The composition of total lipid extracted with chloroform-methanol from the AV12-induced tumor was investigated by thin-layer and paper chromatography. The content of lecitin and sphingomyelin was somewhat decreased and a cerebroside was characteristically detected in this tumor.</p

Kanemasa, Yasuhiro

Ohmori, Masaki

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 23, Issue 5 1969 Article 9OCTOBER 1969Lipid composition of adenovirus-inducedtumorMasaki Ohmori∗ Yasuhiro Kanemasa†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Lipid composition of adenovirus-inducedtumor∗Masaki Ohmori and Yasuhiro KanemasaAbstractThe composition of total lipid extracted with chloroform-methanol from the AV12-inducedtumor was investigated by thin-layer and paper chromatography. The content of lecitin and sphin-gomyelin was somewhat decreased and a cerebroside was characteristically detected in this tumor.∗PMID: 4312769 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 23, 449-452 (1969)LIPID COMPOSITION OF ADENOVIRUS.INDUCED TUMORMasaki OHMORI and Yasuhiro KANEMASADepartment of Pathology, and Department oj Microbiology, Okayama UniversityMedical School, Okayama, Japan (Director. Prof. K. Ogawa)Received for publication, July 28, 1969The present report deals with the results obtained In the study con-ducted on the problem whether or not there is a specificity in the lipidcomposition of AVl2-induced tumor (I) which morphologically possesses aspecificity as the neuroectodermal origin (2) differing from many othervirogenic tumors.MATERIALS AND METHODSVirus and animalsAt the two-week latent period after the intraperitoneal inoculation of 0.1 mlof 102.5TCID 50jO.1 ml of adenovirus type 12 (AVI2) (Huie strain) into newbornSyrian hamsters of less than 24 hours old, tumor developed in the peritonealcavity were removed and used for the experiment.Preparation of lipid fractionThis was conducted by FOLCH'S method (3): namely, 25.6 g of the extirpatedtumors were suspended in 500 ml of chloroform-methanol (2: 1, vjv) and homo-genized by Waring blender, allowed to stand at.room temperature with stirringfor 4 hrs. The extract was obtained after filtration through the sintered glassfunnel. The extraction was repeated twice, the first being at room temperatureovernight and the second at 37 c C overnight. The whole series of filtrates waspooled together and brought to dryness by a rotary evaporator. The extract thusobtained was subjected to FOLCH'S partition dialysis (1) in order to remove in-organic materials and other organic comtaminants.Thin-layer chromatographyThe thin-layer chromatography was carried out on glass plate with a layerof silicic acid (Silica Gel G, Merck). The isolation was conducted mainly forphospholipids, using the solvent system of chloroform-methanol-water (70: 25: 4,vjv jv) for the primary chromatography and chloroform-methanol-7N ammonia(60 : 35: 5, vjvjv) for secondary. As for the coloration iodine vapor was usedfor organic compounds, DITTMER reagent (5) for general phospholipids, ninhyd-rine reagent (6) for lipids containing amino-group, DRAGENDORFF reagent (6) forlipids containing choline and anthrone reagent (7) or diphenylamine reagent (6)for glycolipids.4491Ohmori and Kanemasa: Lipid composition of adenovirus-induced tumorProduced by The Berkeley Electronic Press, 1969450 M.OHMORI and Y. KANEMASAMild alkaline hydrolysis ofphospholipidsThe water soluble components obtained by a mild alkaline hydrolysis oflipid according to DAWSON'S method (8) were chromatographed on Whatman No.1 paper using the mixture of phenol-water-acetic acid-ethanol (80 : 20: 10: 12,vIvIvIv) as the solvent system. Each spot was detected with ninhydrin reagentand HANES-IsHERWOOD reagent (9). As reference compounds for comparison ofthe RF value, hydrolyzates of phosphatidyl glycerol and phosphatidyl ethanola-mine from E. coli, cardiolipin from bovine and lecithin from egg were used.RESULTSBy exposing the thin-layer chromatogram to iodine vapor 17 spotswere obtained as shown in Fig. 1. Six of them ware identified as lysophos.phatidyl choline, sphingomyelin, phosphatidly ethanolamine, cardiolipinSF ::::::::1~X8CL PE XSc::;: ~ ~~ PCX7 ~:~ ~SM~.