EXPERIMENTAL AND THERAPEUTIC MEDICINE 5: 89-94, 2013 Combined nlysis of serum γ-glutmyl trnsferse isoenzyme II, α-l-fucosidse nd α-fetoprotein detected using commercil kit in the dignosis of heptocellulr crcinom JING ZHU 1*, FENG JIANG 1*, HONG-BING NI 2*, MING-BING XIAO 1, BU-YOU CHEN 3, WEN-KAI NI 1, CUI-HUA LU 1 nd RUN-ZHOU NI 1 Deprtments of 1 Gstroenterology, 2 Medicl Lbortory Center nd 3 Rdiochemotherpy, Affilited Hospitl of Nntong University, Nntong, Jingsu 226001, P.R. Chin Received September 20, 2012; Accepted October 29, 2012 DOI: 10.3892/etm.2012.783 Abstrct. γ glutmyl trnsferse isoenzyme II (GGT II) is sensitive biomrker of heptocellulr crcinom (HCC). However, numerous disdvntges of the trditionl mnul method ffected its ppliction. The commercil kit provided convenient nd fst method for the determintion of GGT II levels. The purposes of the present study were to compre the reproducibility nd sensitivity between the mnul nd commercil kit methods nd to evlute the dignostic efficiency for HCC with the combined nlysis of GGT II, α L fucosidse (AFU) nd α fetoprotein (AFP). In ptients with vrious liver diseses (HCC, liver cirrhosis nd chronic heptitis) nd norml subjects, GGT II ws detected by mnul nd commercil polycrylmide gel electrophoresis (PAGE). The levels of AFU nd AFP were ssyed by colorimetry nd chemiluminescence immunossy, respectively. The commercil PAGE hd equl dignostic efficiency with trditionl mnul PAGE nd no significnt differences were observed in intr nd verge gel reproducibility nd GGT Ⅱ sensitivities between the mnul nd commercil PAGE (P>0.05). The incidence of GGT II detected by commercil PAGE in HCC ptients ws 84.1% nd <8% in benign liver disese. The levels of AFU nd AFP in the benign liver diseses nd norml subjects were lower thn those in HCC. According to the cut off vlue obtined by receiver operting chrcteristic curves, totl of 56.6 nd 59.3% of HCC ptients (64 out of 113 nd 67 out of 113) hd AFU >636.5 µmol/l h nd AFP >44.0 µg/l, respectively. There were no significnt correltions Correspondence to: Professor Bu-You Chen, Deprtment of Rdiochemotherpy, Affilited Hospitl of Nntong University, 20 XiSi Rod, Nntong, Jingsu 226001, P.R. Chin E-mil: chenby2005@yhoo.com.cn * Contributed eqully Key words: γ-glutmyl trnsferse isoenzyme II, α-l-fucosidse, α-fetoprotein, heptocellulr crcinom, dignosis between GGT Ⅱ nd AFU or AFP. Combined detection of GGT II with AFU or AFP incresed the dignostic sensitivity to 92.9 nd 93.8%, respectively. These results suggest tht commercil PAGE provides simple nd reproducible method for GGT Ⅱ detection. Combined determintion of GGT II with AFU or AFP exhibited superior sensitivity nd specificity for the dignosis of HCC. Introduction Heptocellulr crcinom (HCC) is the second nd the fifth most frequent type of cncer in Chin nd the world, respectively. The five yer survivl rte is <12% nd mortlity remins equl with morbidity (1,2). The occurrence nd development of HCC is complex multifctor nd multistep process, minly ssocited with chronic nd persistent infection with the heptitis virus, prticulrly the heptitis B virus (HBV) nd heptitis C virus (HCV) (3,4). HCC is highly mlignnt nd metstsizes redily t the erly stges nd its prognosis depends minly on erly dignosis. Imging (BUS, CT, MRI) nd the detection of serum tumor mrkers re fundmentl methods of identifiction in symptomtic ptients with HCC. However, t present no single dignostic method is ble to meet the sensitivity