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Polymorphic Enzyme Systems in Human Hair Sheath Cells
*J.F.S.S. ORIGINAL PAPERS
Polymorphic Enzyme Systems in Human Hair Sheath Cells J. G. SUTTON and C. M. BOSLEY Home Ofice Central Research Establishment, Aldermaston, Reading, Berkshire, United Kingdom RG7 4PN and P. H . WHITEHEAD Home O$ce Forensic Science Laboratory, Sandbeck Way, Audby Lane, Wetherby, W. Yorkshire, United Kingdom LS22 4 D N Abstract Using starch gel electrophoresis, the enzymes Phosphoglucomutase (PGM, locus), Adenylate kinase, Esterase D, Glyoxalase, 6-Phosphogluconate dehydrogenase, Glucose-6phosphate dehydrogenase and Phosphohexose isomerase could be detected in fresh hair sheath cells. Of these, all but Adenylate kinase could be detected in three-week-old sheath cells and of the remainder, all but Esterase D could be detected in seven-weekold sheath cells. Acid phosphatase and Adenosine deaminase enzyme activity was also detected but reliable typing was not possible. No Peptidase A activity could be detected.
Journal of the Forensic Science Society 1982; 22: 199-202 Received 6 May 1981 Introduction A number of attempts at grouping human hair based on the ABO system have been reported over the years. One of the earliest attempts was conducted by Saki in 1951 who macerated the hair by chemical treatment in an attempt to expose the antigenic sites. I n more recent work conducted by Yada et al. (1966) the hairs were physically broken open by hammering them on an anvil. After the initial disruptive stage the samples were treated with anti A or anti B serum followed by the normal absorption-elution technique. Using this procedure they claimed to be able to attain a good correlation with the known blood groups of the individuals from whom the hair samples were initially obtained. However, other workers (Lincoln and Dodds, 1968) have revealed that although hairs show antigenic activity the results are not conclusive enough for routine typing and Court production. Alternative grouping methods based on the various polymorphic enzyme systems were investigated by Twibell and Whitehead (1978) who demonstrated the presence of phosphoglucomutase (PGM), adenosine deaminase (ADA), adenylate kinase (AK) and esterase D (E5D) in the sheath cells associated with plucked hairs. Of these only PGM was reported as being in sufficient quantity to be typed by starch gel electrophoresis. Parallel studies conducted by Yoshida et al., and Ova et al., in 1978 also demonstrated the presence of PGM and EsD and added 6-phosphogluconate dehydrogenase (6PGD) to the list of sheath cell enzymes. I n all instances the phenotypic patterns obtained matched those obtained from the corresponding blood samples. Yoshida et al. (1978)
were, in addition, able to demonstrate PGM, locus activity which cannot readily be detected in the red cell. I n this paper we confirm all the initial observations made by these authors and report the discovery of further polymorphic enzyme systems not previously described.
Materials and Methods Plucked Hairs Hairs were plucked a t random from various regions of the scalp from donors whose various red cell enzyme phenotypes were known.
Ageing Studies Plucked hairs, bearing sheath cells, were mounted on a glass plate with clear adhesive tape in such a way that the sheath cells were fully exposed. The plate was kept in a drawer a t room temperature (20°C-25°C) and only withdrawn when sheath cell samples were required; this was done after a 3- and 7-week period. Electrophoresis and Isoenzyme Visualisation Electrophoresis was conducted in thin starch gels, using well documented methods (Culliford, 1971; Hopkinson et al., 1973; Kompf et al., 1975; Wraxall and Emes, 1976).
Results Table 1 shows the enzymes which can readily be detected in freshly plucked hairs. Although the presence of most of these enzymes has already been reported we have also detected the presence of glyoxalase (GLO), glucose-6phosphate dehydrogenase (G6PD) and phosphohexose isomerase (PHI) in sufficient quantities for successful typing by starch gel electrophoresis. I n addition we found that both AK and EsD could be demonstrated using this technique. Some examples of the different polymorphic enzyme systems investigated are shown in Figures 1 to 3.
