Serum y-glutamyltransferase isoenzymes in extrahepatic biliary obstruction

Transcription

1 Journl of Clinicl Pthology, 1979, 2, Serum y-glutmyltrnsferse isoenzymes in extrheptic biliry obstruction P. R. WNHAMW, C. P. PRC2, AN H. G. SAMMONS From the eprtment of Clinicl Chemistry, st Birminghm Hospitl, Bordesley Green st, Birminghm B9 SST, UK SUMMARY The y-glutmyltrnsferse isoenzymes in the ser of ptients with extrheptic biliry obstruction hve been studied, using electrophoretic, gel filtrtion, nd ultrcentrifugtion techniques, nd compred with those present in norml ser. Five isoenzymes were shown to exist in ptients' ser, three of which were not demonstrted in norml ser. The observtions re discussed in reltion to the influence of biliry regurgittion nd the possible solubilistion of membrne-bound enzymes. The results re compred with those of previous studies on lkline phosphtse. The elevtion of y-glutmyltrnsferse (ygt) (.C ) ctivity in the ser of ptients with obstructive liver disese is well documented, but the reson for the chnge is unknown. t hs been suggested tht the liver normlly elimintes the enzyme into the bile, but when the bile duct is obstructed this process results in the ccumultion of the enzyme in the blood (Szczeklik et l., 1961; Orlowski, 196; Rutenburg et l., 196; Luksik nd Richterich, 1965; Whitfield et l., 1972). The possibility of the bile being n excretory route for ygt produced from non-heptic sources hs lso been considered (Kryszewski et l., 197). Previous work hs shown tht the rise in serum lkline phosphtse (.C..1..1) observed in biliry obstruction my in prt rise by regurgittion of the biliry enzyme (Price nd Smmons, 1974). A study of the isoenzymes of ygt in the ser of ptients with extrheptic biliry obstruction ws therefore undertken, in reltion to those isoenzymes lredy described in humn bile (Wenhm et l., 1978), with view to finding n explntion of the elevtion in serum ygt ctivity observed in this group of ptients. 'Present ddress: eprtment of Clinicl Chemistry, Western Generl Hospitl, Crewe Rod, dinburgh H4 2XU 2Present ddress: eprtment of Chemicl Pthology, Southmpton Generl Hospitl, Tremon Rod, Southmpton S9 4XY Received for publiction 27 Februry 1979 Mteril nd methods Ser were obtined from 1 ptients with extrheptic biliry obstruction, dignosed t lprotomy or necropsy, nd from nine lbortory stff who cted s norml controls. Agr gel electrophoresis ws performed on the Multiphor electrophoresis equipment (LKB Produkter, Bromm, Sweden) by method lredy described (Wenhm et l., 1978). Polycrylmide gel disc electrophoresis ws crried out using the Shndon electrophoresis equipment nd the method of Azzoprdi nd Jyle (197). Before ppliction of the smples, bromophenol blue ws dded to the serum so tht the mobilities of the bnds of enzyme ctivity could be relted to lbumin. The mobilities were expressed s frction of the mobility of lbumin. The ygt isoenzymes frctionted by these two techniques were visulised by mens of incubtion with y-l-glutmyl ct-nphthylmide, coupling the liberted cx-nphthylmine with Fst Blue B, by the optimised method of Wenhm et l. (1978b). The isoenzymes were lso quntitted fter polycrylmide gel electrophoresis by cutting the gel into 5 cm segments nd incubting the homogenised gel segments in buffered substrte ccording to the method of Roslki et l. (197). Gel filtrtion chromtogrphy using Sephdex G2 (Phrmci, Uppsl, Sweden) nd buoynt density ultrcentrifugtion were both performed by methods previously described (Wenhm et l., l 978), nd enzyme ctivity in the frctions obtined ws determined by the method of Roslki nd Trlow (1974). 92