nd specificity criteri required. In terms of serum mrkers, α fetoprotein (AFP) is the preferred serum mrker for the dignosis nd monitoring of HCC but it is negtive in ~40% ptients with erly stge HCC. Even in dvnced HCC, the concentrtions of AFP my be norml in 15 30% of ptients (5). Additionl HCC mrkers, including γ glutmyl trnsferse isoenzyme II (GGT II), α L fucosidse (AFU), Golgi protein 73 (GP73) nd trnsforming growth fctors α nd β (TGF α nd TGF β), hve been used to elevte the dignostic sensitivity, specificity nd erly detection levels. However, there is no bsolute positive nd universl dignostic mrker for HCC. Therefore, it is suggested tht mrkers be simultneously evluted in order to enhnce the detection of HCC (6 8). γ glutmyl trnspeptidse (GGT; EC 2.3.2.2) is membrne bound enzyme which hydrolyzes γ glutmyl or trnsfers it to suitble cceptor to degrde glutthione nd its conjugtes. Serum GGT hs been used in the dignosis of liver
90 ZHU et l: γ-glutamyl TRANSFERASE ISOENZYME II AND HEPATOCELLULAR CARCINOMA diseses, s it exhibits tissue-specific expression. In dmged heptocytes, prticulrly in heptocrcinogenesis, since GGT is relesed into the blood from heptic tissues, significnt chnges occur in serum GGT ctivity (9). However the totl ctivity of GGT hs significnt overlp with vrious liver diseses which limits its vlue in dignosis. The identifiction of heptom-specific GGT (GGT II) provides n efficcious serum mrker for HCC which is regrded s n erly enzyme mrker of precncerous nd cncerous processes. In the serum of HCC ptients, GGT II s heterodimeric glycoprotein hs different electrophoretic mobility nd my be seprted from norml GGT isoenzymes by polycrylmide gel electrophoresis (PAGE) (10 12). However, more disdvntges ffect trditionl mnul PAGE nd prevent its wider ppliction. It hs been difficult to mintin uniform intr nd inter lbortory reproducibility since ll regents of the experiment re rtificilly prepred in ech lbortory. Moreover, cre must be tken when prepring the gel since crylmide is strong neurotoxin in its powdered nd liquid forms. Commercil PAGE kits hve solved these problems nd shortened the electrophoretic run time to only 4 h. The min objective of the present study ws to compre reproducibility nd sensitivity between mnul nd commercil PAGE for the detection of GGT II nd evlute the dignostic efficiency of combined ssy of GGT II, AFU nd AFP. Ptients nd methods Ptients nd specimens. The serum specimens of the HCC group were obtined from 113 ptients (medin ge, 55.3 yers; 95 mles nd 18 femles) from the Affilited Hospitl of Nntong University (Nntong, Chin). Of the ptients, 85.0% (96/113) were heptitis B surfce ntigen (HBsAg) crriers, 9.7% (11/113) were HCV ntibody-positive, 2.7% (3/113) hd lcoholic heptitis nd 2.7% (3/113) hd utoimmune heptitis. The other study groups included liver cirrhosis (LC; 51 ptients; 33 mles, 18 femles; medin ge, 53 yers), chronic virl heptitis (CH; 21 ptients; 16 mles, 5 femles; medin ge, 44 yers) nd norml control subjects group (NC; 14 mles, 14 femles; medin ge, 25 yers). All studied cses were dignosed by heptitis mrkers, blood biochemicl tests, imging or pthohistologicl exmintion. No ptients received ny therpy before the blood specimens were collected. GGT II detected by mnul PAGE. The seprtion of GGT Ⅱ ws performed by mnul PAGE on verticl slb pprtus (12,13). In short, polycrylmide stging gel contining three lyers of seprtion gel (7.7, 11.5 nd 15.4%) nd one lyer of concentrtion gel (4.2%) on top ws loded into verticl slb electrophoresis bth (Model JSZH I, Jingsu Zongheng Co., Ltd., Nntong, Chin). After infusion with negtive nd positive buffer solution, 20 µl of serum ws mixed with 20 µl of 40% sucrose bromophenol blue solution nd introduced into the smple holes of the concentrtion gel, then electrophoresed t constnt voltge of 60 V for 4 h, then 100 V for 12 h. Subsequently, the polycrylmide gel removed from the electrophoresis bth ws covered over with cellulose cette sheet soked with GGT substrte solution (contining 24 mg γ L glutmyl P nitronilide monohydrte, 100 µl of 20% Tween-20, 100 µl of 5% polyvinyl pyrrolidone, 5.0 ml of 0.1 mol/l tromethmine glycylglycine buffer nd 100 µl of 10% NNO 2 ) nd incubted t 37 C for 60 min. The cellulose cette sheet ws then soked with 5 ml stining solution (contining 10% trichlorocetic cid nd 25% glycerol) nd within ~2 min the red bnds of GGT isoenzyme ppered on the cellulose cette sheet. GGT II detected by commercil PAGE kit. The seprtion technique of the commercil kit (Jingsu Zongheng Co., Ltd.) for GGT II ws lso PAGE nd ws performed ccording to the mnufcturer's instructions. Reproducibility studies of mnul PAGE nd commercil PAGE for GGT II. To determine the reproducibility of the mnul PAGE nd commercil PAGE for GGT II, positive nd negtive controls of GGT II (Jingsu Zongheng Co., Ltd.) were electrophoresed nine times on the sme nd different gels from the mnully prepred or commercil gels to determine intr nd verge gel reproducibility. Serum AFU concentrtion. The serum AFU ctivities were ssyed colorimetriclly using semi utomtic biochemistry nlyzer (BA 88). The cut-off vlue of serum AFU for HCC ws obtined using receiver operting chrcteristic (ROC) curve. Serum AFP concentrtion. According to the mnufcturer's instructions, the chemiluminescence immunossy (Abbott, Chicgo, IL, USA) ws used to detected the serum levels of AFP. Sttisticl nlysis. The Chi squre or Fisher's exct test were used for ny 2x2 tbles. Serum AFU concentrtions re presented s the men ± SD nd nlyzed with F tests. Serum AFP concentrtions in ptients with liver disese re presented s medin (rnge) nd nlyzed with the rnk sum test with regrd to the skewness of the distribution of AFP concentrtions. The dignostic vlues of serum AFU nd AFP were evluted using n ROC curve. Spermn's rnk correltion ws used to test the correltions between serum GGT II, AFU nd AFP. P<0.05 ws considered to indicte sttisticlly significnt differences in ll nlyses. All sttisticl nlysis ws performed using the SPSS 17.0 softwre pckge (SPSS, Inc., Chicgo, IL, USA). Results Reproducibility comprison between mnul PAGE nd commercil PAGE. Positive nd negtive controls of GGT II were repetedly electrophoresed on the sme nd different gels from the mnully prepred or commercil gels. The results showed (Tble Ⅰ) tht lthough the verge reproducibility of the commercil PAGE (100%) ws higher thn tht of the mnul PAGE (94.4%), the intr nd verge gel reproducibility differences between the mnul nd commercil PAGE (P>0.05) were not sttisticlly significnt. Serum GGT Ⅱ determined by mnul nd commercil PAGE. Serum GGT Ⅱ ws determined by mnul nd commercil