TABLE 1 DETECTION O F HAIR SHEATH CELL ENZYMES AFTER A 3 AND 7 WEEK INTERVAL System AK EsD GLO G6PD PHI PGM 6PGD
Fresh f f
f f
+
3 Weeks -
++ ++
7 Weeks -
-
++ +
DP 0.139 0.327 0.629
0.02 0.526 f f . f -Ifaint 0.081 - = not detectable = typeable DP = "discriminating power" (Jones, 1972)
+
+
Other enzyme systems which were examined in this study were acid phosphatase (AP), ADA and Peptidase A (PEP A). Although there appeared to be considerable amounts of AP activity we found that the electrophoretic patterns were not distinct enough for reliable typing. ADA could also be demonstrated in sheath cells but its activity appeared to be consistently low. As yet we have not detected PEP A.
PGM
PGM,
d-
PGM
Origin
Figure 1. Starch gel electrophoretic patterns of some of the PGM, and all the PGM, variants observed in human hair sheath cells using a Tris, maleic acid, MgCL,, EDTA buffer system pH7.4 (Spencer et al., 1968).
The results of the ageing studies are summarised in Table 1. This study revealed that all the enzymes which could readily be detected in fresh sheath cells could, with the exception of AK, be detected after 3 weeks. Essentially the same results were obtained after 7 weeks except that EsD activity had decreased to such a point that reliable typing could not be undertaken. Probably the most significant feature of this ageing study has been the discovery that both GLO and PGM,, which have the highest discriminating power, could be detected and successfully typed after a 7-week period.
Blood II
t
1
Hair Blood
Origin
Origin
Figure 2. The starch gel electrophoretic patterns of AK 1 and AK 2 - 1 phenotypes obtained from paired blood and human hair sheath cells samples using a histidine buffer system pH5 (Fildes and Harris, 1966).
111
,a* -2
Blood
1
6PGD A
Hair Hair
1
GPGD A
Blood Origin
Origin
Figure 3. The starch gel electrophoretic patterns of GPGD A phenotypes obtained from paired blood and human hair sheath cell samples using a phosphate buffer system pH7 (Fildes and Parr, 1963).
Discussion The discovery of GLO activity in hair sheath cells has provided a further means of discriminating between different hair samples. The discovery of GLO activity in human sheath cells is particularly valuable since it has also been detected in semen (Emes and Parkin, 1980). In addition, the discovery of both G6PD and PHI, both of which are stable enough to allow successful typing after a 7-week period, is reported. The application of enzyme typing of hair roots in case work should be of most value in cases of assault or murder in which hair is snatched or pulled from the scalp during the offence. References CULLIFORD, B. J., 1971, The Examination and Typing of Bloodstains in the Crime Laboratory, Published by US Department of Justice Law Enforcement Assistance Administration. B., 1980, Forensic Sci. Znt., 15, 265. EMES,E. G. and PARKIN, FILDES, R. A. and PARR,C. W., 1963, Nature, 200, 890. FILDES,R. A. and HARRIS,H., 1966, Natzire, 209, 261. D. A., MESTRINER, M. A., CONTNER, J. and HARRIS,H., 1973, HOPKINSON, Ann. Hum. Genet., 37, 119. JONES,D. A., 1972, J. Forens. Sci. Soc., 12, 335. S. and RITTER,H., 1977, Humangenetik, K ~ M P F*I., , BISSBORT, S., GUSSMANN, 27, 141. LINCOLN, P. J. and DODDS, B. E., 1968, Med. Sci. Law, 8, 38. O., KATSUMATA, Y. and YADA,S., 1978, OYA,M., ITO,H., KIDO,A., SUZAKI, Forens. Sci., 11, 135. SAKI,T., 1951, Jap. J. I,e*c. Med., 5, 19. SPENCER, N., HOPKINSON, D. A. and HARRIS,H., 1968, Ann. Hum. Genet., 32, 9. J. M. and WHITEHEAD, P. H., 1978,J. Forens. Sci., 23, 356. TWIBELL, B. G. D. and EMES,E. G., 1976, J . Forens. Sci. Soc., 16, 127. WRAXALL, YADA,S., OKANE,M. and SANO,Y., 1966, Acta Criminological et Medicinae Legales Japonica, 32, 7. YOSHIDA, ,I., ABET, T. and NAKAMIJRS, K., 1978, Medico-Legal Soc., 21, 7. 202
Polymorphic Enzyme Systems in Human Hair Sheath Cells J. G. SUTTON and C. M. BOSLEY Home Ofice Central Research Establishment, Aldermaston, Reading, Berkshire, United Kingdom RG7 4PN and P. H . WHITEHEAD Home O$ce Forensic Science Laboratory, Sandbeck Way, Audby Lane, Wetherby, W. Yorkshire, United Kingdom LS22 4 D N Abstract Using starch gel electrophoresis, the enzymes Phosphoglucomutase (PGM, locus), Adenylate kinase, Esterase D, Glyoxalase, 6-Phosphogluconate dehydrogenase, Glucose-6phosphate dehydrogenase and Phosphohexose isomerase could be detected in fresh hair sheath cells. Of these, all but Adenylate kinase could be detected in three-week-old sheath cells and of the remainder, all but Esterase D could be detected in seven-weekold sheath cells. Acid phosphatase and Adenosine deaminase enzyme activity was also detected but reliable typing was not possible. No Peptidase A activity could be detected.
