. R A., Tamburro. C. JL, and Kuprhella, (1 K A prospective medical sum*illaner program for the detrs ium .md prrveimon of orrupulumallv-rrl.iied cancer, In Prrvrnnon and Detection of Cancer. II K, Nirbunp*, l.ditor. Part I. Volume 2. Murrrl Drkkrr, Im.t NY (In press) I ] Hutterer, V, and Rubin, E.: Mucopolysaccharides in reversible .md irreversible experimental hepatic- fibrosis In ('oil.men MetalNiiisnt m the LivtT, II. Popper and K. lirrker, Eds. New York, Stratton Intercontinental Medical Book Corjwiration, 1973. pp. 53-56. 12. Kao. K. Y, T,, and Leslie, J C.: Micro fractionation and determination of urinarv glycosaminoglycans. Rxockem. A/*/. 9.317-326* 1974, 13, Koizumi. T,,, Nakamura. N., and Abe. H.; Chances in .icid mucopoivsaceharide in the liver in hepatic fibrosis. Huh him. Hutphji* Acta. 148-749-756. 1967. 14 Korn. K. I).: Isolation of heparin from mouse mast cell tumor, J Hud. Chm, 234:1325-1.329, 1959. 15, Kupciirlla. O. K.. and Tamburro, C. IL: Urinary and tissue idvrosaminocclycnn patterns in hqintic nnfttnxareomu. In Prevention and Detection of < Mincer. M. K. Niebtinfs, Editor. Part 1. Volume 1. Marcel Dekkrr, Inc.. NY (In press), 16 Makk. I.., ("rrerh, |. I*,. Whelan. J (i., and Johnson, M, N.: Livrr damage and angiosarcoma in vinyl chloride workers: A systematic detection program. JAMA 2.30:64-68, 1974. |7 Popficr. IL. and Thomas, L. B.: Alterations of liver and spleen amomr workers exposed to vinyl chloride. Arm. Aiad. AW. 246:172-194, 1975, 18, Rich. (`, and Myers. W P. I-: Excretion of acid mucopolysaccharides in the urine of patients with malignant neoplastic diseases. J. Ijib. and C.hn. Med. 54:22.3-228. 1959. 19 Schiller. S., Slower. (V A,, and ITorfman, A.. A method for the separation of aetd mucopolysaccharides: Its apphe anon to the isolation of hrparm from the skin of rats. J. IUuU f-W_^36:98.3-9K7( |9A|. 2b. Sharon, N.CCSimplex (larhnhvdratcs: Their Chem istry. Biosynthesis, and l'unmons. Reading. Massachusrtts. AddisomWexley Publislumt (-ompany. 1975, 21 Varadt. I). P., (afnnrlli, J \.w and Dorfman. A.. The ac id mucopolysaccharides in normal urine, tiuxhim. Bwphys. Arta. 141:1(13-117. 1967 22. Yamamoto, K., and Trravnma, H.: Comparison of cell coat acid muco(K>iysacehandes of normal liver and vari* ous 4>ciics hqiuioma cells (.'anerr Res, 33:2257-2264, 1973,