EXPERIMENTAL AND THERAPEUTIC MEDICINE 5: 89-94, 2013 91 Tble I. Reproducibility comprison between mnul nd commercil PAGE. Positive control reproducibility Negtive control reproducibility (positive/totl, %) (negtive/totl, %) Averge ------------------------------------------------------------------------- ------------------------------------------------------------------------ reproducibility Method Gel 1 Gel 2 Gel 3 Gel 1 Gel 2 Gel 3 (%) Mnul PAGE 7/9 (77.8) 9/9 (100) 8/9 (88.9) 9/9 (100) 9/9 (100) 9/9 (100) 51/54 (94.4) Commercil PAGE 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 54/54 (100) P>0.05, ll vlues of mnul PAGE compred with those in commercil PAGE (Fisher's exct test). PAGE, polycrlymide gel electrophoresis. Figure 1. GGT-Ⅱ-positive stining ws shown in heptocellulr crcinom. (1) Liver cirrhosis; (2) helthy control; (3, 5 nd 6) heptocellulr crcinom; (4) chronic heptitis. GGT II, γ glutmyl trnsferse isoenzyme II. PAGE in ll cses. Severl bnds (up to 9) of the serum GGT isoenzymes were seprted nd lbeled GGT I, GGT II nd GGT III IX, from the positive to negtive pole ccording to the decresing order of their electrophoretic mobility (Fig. 1). The dignostic vlues of GGT II in the ser of ptients with mlignnt nd benign liver diseses re shown in Tble Ⅱ. By mnul nd commercil PAGE, there were few flse positives of GGT II mong the ptients with benign liver diseses nd none mong the norml subjects. The positive GGT II ws predominntly exhibited by HCC ptients with sensitivities of 74.3 nd 84.1% by mnul nd commercil PAGE, respectively, which were significntly higher thn in the benign liver diseses nd helthy subjects (P<0.05). However, no significnt differences (P>0.05) were observed between mnul nd commercil PAGE. Serum levels of AFU nd AFP. The results demonstrted tht the levels of AFU (726.4±258.4 µmol/l h) nd AFP [321.1 (0.7 10000.0) µg/l] were higher in the HCC group thn in the other groups (Fig. 2, Tble III, P<0.05). Figure 2. Sctterplot of serum levels of AFU nd AFP in vrious liver diseses nd norml control subjects. AFU, α L fucosidse; AFP, α fetoprotein. The cut off vlues for optiml dignostic efficiency determined by ROC curve nlysis (Fig. 3) were 636.5 µmol/l h for AFU nd 44.0 µg/l for AFP nd the res under the ROC curves were 0.772 (95% CI, 0.709 0.834; P<0.05) nd 0.746 (95% CI, 0.677 0.814; P<0.05), respectively. According to the
92 ZHU et l: γ-glutamyl TRANSFERASE ISOENZYME II AND HEPATOCELLULAR CARCINOMA Tble II. Comprison of dignostic sensitivity of GGT-II detected by mnul or commercil PAGE. GGT-II (mnul PAGE) GGT-II (commercil PAGE) ------------------------------------------------------ ------------------------------------------------------ Groups n n % n % HCC 113 84 74.3,b 95 84.1 LC 51 3 5.9 b 4 7.8 CH 21 1 4.8 b 1 4.8 NC 30 0 0.0 b 0 0.0 P<0.05, HCC group compred with the other groups; b P>0.05, the sensitivities of GGT-II in ll groups detected by mnul PAGE were compred with those detected by commercil PAGE (Chi-squre test or Fisher's exct test). GGT II, γ glutmyl trnsferse isoenzyme II; HCC, heptocellulr crcinom; LC, liver cirrhosis; CH, chronic virl heptitis; NC, norml control; PAGE, polycrylmide gel electrophoresis. Tble III. Serum levels of AFU nd AFP in vrious liver diseses nd norml control subjects. AFU (µmol/l h) AFP (µg/l) ---------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------- Groups n Men ± SD F P-vlue 636.5 (%) Medin (rnge) Z b P-vlue b 44.0 (%) HCC 113 726.4±258.4 64 (56.6) c 321.1 (0.7-10000.0) 67 (59.3) c LC 51 562.7±208.6-163.71 0.001 15 (29.4) 7.3 (0.4-860.7) 4.723 0.000 14 (27.5) CH 21 554.4±222.4-172.01 0.021 3 (14.3) 14.2 (0.7-240.3) 4.839 0.000 5 (23.8) NC 30 382.4±133.0-344.05 0.000 0 (0.0) 2.3 (0.2-9.7) 5.898 0.000 0 (0.0) F nd P vlues were clculted by F-tests; b