~ ~~. X3 ~LPC-.,~ X2Xl .::.!840Fig. I Two-dimentional thin.layer chromatography of lipidextracted from the AV 12-induced tumor.(.;~ : Positive for Dittmer reagent® : Positive for ninhydrine reaction® : Positive for Dragendorff reagent@ : Positive for anthrone reagentThe chromatogram was primary developed from right to left with chloroform-methanol-water (70 : 25: 4, v/v/v) and then in the vertical direction with chloro-form-methanol-7N ammonia (60: 35: 5, v/v/v). 0, origin; SF, solvent front;LPC, lysophosphatidyl choline; SM, sphingomyelin; PC, phosphatidyl choline;PE, phosphatidyl ethanolamine; CL, cardiolipin; C, cerebroside; NL, neutrallipid; and unknown substances listed as Xl-XII. Lashed lines indicate faint spots.X 3, X 5, X 8 and X 9 have been tentatively identified as phosphatidyl inositol,phosphatidyl grycerol, n-n-acethyl-phosphatidyl ethanolamine and fatty acid, res-pectively.2Acta Medica Okayama, Vol. 23 [1969], Iss. 5, Art. 9http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss5/9Lipid Composition of AV-induced Tumor 451and cerebroside, and another 4 spots were tentatively identified as phos.phatidyl inositol, phosphatidyl grycerol, n-n-acetyl-phosphatidyl ethano-lamine and fatty acid, respectively. The remaining 7 spots could not beidentified by the methods employed.'In order to make further identification of these compounds, theirdeacylated products of mild alkaline hydrolysis were characterized bymeans of the comparison of their RF values on a paper chromatogramwith those of the reference compounds. The hydrolyzates derived fromthe major components were identified by the color detections and thequantitative relationship in addition to the comparison of RF values.DISCUSSIONIn the present experim~nt with AV12-induced tumor the compositionratio of each component in the phospholipids did not show any appreciabledifference as in other tumors, (10, 11, 12, 13) but there can be observeda decrease in lecitin and sphingomyelin content.What appears to be somewhat specific in the AV12-induced tumorwas the presence of a fairly large quantity of glycolipid. Judging fromthe color reaction and the RF values of that glycolipid, it seemed to con-tain an abundant neutral sugar, which gave a suspicion of cerebroside.These findings seem to be interesting in view of the fact that AV12-inducedtumor is morphologically said to have originated from the undifferentiatedneuro-ectodermal tissue (2).SUMMARYThe compOSitIOn of total lipid extracted with chloroform-methanolfrom the AV12-induced tumor was investigated by thin-layer and paperchromatography. The content of lecitin and sphingomyelin was somewhatdecreased and a cerebroside was characteristically detected in this tumor.ACKNOWLEDGEMENTThe authors wish to e1Cpress profound thanks for the kind guidance of this work to Prof.OGAWA of Department of Fathology.REFERENCES1. TRENTIN, J. J., YABE, Y., TAYLOR, G.: Science 137, 835, 19622. OGAWA, K., TSUTSUMI, A., IWATA, K., FUJII, Y. OHMORI, M., TAGUCHI, K, and YABE,Y.: Conn, 57, 43, 19663. FOLCH, j., LEES, M., SLOANE STANLEY, G. H.: J. Bioi. Chern. 226, 497, 19573Ohmori and Kanemasa: Lipid composition of adenovirus-induced tumorProduced by The Berkeley Electronic Press, 1969452 M. OHMORI and Y. KANEMASA4. FOLCH, J., ASCOLI, I., LEES, M., MEATH, J. A., LE!3ARON, F. N.: j. Biol. Chern. 191,833, 19515. DITTMER, J. G., LESTER, R. 1.: j. Lipid Res. 5, 126, 19646. WAGNER, H., HORHAMMER, L., WOLFF, P.: Biochern, Z. 334, 175, 19617. ROE, J. H.: j. Biol. Chern. 212, 335, 19558. DAWSON, R. M. C.: Biochern. J. 75, 45, 19609. HANES, C. S., ISHERWOOD, F. A.: Nature, 164, 1I0?, 194910. FIGARD, P. H., GREENBERG, D. M.: Cancer Res. 22, 361, 1962II. WALLACH, D. F. H., SODERBERG, J., BRICKER L.: Cancer Res. 20,397,196012. GRAY, G. M.: Biochern. j. 86, 350, 196313. KAWANAMI, J., TSUJI, T., OTSUKA, P.: j. Biochern. 59, 151, 19664Acta Medica Okayama, Vol. 23 [1969], Iss. 5, Art. 9http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss5/9

Lipid composition of adenovirus-induced tumor

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