Transferrin (TF) subtyping in various human organ tissues (spleen, pancreas, heart,liver, muscle, lung, kidney, skin, brain, prostate, testis, ovary and uterus) was carried out using isoelectric focusing and immunoblotting. TF subtypes were demonstrated in fresh tissue samples of almost all the organs examined except a few samples of the pancreas and skin. In tissue samples of the heart, muscle, lung, kidney, brain, prostate, testis, ovary and uterus that were stored at room temperature, TF subtyping was possible for up to 1 week. The findings showed that the TF subtype system is useful for medicolegal individualization of human tissues.journal articl

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INOUE, Toshio

KIDO, Akira

KIMURA, Yukiko

NISHIZONO, Toshiko

OYA, Masakazu

English

University of Yamanashi Academic Repository

Yamanashi Med. J. 8(S), 121--l24, 1993Determination ofTF Subtypes in Human Organ Tissues Using            Isoelectric Focusing and ImmunoblottingAkira Kmo, Yukike KiMuRA, Toshike NisHizoNo, Toshio INouE,                   and Masakazu OyA      Department ofSLegalMedicine, Yamanashi Medical UniversdyAbstract: Transferrin (TF) subtyping in various huinan organ tissues (spleen, pancreas, heart,llver, muscle, lung, kidney, skin, brain, prosta£e, testis, ovary and uterus) was carried out usingisoelectric focusing and immunoblotting. TF subtypes were demonstrated in fresh tissue samplesof almost all the organs examined except a few samples of the paltcreas and skiR. In tissue samplesof the heart, mgscle, lung, kidney, brain, prostate, testis, ovary and uterus that were stored at roomtemperature, TF subtyping was possible fbr up to 1 week. The findings showed tha£ the TFsubtype system is useful for medicolegal individualization of human tissues.Key words: TF subtyping, Isoelectric focusing, Immunoblotting, Individualization, Human           tlssgesINTRODUCTION  Genetic variation of transferrin (TF), theiron-binding 6-globulin fraction of the humanserum, was first reported by Smithiesi) usingstarch gel electrophoresis for separation. Thecommon type was designated TF C, moreanodal variants were TF B and more cathodalvariants TF D.  KUhnl and Spielmann2) as well asThymann3), using isoelectric fecusing andCoomasie staining, demonstrated that the TFC type can be further separated into threesubtypes (TF CI, C2:1 and C2) controlled bytwo autosomal codominan{ alleles (TF*C1 andTF*C2). Previously, we examined the distribu-tion of TF subtypes in aJapaRese populationin YamaRashi Prefecture and estimated theailele frequencies to be TF*Cl =O.743, TF"C2=O.251 and TF*D=O.O06, but the results ofthe demonstration from bloodstains wereOctober 25, 1993November IO, 1993Nakakoma, Yamanashi 409-38, Japanunsatisfactory4).  Recent intreduction of the technique ofimmunoblotting has provided much bettervisualization of focused proteins. Using thistechnique, the TF subtypes have been demon-strated also in bloodstains5), semen5), urine6)and dental pulps7) for medicolegal indi-vidualization.  This study is concerned with the possibilitiesof determinatien of TF subtypes for identify-ing human organ tissues in forensic practiceusing isoelectric focusing followed by im-munoblotting.MATERIALS AND METHODSReceivedAcceptedTamaho,  The foIlowing tissue materials were obtaiRedfrom 13 cadavers (11 males, 2 females) whowere medicolegally autopsied within 48hrafter death: spieen, pancreas, heart (cardiacmuscle), liver, muscle (m. rectus abdominis),lung, kidney, skin includiRg adipose tissue,brain, prostate, testis, ovary and uterus.Approximately 5g of the tissue sample wasI22 A. Kido et al.stored in a glass bottle at roem temperatureand examined at 1-week intervals. The tissueseach weighing about O.5 g were homogenizedin O.5 ml of distilled water using an Ultra-Turrax homogenizer Uanke & Kunkel KG,Staufen im Breisgau, Germany). Thehomogenates were centrifuged at 10,OOO rpmfor 60 min and the supernatants were retainedfor analysis. Blood samples were also takenfrom the same subjects. Eight pt of the super-natant or serum was treated with 2 pt of 1 Mpotassium phosphate buffer (pH 7.0) contain-ing 50U/ml neuraminidase from ClostridiumPeptinge?zs (type V, Sigma, St. Louis, Mo.,U.S.A.) fbr 18 hr at room temperature. Thedesialated supernatant was diluted 1:20 andthe desialated serum 1:IOO with distilled water.They were applied to the gel using 5×6 mmfilter paper strips (Whatman No. 3, MaidstoRe,U K.).  