Z nd P vlues were clculted by rnk sum tests; c P<0.05 nd clculted by Chi-squre or Fisher's exct tests, while the HCC group ws compred with the other groups. AFU, α L fucosidse; AFP, α fetoprotein; HCC, heptocellulr crcinom; LC, liver cirrhosis; CH, chronic virl heptitis; NC, norml control. Figure 3. ROC curve of serum levels of AFU nd AFP for the dignosis of heptocellulr crcinom. ROC, receiver operting chrcteristic; AFU, α L fucosidse; AFP, α fetoprotein. cut off vlues, the sensitivities of AFU nd AFP for the dignosis of HCC were 56.6 nd 59.3%, respectively (Tble III). Serum positive GGT Ⅱ is complementry to the serum levels of AFU nd AFP. Positive GGT Ⅱ ws observed in 95 of 113 ptients with HCC (84.1%) nd mong the 18 ptients with negtive GGT Ⅱ, 10 hd elevted concentrtions of AFU ( 636.5 µmol/l h). No significnt correltion ws observed between GGT Ⅱ nd the serum levels of AFU in HCC ptients (r=0.142, P=0.383). Of 18 HCC ptients with negtive GGT II, 11 hd n AFP concentrtion bove 44.0 µg/l. Also, no significnt correltion ws observed between GGT Ⅱ nd the serum levels of AFP (r=0.102, P=0.517). The dignostic sensitivity ws significntly incresed by combining the informtion on GGT II, AFU nd AFP levels (Tble IV). The combintion of GGT II with AFU (+b), GGT II with AFP (+c) nd the three mrkers (+b+c) hd superior dignostic sensitivities (92.9, 93.8 nd 97.3%) to tht (83.2%) from the combintion of AFU with AFP (b+c) [P<0.05, (b+c) vs. (+b), (+c) nd (+b+c)]. However, the combintion
EXPERIMENTAL AND THERAPEUTIC MEDICINE 5: 89-94, 2013 93 Tble IV. Complementry vlue of GGT-II, AFU nd AFP to dignose heptocellulr crcinom. Mrkers Sensitivity (%) Specificity (%) Accurcy (%) Positive GGT-II () 84.1 (95/113) 95.1 (97/102) d 89.3 (192/215) Positive AFU (b) 56.9 (64/113) b 82.4 (84/102) e 68.8 (148/215) Positive AFP (c) 59.3 (67/113) b 81.4 (83/102) e 69.8 (150/215) +b 92.9 (105/113) 78.4 (80/102) f 86.0 (185/215) +c 93.8 (106/113) 77.5 (79/102) f 86.0 (185/215) b+c 83.2 (94/113) c 68.8 (70/102) 76.3 (164/215) +b+c 97.3 (110/113) 64.7 (66/102) 81.9 (176/215) P<0.05, vs. b, c, (+b), (+c) nd (+b+c); b P<0.05, b nd c vs. (+b), (+c), (b+c) nd (+b+c); c P<0.05, (b+c) vs. (+b), (+c) nd (+b+c); d P<0.05, vs. other groups; e P<0.05, b nd c vs. (b+c) nd (+b+c); f P<0.05, (+b) nd (+c) vs. (+b+c). Sttisticl nlyses were performed using Chi-squre or Fisher's exct tests. GGT II, γ glutmyl trnsferse isoenzyme II; AFU, α L fucosidse; AFP, α fetoprotein. of three mrkers hd the lowest dignostic specificity (64.7%), which ws significntly different from the combintions of AFU (78.4%) nd GGT II with AFP (77.5%) [P<0.05, (+b) nd (+c) vs. (+b+c)]. The combintions of GGT Ⅱ with AFU nd GGT II with AFP hd the optiml dignostic efficiency. Discussion The level of AFP hs been widely estblished s clssic HCC mrker, but n elevted AFP level is lso observed in certin benign liver diseses nd other mlignncies. Furthermore, flse negtive or positive results often occur for number of resons, including geogrphicl nd ethnic vritions nd different techniques being employed. For these resons, it is necessry to perform combined detection of vrious mrkers for the dignosis of HCC. As mentioned previously, GGT II nd AFU re frequently used. As n importnt nd extensively distributed converting enzyme closely correlted with nucleic cid metbolism nd biotrnsformtion, chnges of GGT sensitively reflect heptocyte prenchymtous lesions (14). Previously, GGT hs been widely studied in