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Esterase D phenotyping of bloodstains and hair roots by low voltage isoelectric focusing
Forensic Science International, 28 (1985) Elsevier Scientific Publishers Ireland Ltd.
63
63-67
ESTERASE D PHENOTYPING OF BLOODSTAINS BY LOW VOLTAGE ISOELECTRIC FOCUSING
AND HAIR ROOTS
I. YUASA, N. TAMAKI, T. INOUE and K. OKADA Department of Legal Medicine, Tottori University School of Medicine, Nishimachi 86, Yonago 683, and Scientific Crime Laboratory, Totton’ Prefectural Police Headquarters, Tottori 680 (Japan) (Received December 20,1984) (Accepted March 5, 1985)
Summary A new isoelectric focusing method is described for phenotyping of esterase D in blood stains and hair roots. It permitted easy and rapid discrimination of six phenotypes determined by ESD*l, ESD*Z and ESD*7. Experiments showed it to be practicable in forensic stain work. In addition, this technique was also usable in phenotyping of ESD 5. Key words: Esterase D; Isoelectric focusing; Blood stains; Hair roots
Introduction Human e&erase D (ESD) has been utilized extensively as a marker for forensic paternity tests and stain work since the initial report of three common phenotypes by Hopkinson et al. [l] . These phenotypes ESD 1, ESD 2-l and ESD 2 are controlled by the codominant autosomal alleles ESD*l and ESD*B. Several other variant alleles have been detected in various populations. Two of them, ESD*5 [ 2-51 and ESD*7 [6] occur with appreciable frequency in Caucasians and Japanese, respectively, but it is difficult to differentiate their products from those of ESD*2 and ESD*l, respectively, by starch gel electrophoresis. The differentiation of the three common phenotypes ESD 1, 2-l and 2 is rather unsatisfactory by isoelectric focusing, even though this permits easy differentiation of ESD 5 and ESD 7 allozymes [3-6]. Our recent work has revealed that the six phenotypes determined by ESD*l, ESD*2 and ESD*7 may be classified by lowering the focusing. In this paper the low voltage isoelectric voltage of isoelectric focusing is applied to the phenotyping of ESD in blood stains and hair roots.
Address all correspondence to: Dr. Isao Yuasa, Department of Legal Medicine, Tottori University School of Medicine, Nishimachi 86, Yonago 683, Japan. 0379-0738/85/$03.30
G 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
64
Materials and methods Samples for testing
Hemolysates, prepared by lysing packed red cells with an equal volume of distilled water, were diluted with 0.05 M dithiothreitol (DTT) and left for at least 15 min. Blood stains, prepared by dripping blood onto clean gauze pieces, were dried and stored at room temperature. Hairs were plucked at random from various regions of the scalp from donors whose ESD phenotypes were known. The plucked hairs bearing sheath cells were kept at room temperature. A l-2-cm blood stained thread and hair were repeatedly compressed with a glass rod to extract ESD activities after addition of the minimum quantity of 0.1 M DTT. Isoelectric
focusing
Isoelectric focusing was performed as follows: Polyacrylamide gels (T = 5%, C = 4.5%) with dimensions of 110 X 200 X 0.5 mm contained 2.4% Ampholine (pH 4-6.5) (LKB, Bromma). Electrolyte strips used 1 M H,PO, for the anode and 0.2 N NaOH for the cathode. Filter papers (Whatman No. 2, 5 X 4 mm) saturated with sample were placed 2 cm from the cathodal electrolyte strip. Isoelectric focusing was carried out at 8°C for 75 min at a maximum of 1000 V, 4 W and unlimited mA without removing the filter papers. The ESD activities were visualized by overlaying a filter paper containing 4-methyl-umbelliferylacetate as a substrate. Results Figure 1 presents ESD band patterns as revealed by isoelectric focusing of hemolysates. Five of six phenotypes determined by ESD*l, ESD*B and ESD*7 are shown. Two homozygote phenotypes gave a double banded isoenzyme pattern, whereas three heterozygote phenotypes had heteromeric bands characteristic of this dimeric enzyme protein. The discrimination of five different phenotypes could be easily recognizable. Figure 2 presents ESD band patterns from l-week-old blood stains. The ESD phenotypes were easily demonstrable. A decrease in intensity of ESD bands and an increase in background fluorescence were observed as the stains aged. ESD 7-2 phenotype became more liable to be mistyped as ESD 1 in older blood stain. ESD 7-2 losing a homomeric band of ESD 7 looked like ESD 1 phenotype whose anodal and cathodal bands were of about the same intensity. In Fig. 3 the results obtained from 3day-old hairs are shown. The intensities of individuals bands were low in comparison with those of blood stains though the patterns were same. The activities obtained on isoelectric focusing depended on the amount of sheath cell adhering to each hair root. It was possible to detect ESD phenotypes in 5-week-old stains and l-week-old hair roots.
