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The determination of glyoxalase I phenotypes in human bloodstains
Forensic Science International, 15 (1980) 265 - 271 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
THE DETERMINATION OF GLYOXALASE BLOODSTAINS
ELIZABETH Metropolitan
265
I PHENOTYPES IN HUMAN
G. EMES and B. H. PARIUN Police Forensic Science Laboratory,
(Received December 12,1979;accepted
109 Lambeth
Road, London
(Gt. Britain)
February 12,198O)
Summary A method is described for the determination of glyoxslase I phenotypes in liquid blood and dry bloodstains. The frequencies of glyoxalase I phenotypes in various populations of S.E. England are included.
Introduction The conversion of methyl glyoxal to lactate in human tissue (the glyoxalase (GLO) reaction) is catalysed by two enzymes, GLO I and GLO II, with reduced glutathione (GSH) as coenzyme.
Methyl GLO I
S-lactoyl glutathione
:,:,:,,,d
GLO I is present in human erythrocytes in high concentration, but GLO II is reported to be absent [ 11. Various workers [ 2 - 41 have reported GLO I to be polymorphic in European and African populations, indicating its potential use as an additional marker in the forensic bloodgrouping laboratory. The method used by Kompf et al. [ 41 to determine the GLO type of blood samples proved unsatisfactory for use with bloodstains. A more sensitive method developed by Parr et al. [ 51, involving the use of iodine to measure the uptake of reduced glutathione in forming S-lactoyl glutathione, has been adapted for the typing of small samples of bloodstained material.
266
Materials and methods Samples Haemolysates are prepared by freezing and thawing packed, washed erythrocytes three times and diluting them 1:l with a solution of 0.18 M fl-mercaptoethanol(/3-mercaptoethanol as supplied by BDH, Poole, Gt. Britain; diluted 1:80 with distilled water). Pieces of bloodstained material (approximately 5 mm X 2 mm) are cut out, moistened in a minimum amount of 0.18 M p-mercaptoethanol and eluted for 1.5 to 2 hours. The resulting eluate is soaked up on one or two cotton threads, approximately 1 cm in length. Bloodstains on non-absorbent surfaces can be swabbed with cotton threads also moistened with 0.18 M /I-mercaptoethanol. The use of dithiothreitol (Cleland’s reagent) increased the mobility of GLO I under our conditions, leading to the distortion of the zones of GLO activity. Its use has therefore been discontinued. Electrophoresis A 12% starch-gel plate, 1 mm thick, was prepared by the method of WraxaII and Culliford [6]. The bridge buffer is 0.2 M phosphate buffer (pH 6.8), and the gel buffer a 1 in 5 dilution of the bridge buffer in distilled water. Electrophoresis is carried out at 12 V/cm for 4.5 hours, after which the zones of GLO I are detected by the technique of Parr et al. [ 51. Location
of enzyme
activity
The GLO I isozymes are visualised by adding solution A, incubating, then adding solution B . Solution A: 10 ml of reaction buffer (as bridge buffer), 20 /.d of methyl glyoxal (Sigma grade II, 40% (w/v) aqueous solution), 12 mg of glutathione. Iodine solution (previously prepared): 2.54 g of iodine, 1.65 g of potassium iodide, 30 ml of distilled water. Dissolve the potassium iodide in about 5 ml of distilled water, add iodine and leave for 24 hours. Add the remainder of the 30 ml of water, filter and place in a dark bottle. Solution B: 0.2 ml of iodine solution, 0.6 g of agarose in 30 ml of distilled water (i.e. 2% agarose solution). The presence of sulphate groups in agar [ 71 inhibits the formation of the starch-iodine complex. Method
Solution A is soaked onto a piece of Whatman the gel surface covering the area between the origin cubated in a moisture chamber for 20 minutes at 37 the agarose should be added to the water and placed
3 mm filter paper, which is laid on and the anode. The gel is then in“c. During this incubation period, in a boiling water bath to melt the
agarose. After incubation, the filter paper is removed carefully from the gel surface; 0.3 ml of the iodine solution is added to the boiling agarose and the mixture thoroughly mixed to dissolve all traces of the iodine. The iodine-agarose mixture is poured over the gel surface previously covered by the filter paper. The areas of GLO I activity are immediately visualised by the formation of a blueblack colour where the iodine has reacted with the starch. At these sites the reduced glutathione has combined with methyl glyoxal (catalysed by GLO I) to produce S-lactoyl
267 glutathione. In the absence of free glutathione, the iodine is able to react with the starch. On the remainder of the gel surface the glutathione has bound the iodine preventing the formation of the starch-iodine complex [ 81.