tumorigenesis, such s the dysfunction of DNA synthesis nd nucleic cid metbolism. Certin reserchers hve observed highly positive correltion between liver RNA level nd heptic GGT gene expression in heptom model (15). The bnorml regultion of genes my be the result of the inctivtion of tumor suppressor genes or the ctivtion of proto oncogenes initited by crcinogens. The seprtion of serum GGT isoenzymes using PAGE works minly on the bsis of their different electrophoretic mobilities. A number of reserchers hve reported different results for the heptom-specific GGT isoenzyme ccording to frctiontion systems. Initilly, the positive rte ws only 27 63% (16 18). Xu et l subsequently reported tht the GGT isoenzymes my be divided into 9 to 11 bnds by verticl slb polycrylmide grdient gel electrophoresis nd number of these bnds (I, II nd II bnds, GGT II) were observed to hve positive rte of 90% (13). In the present study, the positive rte of GGT II in HCC ws 74.3% using the trditionl mnul PAGE described by Xu et l, similr to the study by Cui et l (77.6%) (19). Previous studies suggest tht GGT Ⅱ is importnt for the dignosis of HCC, but certin disdvntges of the trditionl mnul method ffected the sensitivity of GGT II. Yo nd Dong lso demonstrted tht GGT II ws good mrker, but there ws no simple nd esy detection method (20). Now, the use of commercil PAGE kits hs solved this problem. In the present study, the reproducibility (100%) nd dignostic sensitivity (84.1%) of GGT II detected by commercil PAGE kit ws higher thn tht of the trditionl method. The commercil PAGE kit provided simple, convenient method for clinicl ppliction. As glycosidse which is widespred in vriety of cell lysosomes in the humn body, AFU is ssocited with cid hydrolysis of vriety of fucose contining fucoglyco conjugtes. The ctivity of this liposoml enzyme is detectble nd elevted ctivities re observed in the ser of HCC ptients compred with chronic liver disese nd helthy individuls (21). Erly studies showed tht the sensitivity nd specificity of AFU for dignosis of primry heptocrcinom were 70 80% (22,23). In contrst to AFP, the ctivity levels of AFU were not correlted with tumor mgnitude nd AFU ws of vlue in the dignosis of HCC ptients with negtive or low serum levels of AFP, prticulrly for smll HCC (<5 cm) (24 26). However, elevted serum AFU is lso detected in colorectl cncer, ovrin cncer nd other mlignncies. In ddition, serum AFU levels chnge in dibetes, pncretitis nd hypothyroidism (27). In the present study, the sensitivity of AFU for HCC ws only 56.6% which ws lower thn previous reports, owing to higher cut-off vlue (636.5 µmol/l h) with higher dignostic specificity (82.4%). To improve the dignostic sensitivity, the combined detection of AFU with other tumor mrkers should be commonly used in clinicl prctice. Spermn's rnk correltion nlysis showed tht positive GGT II ws not significntly correlted with either AFU or AFP, suggesting tht these three mrkers re complementry in the dignosis of HCC. The sensitivity of HCC detection using GGT II ws 84.1%. This sensitivity incresed to 92.9 nd 93.8% when combined with AFU or AFP, respectively. The combintion of three mrkers yielded 97.3% detection sensitivity but the lowest dignostic specificity (64.7%). In summry, the combintions of GGT II with AFU or AFP hd optiml dignostic efficiency. We propose tht GGT Ⅱ be mesured with AFU or AFP to improve the detection sensitivity of HCC.
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