Journal of the Forensic Science Society 1982; 22: 199-202 Received 6 May 1981 Introduction A number of attempts at grouping human hair based on the ABO system have been reported over the years. One of the earliest attempts was conducted by Saki in 1951 who macerated the hair by chemical treatment in an attempt to expose the antigenic sites. I n more recent work conducted by Yada et al. (1966) the hairs were physically broken open by hammering them on an anvil. After the initial disruptive stage the samples were treated with anti A or anti B serum followed by the normal absorption-elution technique. Using this procedure they claimed to be able to attain a good correlation with the known blood groups of the individuals from whom the hair samples were initially obtained. However, other workers (Lincoln and Dodds, 1968) have revealed that although hairs show antigenic activity the results are not conclusive enough for routine typing and Court production. Alternative grouping methods based on the various polymorphic enzyme systems were investigated by Twibell and Whitehead (1978) who demonstrated the presence of phosphoglucomutase (PGM), adenosine deaminase (ADA), adenylate kinase (AK) and esterase D (E5D) in the sheath cells associated with plucked hairs. Of these only PGM was reported as being in sufficient quantity to be typed by starch gel electrophoresis. Parallel studies conducted by Yoshida et al., and Ova et al., in 1978 also demonstrated the presence of PGM and EsD and added 6-phosphogluconate dehydrogenase (6PGD) to the list of sheath cell enzymes. I n all instances the phenotypic patterns obtained matched those obtained from the corresponding blood samples. Yoshida et al. (1978)
were, in addition, able to demonstrate PGM, locus activity which cannot readily be detected in the red cell. I n this paper we confirm all the initial observations made by these authors and report the discovery of further polymorphic enzyme systems not previously described.
Materials and Methods Plucked Hairs Hairs were plucked a t random from various regions of the scalp from donors whose various red cell enzyme phenotypes were known.
Ageing Studies Plucked hairs, bearing sheath cells, were mounted on a glass plate with clear adhesive tape in such a way that the sheath cells were fully exposed. The plate was kept in a drawer a t room temperature (20°C-25°C) and only withdrawn when sheath cell samples were required; this was done after a 3- and 7-week period. Electrophoresis and Isoenzyme Visualisation Electrophoresis was conducted in thin starch gels, using well documented methods (Culliford, 1971; Hopkinson et al., 1973; Kompf et al., 1975; Wraxall and Emes, 1976).
Results Table 1 shows the enzymes which can readily be detected in freshly plucked hairs. Although the presence of most of these enzymes has already been reported we have also detected the presence of glyoxalase (GLO), glucose-6phosphate dehydrogenase (G6PD) and phosphohexose isomerase (PHI) in sufficient quantities for successful typing by starch gel electrophoresis. I n addition we found that both AK and EsD could be demonstrated using this technique. Some examples of the different polymorphic enzyme systems investigated are shown in Figures 1 to 3.