Isoelectric focusing was performed with aBio-Phoresis Electrophoresis Cell (Bio-Rad,Richmond, Calif., U.S.A.) and a Model S,OOOXi Power Supply (Bio-Rad) on polyacrylamidegel plates (23eXllOxO.5 mm). The gel platewas composed of 20ml of stock solutien(5.25% acrylarnidelO.25% N,N'-methyleAebis-acrylamide) containiRg 1 ml of Ampholine pH5-7 (Pharmacia, Uppsala, Sweden), O.3 ml ofe.Ol% ribofiavin and 2.5 g of sucrose. Theelectrode paper strips were soaked with 1 Mphosphoric acid for the anode and with l Msodium hydroxide fbr the cathode. After pre-focusiRg at 1,OOO Vmax and 5 mAmax fbr 60min, the specimens were applied to the gelsurface 2cm from the cathode. Electro-focusing was conducted at a constant voltage of1,OOOV for I80 min. The specimens wereremoved after 60 miR of focusing. Duringfocusing the gel was coeled by circulatingwater at 40C.  A sheet of nitrocellulose membrane (Bio-Rad) was placed onto the gel surface and leftfor 60 milt with a lkg weight. After theblotting the membrane was rinsed in 20 mMtris/500 mM sodium chloride buffer, pH 7.5(TBS) for 10min and immersed in TBScontaining 3% gelatin for 30 min. Afterwashing in TBS for 15 min, the membrane wasincubated for 60 min in rabbit anti-human TFserum (DAKO, Denmark) that was diluted1000-fo}d with TBS containing O.05% Tween20 (TTBS). Then the membrane was washed 3times in TTBS for 30 min and incubated for60 min in goat anti-rabbit IgG sertim coaju-gated with alkaline phosphatase (Sigma) thatwas diluted 1000-fold with TTBS. After 3washes in TTBS fbr 30 min, the membranewas incubated in 50 ml of staining solution(l.8 g sodium hydroxide and 3.7 g boric aciddissolved in l,OOO ml distilled water) contain-ing 25 mg 6-naphthyl phosphate, 25 mg FastBlue BB salt and 60 mg magnesium sulfate fbra few minutes.         RESULTS AND DISCUSSION  By the present isoelectric focusing and im-munoblotting clear TF subtype patterns wereobtained in fresh tissues of almost all theorgans examiRed (Fig. 1). However, the bandswere so faint and indistinct that in 7 samples ofpancreas and 3 samples ofskin taken from the13 cadavers the subtyping was unsuccessful.Beth TF C1 and TF C2 types exhibited a singleband: TF CI migrated more anodally and TFC2 more cathodally. The TF C2-1 type wasrepresented by the combination of TF C1 andC2 baRds. The pattem seen in the organtissues was in complete agreement with that inthe corresponding serum samples.  After storage for 1 week, the TF subtypeswere correctly determined from tissue samplesof heart, muscle, lung, kidney, brain, prostate,testis, ovary altd uterus, but phenotyping wasnot possible in 4 samples ofspleen, 10 samplesof pancreas, 5 samples of liver and 9 samplesofskin taken from the l3 cadavers. The bandsbecame fainter and mere indistinct with anincreasing period of sterage. However, nosignificant difllerence in loss of intensity wasobserved among the allelic products. StorageDetermination of TF Subtypes in Human Tissues 123(M･ N"il1/ /!:   tu -F-     AB,2}alPNDst,'-'"--T.---  su .q!Nr,    i. si' ii),g- 'Y;iEt-"b g-s(3}AFig. 1ABcici--"XW G   "pwgzz･   Be"Isoelectric focusingmale, TF C1; (2) femaleC: heart; D: liver;prostate; K: testis; L: ovaryCl; C2-1: control serum sample for TF C2-1;fbr TF C2. The X pa GM"     e ,",- i･                ･e,                s rc YG re  patterns of TF subtypes    ,TF C2-1; (3) male E: muscle; F: lung;     ; M: uterus; Cl: control serum sample for TFanode is at the top.               tg･            .g'             .I      tt X cr K CIee-･3 ¢Z･         e    "･   1         .        