2 Serum y-glutmyltrnsferse isoenzymes in extrheptic biliry obstruction Results LCTROPHORTC STUS ch of the ptient's ser showed three zones of enzyme ctivity fter gr gel electrophoresis, in the l-, Y2-, nd fl-globulin frctions. n norml ser, only two bnds of ctivity were obtined, in the cvj- nd CX2-globulin frctions. Polycrylmide gel disc electrophoresis of norml ser lso resulted in two zones of ctivity with mobilities of 2 % nd 77 % of lbumin (Rlb vlues of 2 nd 77). Ser from ptients with extrheptic biliry obstruction gve rise to four zones of ctivity fter polycrylmide gel electrophoresis, with Rlb vlues of, 2, 55, nd 77. The origin bnd nd the bnd with n Rlb vlue of 55 ppered to contribute the most towrds enzyme ctivity, nd this ws confirmed using the quntittive technique (Fig. 1). -1._ 5 N c L o 19s / 1 f,'n / 4s 7s % 751 t Frction number Fig. 2 Gel filtrtion chrcteristics of ygt isoenzymes in norml serum: --- serum proteins; - enzyme ctivity (fu/l). 1 ~~~~z - r ' B} co - C Ln < U (U~~ C?-- ~ -- ~ L-- ~ ~ ~ A Origin Segment number Fig. 1 Quntittive loclistion of ygt isoenzymes fter polycrylmide gel electrophoresis of ser from two ptients with extrheptic biiry obstruction. nzyme ctivity expressed s bsorbnce 45 nm: --- ptient CC; - ptient PM. GL FLTRATON STUS Frctiontion of serum yielded three min protein peks, s described by Flodin nd Killnder (1962). The norml ser ech yielded two peks of ygt ctivity, eluting between the 19S nd 7S nd between the 7S nd 4S proteins, respectively (Fig. 2). The men recovery of enzyme ctivity ws 95%. Three frctions were obtined from ech of the ser from ptients with extrheptic biliry obstruction, one eluting with the void volume, one between the 19S nd 7S, nd the other between the 7S nd 4S proteins (Fig. ). n ll smples studied, the first two peks lwys possessed much more ctivity thn the third, whose ctivity did not differ significntly from the level observed in the norml ser (Tble 1). The men recovery of enzyme ctivity ws 98 %. :> N c ll Frction number Fig. Gel filtrtion chrcteristics of ygt isoenzymes in serum from ptient (PM) with extrheptic biiry obstruction: --- serum proteins; enzyme ctivity (Ull). Tble 1 ygt ctivity in differed frctions obtined by gel filtrtion nd ultrcentrifugtion ofser from ptients with extrheptic biliry obstruction, expressed s percentge of totl ctivity recovered Smple Gelfiltrtion Ultrcentrifugtion Void 19S-7S 7S-4S 4S Low density volume frction PM CC NS MW FG MG AM AB Norml ser Zero Zero ULTRACNTRFUGATON STUS Buoynt density ultrcentrifugtion of norml ser showed only one pek of ygt ctivity, sedimenting with the 4S proteins (Fig. 4). nzyme recovery V fl

3 94 P. R. Wenhm, C. P. Price, nd H. G. Smmons 4 Albumin - ~1gG-1 / 1gMH,' 7S // 4S Fig. 4 Ultrcentrifugtion chrcteristics of ygt isoenzymes in norml serum: --- serum proteins; * enzyme ctivity (U/). -4 > 8', - / / N11 /, H.> B N c,,"'1,j /N X wd 19S-' 'gir-" N c // uj v Frction number Frction number Fig. 5 Ultrcentrifugtiorn chrcteristics of ygt isoenzymes in serum from ptient (PM) with extrheptic biliry obstruction: --- serum proteins: * * enzyme ctivity (U/i) n 1 Cn co verged 95%. All of the ptients' smples studied showed two peks of ygt ctivity, one sedimenting with the 4S proteins nd the other floting t the top of the grdient (Fig. 5). The men recovery of enzyme ctivity ws 79 %. The reltive contributions of the 4S nd the low density peks towrds totl ctivity were not lwys constnt (Tble 1), but the ctivity of the 4S pek in the ptients' smples ws lwys higher thn the 4S pek observed in the norml ser. COMPARSON OF SONZYM CHARACTRSTCS OBTAN BY FRACTONATON TCHNQUS Frctions representing peks of ctivity, obtined by gel filtrtion, were pooled, concentrted using Lyphogel (Hwksley nd Sons Ltd, Lncing, Sussex, UK), then subjected to buoynt density ultrcentrifugtion, nd gr nd