Results Figure 1 shows the three common phenotypes of GLO I, viz. GLO 1, GLO 2-l and GLO 2 [4]. After electrophoresis and subsequent staining, the GLO 1 and 2 phenotypes each appear as a single, intensely staining band of activity with GLO 1 being the slower anodal component. The GLO 2-l phenotype appears as three bands, with the two lighter bands having the same mobility as the GLO 1 and GLO 2 isozymes. The third band, which is the most intense, possesses intermediate mobility, indicating the dimeric structure of the GLO I molecule [ 91. Work on bloodstains prepared in the laboratory showed that the GLO I isozymes survived drying and could be demonstrated in an unaltered form for up to 12 weeks after the stain was made (Fig. 2). Although the isozymes demonstrated in bloodstains are not so distinct as those obtained from fresh haemolysates, the GLO I phenotypes can still be unambiguously determined. The streaking effect observed at the edges of the zones of GLO activity, particularly in the 2-1 phenotype, is probably produced by pooling of the eluate from the bloodstained material after it is inserted in the gel. This observation was further supported by blind trials, in which 150 bloodstains made on cotton or woollen material were typed (the results are summarised in Table 1). Only where the band pattern was of sufficient intensity to be read clearly was a result recorded. Of the 150 stains, 136 were correctly typed, 14 stains gave unrecordable results and no results were incorrect. The blind trials were followed by typing bloodstains from casework material, selected from clothing, etc., of injured persons, where, by grouping the blood in a number of other systems there could be little doubt that the blood was from the victim. Again, the results showed that the GLO I phenotype of a blood sample could be reliably determined from a bloodstain. To date, 8364 blood samples have been typed for GLO I. The bloods are from criminals and victims of crimes of violence in South-East England. The frequencies of the phenotypes of GLO I observed in various ethnic groups in this population are shown in Table 2. Discussion The distribution of GLO I phenotypes in the British population (see Table 2) is very favourable for use in forensic blood typing. The Discriminating Power (DP) [lo] of GLO I has been calculated from these frequency figures to be 0.623, which is higher than that of ABO (DP = 0.600) and Hp
268
t
w
2
1
2
2-1
Fig. 2. The common glyoxalase phenotypes in bloodstains.
GLO
f
yr
-r,
2
2-1
.
t
270 TABLE 1 Summary of blind trials on bloodstains of known GLO I phenotypes No. of stains
Stain substrate
Age of stains
Correctly typed
Unreportable results
Incorrectly typed
150
Cotton or wool
4 to 92 days
136
14
0
TABLE 2 GLO I frequencies in S.E. England Population
Number tested
Caucasian
6806
British Negroes British Asians
1
2-1
2
GLO’
GL02
obs exp
21.61 21.60
49.72 49.75
28.67 28.65
0.4647
0.5353
1098
obs exp
11.75 11.29
43.72 44.63
44.54 44.08
0.3361
0.6639
460
obs exp
5.87 6.25
38.26 37.50
55.87 56.25
0.2500
0.7500
(DP = 0.616) but lower than the DP for EAP (0.672)
and Rhesus (DP = 0.787). This paper describes a method for determining GLO I phenotypes from bloodstains up to 12 weeks’ old. The method utilizes electrophoresis in a starch gel 1 mm thick and is suitable for typing the small portions of bloodstained material found in forensic casework. The method has been used routinely in the Metropolitan Police Forensic Science Laboratories for the last two years.