65
1
2
3 4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
Fig. 1. ESD phenotypes in hemolysates from 18 individuals. Anode at top. Sample?S1,7, 10, 15, 16: ESD 1; 2, 4, 8, 13, 14, 18: ESD 2-1; 3,6,12,17: ESD 2; 9: ESD 7-1; 5,ll: ESD 7-2.
1
2
3 4
5
6
7
8 9 101112
Fig 2. ESD phenotypes in l-week-old Samples are the same as in Fig. 1.
blood
131415
16 1718
stains from 18 individuals. Anode at top.
1234567 Fig. 3. ESD phenotypes in 3-dayald 1,7:ESD1;6:ESD2-1;4:ESD2;2:ESD7-1;3,5:ESD7
12345
hair roots
from
7 indiv iduals. -2.
Anode
at top. Samples
67891011
Fig. 4. Separation of ESD 5-l focusing. Anode at top. Samples 6: ESD 5-1.
phenotype in blood stail ns by low voltage isoelectric ESD 2; 7, 10: ESD 1; 1, 2, 4, 5, 8, 9: ESD 2-1; 3,ll:
67
Discussion The initial attempt at phenotyping of ESD in blood stains was made by Horscroft and Sutton [7] . Their work was incomplete since it did not include the use of ESD 2. This was remedied by White [8] but he also was unsuccessful in differentiating the three common phenotypes ESD 1, ESD 2-l and ESD 2. Olaisen et al. [3] showed the differences between ESD 1 and ESD 2 to be marginal by isoelectric focusing on gels with a narrow pH gradient and a wide focusing area. Our results suggest that the isoelectric focusing method described above is capable of discriminating the six phenotypes determined by ESD*l, ESD*B and ESD*7 as well as ESD 5-l. Nishigaki and Itoh [6] pointed out that the mean values of ESD activity varies significantly among the phenotypes. The enzyme activity of ESD 7 is l/3.5 of ESD 1 and l/1.5 of ESD 2, suggesting that good care must be taken in ESD phenotyping in forensic materials. Acknowledgement We are grateful Bank, Minneapolis of ESD 5-l.
to Mr. Dale D. Dykes, Minneapolis War Memorial Blood for his provision of dried blood stain with a phenotype
References 1 D.A. Hopkinson, M.A. Mestriner, J. Cortner and H. Harris, Esterase D: a new human polymorphism. Ann. Hum. Genet., 37 (1973) 119-137. 2 W. Martin, Neue Elektrophoresemethoden zur Darstellung von Serum- und Enzympolymorphismen: technische Verbesserungen, Hinweis auf weiteres ESD-Allel. Arztl. Lab., 25 (1979) 65-67. 3 B. Olaisen, A. Siverts, R. Jonassen, B. Mev& and T. Gedde-Dahl, The ESD polymorphism: further studies of the ESD 2 and ESD 5 allele products. Hum. Genet., 57 (1981) 351-353. 4 D.D. Dykes, H.F. Polesky and S. Miller, Frequency of the ESD 5 allele in three ethnic groups in Minnesota. Hum. Genet., 62 (1982) 162-163, 5 A. Gunther, M. Basler, J. Henke and H.-G. Scheil, Zur Hiiufigkeit des Allels EsD’ (Esterase D, E.C. 3.1.1.1) in einer Westdeutschen Population (Diisseldorfer Raum). Arztl. Lab., 28 (1982) 355-356. 6 I. Nishigaki and T. Itoh, Isoelectric focusing studies of human red cell esterase D: evidence for polymorphic occurrence of a new allele ESD’ in Japanese. Hum. Genet., 66 (1984) 92-95. 7 G. Horscroft and J.G. Sutton, An evaluation of ESD typing by isoelectric focusing. J. Forensic Sci. Sot., 23 (1983) 139-142. 8 J.M. White, An evaluation of ESD typing by isoelectric focusing. J. Forensic Sci. SOC., 24 (1984) 177-178.