TABLE 1 DETECTION O F HAIR SHEATH CELL ENZYMES AFTER A 3 AND 7 WEEK INTERVAL System AK EsD GLO G6PD PHI PGM 6PGD
Fresh f f
f f
+
3 Weeks -
++ ++
7 Weeks -
-
++ +
DP 0.139 0.327 0.629
0.02 0.526 f f . f -Ifaint 0.081 - = not detectable = typeable DP = "discriminating power" (Jones, 1972)
+
+
Other enzyme systems which were examined in this study were acid phosphatase (AP), ADA and Peptidase A (PEP A). Although there appeared to be considerable amounts of AP activity we found that the electrophoretic patterns were not distinct enough for reliable typing. ADA could also be demonstrated in sheath cells but its activity appeared to be consistently low. As yet we have not detected PEP A.
PGM
PGM,
d-
PGM
Origin
Figure 1. Starch gel electrophoretic patterns of some of the PGM, and all the PGM, variants observed in human hair sheath cells using a Tris, maleic acid, MgCL,, EDTA buffer system pH7.4 (Spencer et al., 1968).
The results of the ageing studies are summarised in Table 1. This study revealed that all the enzymes which could readily be detected in fresh sheath cells could, with the exception of AK, be detected after 3 weeks. Essentially the same results were obtained after 7 weeks except that EsD activity had decreased to such a point that reliable typing could not be undertaken. Probably the most significant feature of this ageing study has been the discovery that both GLO and PGM,, which have the highest discriminating power, could be detected and successfully typed after a 7-week period.
Blood II
t
1
Hair Blood
Origin
Origin
Figure 2. The starch gel electrophoretic patterns of AK 1 and AK 2 - 1 phenotypes obtained from paired blood and human hair sheath cells samples using a histidine buffer system pH5 (Fildes and Harris, 1966).
111
,a* -2
Blood
1
6PGD A
Hair Hair
1
GPGD A
Blood Origin
Origin
Figure 3. The starch gel electrophoretic patterns of GPGD A phenotypes obtained from paired blood and human hair sheath cell samples using a phosphate buffer system pH7 (Fildes and Parr, 1963).
Discussion The discovery of GLO activity in hair sheath cells has provided a further means of discriminating between different hair samples. The discovery of GLO activity in human sheath cells is particularly valuable since it has also been detected in semen (Emes and Parkin, 1980). In addition, the discovery of both G6PD and PHI, both of which are stable enough to allow successful typing after a 7-week period, is reported. The application of enzyme typing of hair roots in case work should be of most value in cases of assault or murder in which hair is snatched or pulled from the scalp during the offence. References CULLIFORD, B. J., 1971, The Examination and Typing of Bloodstains in the Crime Laboratory, Published by US Department of Justice Law Enforcement Assistance Administration. B., 1980, Forensic Sci. Znt., 15, 265. EMES,E. G. and PARKIN, FILDES, R. A. and PARR,C. W., 1963, Nature, 200, 890. FILDES,R. A. and HARRIS,H., 1966, Natzire, 209, 261. D. A., MESTRINER, M. A., CONTNER, J. and HARRIS,H., 1973, HOPKINSON, Ann. Hum. Genet., 37, 119. JONES,D. A., 1972, J. Forens. Sci. Soc., 12, 335. S. and RITTER,H., 1977, Humangenetik, K ~ M P F*I., , BISSBORT, S., GUSSMANN, 27, 141. LINCOLN, P. J. and DODDS, B. E., 1968, Med. Sci. Law, 8, 38. O., KATSUMATA, Y. and YADA,S., 1978, OYA,M., ITO,H., KIDO,A., SUZAKI, Forens. Sci., 11, 135. SAKI,T., 1951, Jap. J. I,e*c. Med., 5, 19. SPENCER, N., HOPKINSON, D. A. and HARRIS,H., 1968, Ann. Hum. Genet., 32, 9. J. M. and WHITEHEAD, P. H., 1978,J. Forens. Sci., 23, 356. TWIBELL, B. G. D. and EMES,E. G., 1976, J . Forens. Sci. Soc., 16, 127. WRAXALL, YADA,S., OKANE,M. and SANO,Y., 1966, Acta Criminological et Medicinae Legales Japonica, 32, 7. YOSHIDA, ,I., ABET, T. and NAKAMIJRS, K., 1978, Medico-Legal Soc., 21, 7. 202