References P. P. Cohen and E. K. Sober, Glyoxahtse activity of erythrocytes from cancerous rats and human subjects. Cancer Res., 5 (1945) 631. I. A. Bagster and C. W. Parr, Human erythrocyte glyoxalase I polymorphism. J. Physiol., 256 (1976) 56. I. A. Bagster, C. W. Parr and S. G. Welch, Erythrocyte glyoxalase I polymorphism in an African and English population. FEBS Lett., 60 (2) (1975) 336. J. Kompf, S. Bissbort, S. Gussman and H. Ritter,Polymorphism of red cell glyoxalase I (EC 4.4.1.5). A new genetic marker in man. Humangenetik, 27 (1975) 141. C. W. Parr, I. A. Bagster and S. G. Welch, Human red cell glyoxalase I polymorphism. Biochem. Genet., 15 (1977) 109. B. G. D. WraxaIl and B. J. Culhford, A thin layer starch gel method for enzyme typing of bloodstains. J. Forensic Sci. Sot., 8 (1968) 81.
271 7 C. Araki, Carbohydrate chemistry of substances of biological interest. Proceedings of the Fourth International Congress of Biochemistry (Vienna), 1958, Vol. 1, Symp. 1, Pergamon, 1959, p. 15. 8 W. L. Hughes, The chemistry of iodination. Ann. N.Y. Acad. Sci., 70 (1957) 3. 9 B. Oray and S. J. Norton, A twostep purification of mouse liver glyoxalase I and evidence of its dimeric constitution. Biochim. Biophys. Acta, 483 (1977) 203. 10 K. W. Smalldon and A. C. Moffat, The calculation of Discriminating Power for a series of correlated attributes. J. Forensic Sci. Sot., 13 (1973) 291.
THE DETERMINATION OF GLYOXALASE BLOODSTAINS
ELIZABETH Metropolitan
265
I PHENOTYPES IN HUMAN
G. EMES and B. H. PARIUN Police Forensic Science Laboratory,
(Received December 12,1979;accepted
109 Lambeth
Road, London
(Gt. Britain)
February 12,198O)
Summary A method is described for the determination of glyoxslase I phenotypes in liquid blood and dry bloodstains. The frequencies of glyoxalase I phenotypes in various populations of S.E. England are included.
Introduction The conversion of methyl glyoxal to lactate in human tissue (the glyoxalase (GLO) reaction) is catalysed by two enzymes, GLO I and GLO II, with reduced glutathione (GSH) as coenzyme.
Methyl GLO I
S-lactoyl glutathione
:,:,:,,,d
GLO I is present in human erythrocytes in high concentration, but GLO II is reported to be absent [ 11. Various workers [ 2 - 41 have reported GLO I to be polymorphic in European and African populations, indicating its potential use as an additional marker in the forensic bloodgrouping laboratory. The method used by Kompf et al. [ 41 to determine the GLO type of blood samples proved unsatisfactory for use with bloodstains. A more sensitive method developed by Parr et al. [ 51, involving the use of iodine to measure the uptake of reduced glutathione in forming S-lactoyl glutathione, has been adapted for the typing of small samples of bloodstained material.