I XL M e3 ¢2-IC2     :    -s k       1' IL.'       l ,iLkue as x es e2-:ce    in fresh human tissues. (1) ,TF C2. A: spleen; B: pancreas;G: kidney; H: skin; I: brain; J:     C2: control serum samplefor 2 weeks yielded much poorer results, and itwas difficult to identify the TF bands in mostorgan tlssues.  The above results are summarized in Table1, which shows that the TF subtyping ispossible from tissue samples of the heart,muscle, lung, kidney, brain, prostate, testis,ovary and uterus that were stored for at most 1week.  In mass disasters such as automobile acci-dents, aircraft crashes or explosions, humanorgan tissues are often submitted as evidencefor personal identification. For this purpose,ABO groups, Gm factors and PGM enzymetypes have been shown to be quite suitable8).We recently reported positive results for thedemonstration of polymorphic enzymes(PGMI, ESD, FUC, DIA3 and PGD) fromhuman tissues and the determination limits ofthose enzymes from tissue samples. stored atroom temperature9ffiB). The TF subtype sys-tem can also be a useful means for medicolegalidentification of human tissues.l24Table 1. Positive results of TF subtyping   A. Kido et al.in various human organ tlssuesPhenotype No. tested Periodof      storageTissueSpleen Pancreas Heart Liver Muscle Lung Kidney Skin Brain Prostate Testis Ovary UterusCl 8 48hours8(M:7,F:1)]week 6         2weeks 2         3weeks 2         4weeks O43o88632853o8853l88648885336411l8822o7733o77l1}11llo111}lC2-1 4 48hours4(M:3,F:1)lweek 2         2weeks 2         3weeks 2         4weeks O2o44llo42o4422l4422l44322so4422138211332211lo11oC2 1(M: l)48 hours 1lweek 12weeks l3weeks O4 weekso 11111l11o1111o11l1l1111llo}l1ol111o}l111M: male, F: female"               REFERENCES}) Smithies O. Variations in human serum 6-   globulins. Nature 1957; l8e: l482-1483.2) KUhnlP,SpielmannW.Transferrin:Evidence   for two common subtypes of £he Tff allele.   Hum Genet l978' 43: 91-95.                  ,3) Thymann M. Identification of a new serum   protein polymorphism as transferrin. Hum   Genet l978' 43: 225-229.             ,4) Kido A, Oya M, Komatsu N. Determination of   Tf C subtypes by isoelectric focusing and its   demonstration from bloodstains. Jpn J Legal   Med 1984' 38: 5-8.            ,5) ¥amaba T, Sawazaki K, Yasuda T, et al.   Transferrin subtyping of bloodstains and se-   men using isoelectric focusing and immuno-   blotting. Forensic Sci Int 1991; 50: l87-194.6) Kishi K, Ikehara Y, Yasuda T, et aL Transfer-   rin polymorpkism detected in human urine   using isoelectric focttsiRg fol}owed by irr}-   munoblotting. Forensic Sci Int l990; 45:   225-230.7) KidoA,KimuraY,OyaM.Transferrinsubtyp-   ing in dental pulps. J Forensic Sci 1993; 38:   I063-I067.8) Oepen I. AB-, Rh-, Inv- und PGM-    Bestimmung an Haut, Muskultur, Milz und    Niere zur Identifizierung von Leichenteilen.    Beitr Gerichtl Med l97S'- 31: 300-306.                         ,9) Oya M, Kido A, Komatsu N, Shibata R. Die    AnweRdung der Histoelektrofok"ssieruRg zur    Bestirnmung der PGMi-Subtypen an mensch-    }ichen K6rpergeweben. Z Rechtsmed 1984; 92:    225-230.10) Komatsu N. Polymorphism of EsD by isoelec-    tric focusing: Phenotyping in humaR tissues,    deRtal pulps, hair roots, and semen. Z Rechts-    med l986' 97: 147-l55.            'H) Kido A, Komatsu N, Ose Y, Oya M. cr-L-    Fucosidase phenotyping in human tissues, den-    tal pulps and hair roots. Forensic Sci Int I987;    33: 53-59.12) KidoA,KomatsuN,OyaM,SekiyamaS.DIA3    phenotyping in human tissues, dental pulps,    and hair roots by isoelectric fbcusing. Z Rechts-    med 1988' 100: 133-l37.            ,13) Komatsu N, Kido A, Kimura Y, Oya M. PGD    phenotypingin bloodstains, organ tissues den-    tal pulps and hair roots by isoelectric focusing.    Forensic Sci Int 1990; 48: 185-194.

<Original Article> Determination of TF Subtypes in Human Organ Tissues Using Isoelectric Focusing and Immunoblotting

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<Original Article> Determination of TF Subtypes in Human Organ Tissues Using Isoelectric Focusing and Immunoblotting

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