63
63-67
ESTERASE D PHENOTYPING OF BLOODSTAINS BY LOW VOLTAGE ISOELECTRIC FOCUSING
AND HAIR ROOTS
I. YUASA, N. TAMAKI, T. INOUE and K. OKADA Department of Legal Medicine, Tottori University School of Medicine, Nishimachi 86, Yonago 683, and Scientific Crime Laboratory, Totton’ Prefectural Police Headquarters, Tottori 680 (Japan) (Received December 20,1984) (Accepted March 5, 1985)
Summary A new isoelectric focusing method is described for phenotyping of esterase D in blood stains and hair roots. It permitted easy and rapid discrimination of six phenotypes determined by ESD*l, ESD*Z and ESD*7. Experiments showed it to be practicable in forensic stain work. In addition, this technique was also usable in phenotyping of ESD 5. Key words: Esterase D; Isoelectric focusing; Blood stains; Hair roots
Introduction Human e&erase D (ESD) has been utilized extensively as a marker for forensic paternity tests and stain work since the initial report of three common phenotypes by Hopkinson et al. [l] . These phenotypes ESD 1, ESD 2-l and ESD 2 are controlled by the codominant autosomal alleles ESD*l and ESD*B. Several other variant alleles have been detected in various populations. Two of them, ESD*5 [ 2-51 and ESD*7 [6] occur with appreciable frequency in Caucasians and Japanese, respectively, but it is difficult to differentiate their products from those of ESD*2 and ESD*l, respectively, by starch gel electrophoresis. The differentiation of the three common phenotypes ESD 1, 2-l and 2 is rather unsatisfactory by isoelectric focusing, even though this permits easy differentiation of ESD 5 and ESD 7 allozymes [3-6]. Our recent work has revealed that the six phenotypes determined by ESD*l, ESD*2 and ESD*7 may be classified by lowering the focusing. In this paper the low voltage isoelectric voltage of isoelectric focusing is applied to the phenotyping of ESD in blood stains and hair roots.
Address all correspondence to: Dr. Isao Yuasa, Department of Legal Medicine, Tottori University School of Medicine, Nishimachi 86, Yonago 683, Japan. 0379-0738/85/$03.30
G 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
64
Materials and methods Samples for testing
Hemolysates, prepared by lysing packed red cells with an equal volume of distilled water, were diluted with 0.05 M dithiothreitol (DTT) and left for at least 15 min. Blood stains, prepared by dripping blood onto clean gauze pieces, were dried and stored at room temperature. Hairs were plucked at random from various regions of the scalp from donors whose ESD phenotypes were known. The plucked hairs bearing sheath cells were kept at room temperature. A l-2-cm blood stained thread and hair were repeatedly compressed with a glass rod to extract ESD activities after addition of the minimum quantity of 0.1 M DTT. Isoelectric
focusing
Isoelectric focusing was performed as follows: Polyacrylamide gels (T = 5%, C = 4.5%) with dimensions of 110 X 200 X 0.5 mm contained 2.4% Ampholine (pH 4-6.5) (LKB, Bromma). Electrolyte strips used 1 M H,PO, for the anode and 0.2 N NaOH for the cathode. Filter papers (Whatman No. 2, 5 X 4 mm) saturated with sample were placed 2 cm from the cathodal electrolyte strip. Isoelectric focusing was carried out at 8°C for 75 min at a maximum of 1000 V, 4 W and unlimited mA without removing the filter papers. The ESD activities were visualized by overlaying a filter paper containing 4-methyl-umbelliferylacetate as a substrate. Results Figure 1 presents ESD band patterns as revealed by isoelectric focusing of hemolysates. Five of six phenotypes determined by ESD*l, ESD*B and ESD*7 are shown. Two homozygote phenotypes gave a double banded isoenzyme pattern, whereas three heterozygote phenotypes had heteromeric bands characteristic of this dimeric enzyme protein. The discrimination of five different phenotypes could be easily recognizable. Figure 2 presents ESD band patterns from l-week-old blood stains. The ESD phenotypes were easily demonstrable. A decrease in intensity of ESD bands and an increase in background fluorescence were observed as the stains aged. ESD 7-2 phenotype became more liable to be mistyped as ESD 1 in older blood stain. ESD 7-2 losing a homomeric band of ESD 7 looked like ESD 1 phenotype whose anodal and cathodal bands were of about the same intensity. In Fig. 3 the results obtained from 3day-old hairs are shown. The intensities of individuals bands were low in comparison with those of blood stains though the patterns were same. The activities obtained on isoelectric focusing depended on the amount of sheath cell adhering to each hair root. It was possible to detect ESD phenotypes in 5-week-old stains and l-week-old hair roots.