266
Materials and methods Samples Haemolysates are prepared by freezing and thawing packed, washed erythrocytes three times and diluting them 1:l with a solution of 0.18 M fl-mercaptoethanol(/3-mercaptoethanol as supplied by BDH, Poole, Gt. Britain; diluted 1:80 with distilled water). Pieces of bloodstained material (approximately 5 mm X 2 mm) are cut out, moistened in a minimum amount of 0.18 M p-mercaptoethanol and eluted for 1.5 to 2 hours. The resulting eluate is soaked up on one or two cotton threads, approximately 1 cm in length. Bloodstains on non-absorbent surfaces can be swabbed with cotton threads also moistened with 0.18 M /I-mercaptoethanol. The use of dithiothreitol (Cleland’s reagent) increased the mobility of GLO I under our conditions, leading to the distortion of the zones of GLO activity. Its use has therefore been discontinued. Electrophoresis A 12% starch-gel plate, 1 mm thick, was prepared by the method of WraxaII and Culliford [6]. The bridge buffer is 0.2 M phosphate buffer (pH 6.8), and the gel buffer a 1 in 5 dilution of the bridge buffer in distilled water. Electrophoresis is carried out at 12 V/cm for 4.5 hours, after which the zones of GLO I are detected by the technique of Parr et al. [ 51. Location
of enzyme
activity
The GLO I isozymes are visualised by adding solution A, incubating, then adding solution B . Solution A: 10 ml of reaction buffer (as bridge buffer), 20 /.d of methyl glyoxal (Sigma grade II, 40% (w/v) aqueous solution), 12 mg of glutathione. Iodine solution (previously prepared): 2.54 g of iodine, 1.65 g of potassium iodide, 30 ml of distilled water. Dissolve the potassium iodide in about 5 ml of distilled water, add iodine and leave for 24 hours. Add the remainder of the 30 ml of water, filter and place in a dark bottle. Solution B: 0.2 ml of iodine solution, 0.6 g of agarose in 30 ml of distilled water (i.e. 2% agarose solution). The presence of sulphate groups in agar [ 71 inhibits the formation of the starch-iodine complex. Method
Solution A is soaked onto a piece of Whatman the gel surface covering the area between the origin cubated in a moisture chamber for 20 minutes at 37 the agarose should be added to the water and placed
3 mm filter paper, which is laid on and the anode. The gel is then in“c. During this incubation period, in a boiling water bath to melt the
agarose. After incubation, the filter paper is removed carefully from the gel surface; 0.3 ml of the iodine solution is added to the boiling agarose and the mixture thoroughly mixed to dissolve all traces of the iodine. The iodine-agarose mixture is poured over the gel surface previously covered by the filter paper. The areas of GLO I activity are immediately visualised by the formation of a blueblack colour where the iodine has reacted with the starch. At these sites the reduced glutathione has combined with methyl glyoxal (catalysed by GLO I) to produce S-lactoyl
267 glutathione. In the absence of free glutathione, the iodine is able to react with the starch. On the remainder of the gel surface the glutathione has bound the iodine preventing the formation of the starch-iodine complex [ 81.
Results Figure 1 shows the three common phenotypes of GLO I, viz. GLO 1, GLO 2-l and GLO 2 [4]. After electrophoresis and subsequent staining, the GLO 1 and 2 phenotypes each appear as a single, intensely staining band of activity with GLO 1 being the slower anodal component. The GLO 2-l phenotype appears as three bands, with the two lighter bands having the same mobility as the GLO 1 and GLO 2 isozymes. The third band, which is the most intense, possesses intermediate mobility, indicating the dimeric structure of the GLO I molecule [ 91. Work on bloodstains prepared in the laboratory showed that the GLO I isozymes survived drying and could be demonstrated in an unaltered form for up to 12 weeks after the stain was made (Fig. 2). Although the isozymes demonstrated in bloodstains are not so distinct as those obtained from fresh haemolysates, the GLO I phenotypes can still be unambiguously determined. The streaking effect observed at the edges of the zones of GLO activity, particularly in the 2-1 phenotype, is probably produced by pooling of the eluate from the bloodstained material after it is inserted in the gel. This observation was further supported by blind trials, in which 150 bloodstains made on cotton or woollen material were typed (the results are summarised in Table 1). Only where the band pattern was of sufficient intensity to be read clearly was a result recorded. Of the 150 stains, 136 were correctly typed, 14 stains gave unrecordable results and no results were incorrect. The blind trials were followed by typing bloodstains from casework material, selected from clothing, etc., of injured persons, where, by grouping the blood in a number of other systems there could be little doubt that the blood was from the victim. Again, the results showed that the GLO I phenotype of a blood sample could be reliably determined from a bloodstain. To date, 8364 blood samples have been typed for GLO I. The bloods are from criminals and victims of crimes of violence in South-East England. The frequencies of the phenotypes of GLO I observed in various ethnic groups in this population are shown in Table 2. Discussion The distribution of GLO I phenotypes in the British population (see Table 2) is very favourable for use in forensic blood typing. The Discriminating Power (DP) [lo] of GLO I has been calculated from these frequency figures to be 0.623, which is higher than that of ABO (DP = 0.600) and Hp
268
t
w
2
1
2
2-1
Fig. 2. The common glyoxalase phenotypes in bloodstains.