polycrylmide gel electrophoresis. Pooled frctions obtined fter buoynt density ultrcentrifugtion were electrophoresed without prior concentrtion. The results of these investigtions in ptients' ser re shown in Tble 2 nd indicte the presence of up to five isoenzymes, of which two re present in norml ser. They re rbitrrily numbered in Tble 2 solely for ese of discussion. iscussion Mny methods hve been used for the frctiontion of serum ygt isoenzymes in heptobiliry diseses, but little comprtive work hs been undertken on the techniques employed. Our results using gr gel electrophoresis show tht, in extrheptic biliry obstruction, zones of enzyme ctivity re present in the cxl-, CX2- nd f8-globulin frctions. These results re in greement with those of Miyzki nd Okumur (1972) using gr, nd lso with Hetlnd et l. (1975) using grose, nother medium tht does not exert moleculr sieving effect. Polycrylmide gel electrophoresis of ptients' ser produced four zones of enzyme ctivity, two of which were not present in norml ser (Tble 2) nd which together constituted lmost ll of the totl ctivity. A high ctivity origin bnd ws lso demonstrted by Azzoprdi nd Jyle (197) using polycrylmide, nd by Orlowski nd Szczeklik (1967) nd Kokot nd Kusk (1968) using strch gel. Sephdex gel filtrtion of ptients' ser produced Tble 2 Physicochemicl chrcteristics of the ygt isoenzymes in ptients' ser with extrheptic biliry obstruction soenzyme lutionfrom Buoynt density lectrophoretic Reltive contribution G2 ultrcentrifugtion mobility towrds totl ctivity Agr Polycrylmide Si Void volume Flots + + Sil Void volume Flots, Sill 19S-7S 4S, SiV* 19S-7S 4S, 2 + SV* 7S-4S 4S, 77 + *Present lso in norml ser

4 Serum y-glutmyltrnsferse isoenzymes in extrheptic biliry obstruction three ygt frctions, eluting with the void volume, 19S-7S, nd 7S-4S protein frctions. The lst two frctions were lso obtined from norml ser. These results gree with the work of Orlowski et l. (1965), Orlowski nd Szczeklik (1967), nd Kokot nd Kusk (1968), except tht these uthors climed to hve shown the 19S frction to be present in norml ser. After buoynt density ultrcentrifugtion of norml ser, ll of the enzyme ctivity sedimented with the 4S protein frction (Fig. 4) in greement with Szewczuk (1966). Ptients' ser, on the other hnd, showed the presence of n dditionl low density frction (Fig. 5). From the results of the comprison study it ws concluded tht five isoenzymes could be identified in the ser of ech ptient, nd tht the increse in the totl serum ygt ctivity observed in these ptients ws due to the ppernce of three isoenzymes, Si, Si1, nd Sill (Tble 2), not normlly present. soenzymes S11 nd S111 pper to contribute eqully towrds most of the ctivity but re very different in their chromtogrphic chrcteristics. soenzyme Sil is low density molecule of high moleculr size, wheres isoenzyme Sll is smller, denser molecule. The remining bnorml isoenzyme, isoenzyme S, is identicl with Sl in every respect except for its electrophoretic mobility on gr gel (Tble 2). t my be tht these isoenzymes differ only in component conferring different electricl chrge t ph 8-6. The high moleculr size of isoenzymes S1 nd Sl my rise from polymeristion of the norml liver enzyme, lthough this is unlikely owing to their very low buoynt density. t is much more likely tht they re the result of the ssocition of ygt with very low density component such s lipid or lipoprotein. Such n ssocition hs been demonstrted for lkline phosphtse (Moss, 1962; unne et l., 1967; Jennings et l., 197; Price nd Smmons, 1974). n the cse of ygt, it hs been suggested tht the biliry enzyme my be ssocited with lipid (Wenhm et l., 1978); furthermore, Beck (1978) suggested tht serum isoenzyme might be ssocited