65
1
2
3 4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
Fig. 1. ESD phenotypes in hemolysates from 18 individuals. Anode at top. Sample?S1,7, 10, 15, 16: ESD 1; 2, 4, 8, 13, 14, 18: ESD 2-1; 3,6,12,17: ESD 2; 9: ESD 7-1; 5,ll: ESD 7-2.
1
2
3 4
5
6
7
8 9 101112
Fig 2. ESD phenotypes in l-week-old Samples are the same as in Fig. 1.
blood
131415
16 1718
stains from 18 individuals. Anode at top.
1234567 Fig. 3. ESD phenotypes in 3-dayald 1,7:ESD1;6:ESD2-1;4:ESD2;2:ESD7-1;3,5:ESD7
12345
hair roots
from
7 indiv iduals. -2.
Anode
at top. Samples
67891011
Fig. 4. Separation of ESD 5-l focusing. Anode at top. Samples 6: ESD 5-1.
phenotype in blood stail ns by low voltage isoelectric ESD 2; 7, 10: ESD 1; 1, 2, 4, 5, 8, 9: ESD 2-1; 3,ll:
67
Discussion The initial attempt at phenotyping of ESD in blood stains was made by Horscroft and Sutton [7] . Their work was incomplete since it did not include the use of ESD 2. This was remedied by White [8] but he also was unsuccessful in differentiating the three common phenotypes ESD 1, ESD 2-l and ESD 2. Olaisen et al. [3] showed the differences between ESD 1 and ESD 2 to be marginal by isoelectric focusing on gels with a narrow pH gradient and a wide focusing area. Our results suggest that the isoelectric focusing method described above is capable of discriminating the six phenotypes determined by ESD*l, ESD*B and ESD*7 as well as ESD 5-l. Nishigaki and Itoh [6] pointed out that the mean values of ESD activity varies significantly among the phenotypes. The enzyme activity of ESD 7 is l/3.5 of ESD 1 and l/1.5 of ESD 2, suggesting that good care must be taken in ESD phenotyping in forensic materials. Acknowledgement We are grateful Bank, Minneapolis of ESD 5-l.
to Mr. Dale D. Dykes, Minneapolis War Memorial Blood for his provision of dried blood stain with a phenotype
References 1 D.A. Hopkinson, M.A. Mestriner, J. Cortner and H. Harris, Esterase D: a new human polymorphism. Ann. Hum. Genet., 37 (1973) 119-137. 2 W. Martin, Neue Elektrophoresemethoden zur Darstellung von Serum- und Enzympolymorphismen: technische Verbesserungen, Hinweis auf weiteres ESD-Allel. Arztl. Lab., 25 (1979) 65-67. 3 B. Olaisen, A. Siverts, R. Jonassen, B. Mev& and T. Gedde-Dahl, The ESD polymorphism: further studies of the ESD 2 and ESD 5 allele products. Hum. Genet., 57 (1981) 351-353. 4 D.D. Dykes, H.F. Polesky and S. Miller, Frequency of the ESD 5 allele in three ethnic groups in Minnesota. Hum. Genet., 62 (1982) 162-163, 5 A. Gunther, M. Basler, J. Henke and H.-G. Scheil, Zur Hiiufigkeit des Allels EsD’ (Esterase D, E.C. 3.1.1.1) in einer Westdeutschen Population (Diisseldorfer Raum). Arztl. Lab., 28 (1982) 355-356. 6 I. Nishigaki and T. Itoh, Isoelectric focusing studies of human red cell esterase D: evidence for polymorphic occurrence of a new allele ESD’ in Japanese. Hum. Genet., 66 (1984) 92-95. 7 G. Horscroft and J.G. Sutton, An evaluation of ESD typing by isoelectric focusing. J. Forensic Sci. Sot., 23 (1983) 139-142. 8 J.M. White, An evaluation of ESD typing by isoelectric focusing. J. Forensic Sci. SOC., 24 (1984) 177-178.