GLO
f
yr
-r,
2
2-1
.
t
270 TABLE 1 Summary of blind trials on bloodstains of known GLO I phenotypes No. of stains
Stain substrate
Age of stains
Correctly typed
Unreportable results
Incorrectly typed
150
Cotton or wool
4 to 92 days
136
14
0
TABLE 2 GLO I frequencies in S.E. England Population
Number tested
Caucasian
6806
British Negroes British Asians
1
2-1
2
GLO’
GL02
obs exp
21.61 21.60
49.72 49.75
28.67 28.65
0.4647
0.5353
1098
obs exp
11.75 11.29
43.72 44.63
44.54 44.08
0.3361
0.6639
460
obs exp
5.87 6.25
38.26 37.50
55.87 56.25
0.2500
0.7500
(DP = 0.616) but lower than the DP for EAP (0.672)
and Rhesus (DP = 0.787). This paper describes a method for determining GLO I phenotypes from bloodstains up to 12 weeks’ old. The method utilizes electrophoresis in a starch gel 1 mm thick and is suitable for typing the small portions of bloodstained material found in forensic casework. The method has been used routinely in the Metropolitan Police Forensic Science Laboratories for the last two years.
References P. P. Cohen and E. K. Sober, Glyoxahtse activity of erythrocytes from cancerous rats and human subjects. Cancer Res., 5 (1945) 631. I. A. Bagster and C. W. Parr, Human erythrocyte glyoxalase I polymorphism. J. Physiol., 256 (1976) 56. I. A. Bagster, C. W. Parr and S. G. Welch, Erythrocyte glyoxalase I polymorphism in an African and English population. FEBS Lett., 60 (2) (1975) 336. J. Kompf, S. Bissbort, S. Gussman and H. Ritter,Polymorphism of red cell glyoxalase I (EC 4.4.1.5). A new genetic marker in man. Humangenetik, 27 (1975) 141. C. W. Parr, I. A. Bagster and S. G. Welch, Human red cell glyoxalase I polymorphism. Biochem. Genet., 15 (1977) 109. B. G. D. WraxaIl and B. J. Culhford, A thin layer starch gel method for enzyme typing of bloodstains. J. Forensic Sci. Sot., 8 (1968) 81.
271 7 C. Araki, Carbohydrate chemistry of substances of biological interest. Proceedings of the Fourth International Congress of Biochemistry (Vienna), 1958, Vol. 1, Symp. 1, Pergamon, 1959, p. 15. 8 W. L. Hughes, The chemistry of iodination. Ann. N.Y. Acad. Sci., 70 (1957) 3. 9 B. Oray and S. J. Norton, A twostep purification of mouse liver glyoxalase I and evidence of its dimeric constitution. Biochim. Biophys. Acta, 483 (1977) 203. 10 K. W. Smalldon and A. C. Moffat, The calculation of Discriminating Power for a series of correlated attributes. J. Forensic Sci. Sot., 13 (1973) 291.