with lipid component. n study resembling our own, Huseby (1978) concluded tht the heterogeneity ofygt found fter gel filtrtion nd electrophoresis ws due to ggregtes of n mphiphilic form of ygt with lipids nd other proteins. This uthor, studying ygt in liver extrct, bile, nd norml serum pool, showed tht ppin digestion nd incubtion with detergent ech produced smller molecule, corresponding to isoenzymes SV nd S111, respectively, in this study. The detergent effect upon biliry ygt confirmed our previous demonstrtion (Wenhm et l., 1978) tht 95 extrction of biliry ygt with n-butnol resulted in the production of smller molecule possessing identicl physicochemicl chrcteristics to isoenzyme Sill (Tble 2), process similr to tht lredy proposed for biliry lkline phosphtse (Price et l., 1972). We propose tht isoenzyme S111 is the min liver isoenzyme, nd in response to the obstruction its rte of relese into the circultion is incresed. t is normlly present in the bile but ttched to lipid component, resulting in lrger molecule with slower electrophoretic mobility. Whether this incresed rte of relese into the circultion is due to n incresed rte of production remins to be determined. Such process hs been ruled out in the guine-pig (Huseby nd Vik, 1978). The origin of the isoenzyme SV remins obscure. Huseby (1978) ws ble to produce it by incubting liver homogente t ph 7-2 nd 7 C s well s by the ction of ppin digestion. This enzyme therefore my well rise by in vivo proteolytic ctivity on isoenzyme Sll. soenzymes S nd S1i my rise by either: (i) regurgittion of biliry ygt bck into the circultion due to obstruction of norml bile flow. uring regurgittion the biliry enzyme my become ssocited with lipoprotein crrier, conferring the low buoynt densities of isoenzymes S nd S1, similr to tht proposed for lkline phosphtse (Price nd Smmons, 1974); or: (ii) solubilistion of liver membrne-bound enzyme into membrnous vesicles by the ction of ccumulted bile slts. Such process hs been suggested in the cse of lkline phosphtse (e Broe et l., 1975; 1978) nd lso ygt during experimentl obstruction in the guine-pig (Huseby nd Vik, 1978). Present evidence cnnot prove which, if not both, processes occur. Nevertheless this study hs demonstrted tht in obstructive liver disese similr process occurs with respect to biliry ygt s occurs with biliry lkline phosphtse, the biliry enzyme ppering in the circultion in modified form. n the cse of lkline phosphtse, fewer isoenzymes hve been demonstrted in the ser of ptients with obstructive liver disese, nd therefore it would be unwise to extend the similrities too fr. However, it is well known tht chnges in serum ygt nd lkline phosphtse re not necessrily the sme in ll ptients with liver disese, nd clerly other fctors my contribute to the elevtion of serumygt ctivity. As the fctors tht ffect levels of enzymes in the blood re determined, the importnce of the isoenzymes of ygt in serum will be better understood. Then the mesurement of the isoenzymes of ygt

5 96 P. R. Wenhm, C. P. Price, nd H. G. Smmons nd lkline phosphtse my prove to be of vlue in the differentil dignosis of liver disese. References Azzoprdi,., nd Jyle, M. F. (197). Formes moleculires multiples de l gmm-glutmyl-trnspeptidse. Clinic Chimic Act, 4, Beck, P. R. (1978). Butnol extrction of serum nd urinry gmm-glutmyltrnsferse nd its ppliction in clinicl dignosis. Annls of Clinicl Biochemistry, 15, e Broe, M.., Borgers, M., nd Wieme, R. J. (1975). The seprtion nd chrcteriztion of liver plsm membrne frgments circulting in the blood of ptients with cholestsis. Clinic Chimic Act, 59, e Broe, M.., Wieme, R. J., Logghe, G. N., nd Roels, F. (1978). Spontneous shedding of plsm membrne frgments by humn cells in vivo nd in vitro. Clinic Chimic Act, 81, unne, J., Fennelly, J. J., nd McGeeney, K. (1967). Seprtion of lkline phosphtse enzymes in humn serum using gel filtrtion (Sephdex G-2) techniques. Cncer, 2, Flodin, P., nd Killnder, J. (1962). Frctiontion of humn-serum proteins by gel filtrtion. Biochimic nd Biophysic Act, 6,4-41. Hetlnd,., Andersson, T. R., nd Gerner, T. (1975). The heterogeneity of the serum ctivity of y-glutmyl trnspeptidse in heptobiliry diseses s studied by grose gel electrophoresis. Clinic Chimic Act, 62, Huseby, N.. (1978). Multiple forms of y-glutmyltrnsferse in norml humn liver, bile nd serum. Biochimic nd Biophysic Act, 522, Huseby, N.., nd Vik, T. (1978). The ctivity of y- glutmyl-trnsferse fter bile duct ligtion in guine pig. Clinic Chimic Act, 88, Jennings, R. C., Brocklehurst,., nd Hirst, M. (197). A comprtive study of lkline phosphtse enzymes using strch-gel electrophoresis nd Sephdex gel filtrtion with specil reference to high moleculr weight enzymes. Clinic Chimic Act,, Kokot, F., nd Kusk, J. (1968). Heterogeneity of serum y-glutmyl trnspeptidse in different internl diseses, studied by strch-gel electrophoresis nd Sephdex filtrtion. nzymologi Biologic et Clinic, 9, Kryszewski, A. J., Nele, G., Whitfield, J. B., nd Moss,. W. (197). nzyme chnges in experimentl biliry obstruction. Clinic Chimic Act, 47, Luksik, S., nd Richterich, R. (1965). Comprison of the dignostic vlue of serum lkline phosphtse nd y-glutmyl trnspeptidse in biliry obstruction. Archivum mmunologie et Therpie xperimentlis, 1, Miyzki, S., nd Okumur, M. (1972). Chnge of serum y-glutmyl trnspeptidse level nd isoenzyme pttern in heptobiliry pncretic disese. Clinic Chimic Act, 4, Moss,. W. (1962). so-enzymes of lkline phosphtse in utolysed nd butnol-extrcted liver preprtions. Nture, 19, Orlowski, M. (196). The role of y-glutmyl trnspeptidse in the internl diseses clinic. Archivum mmunologie et Therpie xperimentlis, 11, Orlowski, M., Szczeklik, A., nd Kolczkowsk, B. (1965). Heterogeneity of humn y-glutmyl trnspeptidse studied by Sephdex gel filtrtion. Archivum mmunologie et Therpie xperimentlis, 1, Orlowski, M., nd Szczeklik, A. (1967). Heterogeneity of serum gmm-glutmyl trnspeptidse in heptobiliry diseses. Clinic Chimic Act, 15, Price, C. P., Hill, P. G., nd Smmons, H. G. (1972). The nture of the lkline phosphtses of bile. Journl of Clinicl Pthology 25, Price, C. P., nd Smmons, H. G. (1974). The nture of the serum lkline phosphtses in liver diseses. Journl ofclinicl Pthology, 27, Roslki, S. B., Ru,., Lehmnn,., nd Prentice, M. (197). etermintion of serum y-glutmyl trnspeptidse ctivity nd its clinicl pplictions. Annls of Clinicl Biochemistry, 7, Roslki, S. B., nd Trlow,. (1974). Optimised determintion of y-glutmyltrnsferse by rection-rte nlysis. Clinicl Chemistry, 2, Rutenburg, A. M., Goldbrg, J. A., nd Pined,. P. (196). Serum y-glutmyl trnspeptidse ctivity in heptobiliry pncretic disese. Gstroenterology, 45, Szczeklik,., Orlowski, M., nd Szewczuk, A. (1961). Serum y-glutmyl trnspeptidse ctivity in liver disese. Gstroenterology, 41, Szewczuk, A. (1966). A soluble form of y-glutmyl trnspeptidse in humn tissues. Clinic Chimic Act, 14, Wenhm, P. R., Price, C. P., nd Smmons, H. G. (1978). y-glutmyl trnsferse isoenzymes in humn bile. Journl of Clinicl Pthology, 1, Wenhm, P. R., Price, C. P., nd Smmons, H. G. (1978b). A short review of techniques for the loclistion of y-glutmyl trnsferse isoenzymes fter electrophoresis. Annls of Clinicl Biochemistry, 15, Whitfield, J. B., Pounder, R.., Nele, G., nd Moss,. W. (1972). Serum y-glutmyl trnspeptidse ctivity in liver disese. Gut, 1, Requests for reprints to: P. R. Wenhm, eprtment of Clinicl Chemistry, Western Generl Hospitl, dinburgh H4 2XU, UK.