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Quantitation of nucleotides, nucleosides and bases in antemortem and postmortem bloodstains by high-performance liquid chromatography
Forensic Science International 71 (1995) 123-130
ELSEVIER
Forensic Science Internihionid
Quantitation of nucleotides, nucleosides and bases in antemortem and postmortem bloodstains by high-performance liquid chromatography H. Sugie* a, T. Nishikawab, T. Funaoa “Department of Legal Medicine, Kitasato University School of Medicine, 1-15-I Kitasato, Sagamihara, Kanagawa 228, Japan bDepartment of Clinical Pathology. Kitasato University School of Medicine, I-15-1 Kitasato, Sagamihara. Kanagawa 228, Japan
Received21 April 1994;accepted28 September1994 Abstract
Ante- and post-mortem bloodstains prepared from the blood of volunteers and corpses were analysed for ATP and its related compounds by reversed-phasehigh-performance liquid chromatography (HPLC). The results showed that (1) ATP was present in a large amount in antemortem bloodstains but not in postmortem stains, (2) AMP, adenosine, inosine, hypoxanthine, xanthine and uracil either were not detected or were detected in smaller amounts in antemortem than in postmortem bloodstains, and (3) ADP was present in both ante- and postmortem bloodstains. These differences suggestthat quantitation of these compounds may be useful in identifying whether bloodstains are ante- or post-mortem. Keywork Bloodstains; Antemortem and postmortem; ATP metabolites; High-performance liquid chromatography
1. Inlmduction It is sometimes necessary to identify bloodstains as ante- or post-mortem. The following tests have been reported for the differentiation of bloodstains:
(1) thin-layer chromatography for quantitative assay of free amino acids [l], (2) immunodiffusion and immunoelectrophoresis using anti-fibrinogen antibody
[219 l
Correspondingauthor.
0379-0738/95/$09.50 0 1995 Elsevier Science Ireland SSDI 0379-0738(94)01650-T
Ltd. All rights reserved
124
H. Sugie et al. /Forensic Sci. ht. 71 (1995) 123-130
(3) enzyme antibody technique using anti-myoglobin antibody or anti-fibrinogen antibody [3], (4) scanning electronic microscopy for the detection of fibrin [4], and (5) enzymatic assay of 2,3-diphosphoglycerate and lactate [5]. The analytes were amino acids, fibrinogen, fibrin, myoglobin or the metabolites of the glycolytic pathway. We have found that amounts of nucleotides, nucleosides and basesdiffer in anteand post-mortem bloodstains, suggesting that quantitation of these compounds will be useful and more reliable than other tests for differentiation. 2. Materials and methods 2.1. Reagents
ATP, ADP, AMP, adenosine (Ado), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA) and uracil (Ura) were obtained from Wako Pure Chemicals (Tokyo, Japan). ATPase, myokinase, alkaline phosphatase, nucleosidase, xanthine oxidase and uricase were obtained from Boehringer Mannheim Co. (Tokyo, Japan). 2.2. Preparation of antemortem blood and bloodstains
Blood specimens from 42 healthy volunteers were collected in heparin-coated tubes. A portion of the blood was dropped onto filter paper immediately after collection to prepare antemortem bloodstains, and the remaining blood was incubated at various temperatures for analysis. 2.3. Preparation of postmortem bloodstains
During medicolegal autopsy (2-240 h after death), blood from the heart was dropped onto filter paper to prepare postmortem bloodstains. These filter papers (n = 554) were stored at room temperature (about 23°C) until extraction. 2.4. Procedure of extraction from bloodstains and blood
About 10 mg of dried mass of the bloodstain was scraped off the paper, weighed on a Shimazu AEG-200 scale,mixed with 0.5 ml of cooled 0.6 M perchloric acid solution and ground in a tube with a glass pestle for 10 min. The suspension was centrifuged at 10 000 rev./min for 10 min at 4°C to obtain supernatant for HPLC analysis. Blood (0.1 ml) from volunteers was mixed with 0.5 ml of the cooled perchloric acid solution and extracted in the same way. 2.5. HPLC conditions
A Waters chromatograph system (a 6000A pump, U6K injector, 441 absorbance detector at 254 nm) with a Hitachi D-2500 integrator was used. A 20-~1aliquot of the supernatant extract was injected into a Waters Radial Cl8 Resolve Cartridge column (100 x 8 mm, 10 pm) and eluted with a 1:99 mixture of ethanol/O.1 M NH4H2P04 (pH 3.0) at a flow rate of 2.0 ml/min.
H. Sugie et al. /Forensic &i. ht. 71 (1995) 123-130
125
2.6. Definition of antemortem index and postmortem index In order to differentiate clearly between ante- and post-mortem bloodstains from the ratio of these metabolites, we defined indices as follows: AM1 (antemortem index, %) = ATP + ADP + AMP x 100 ATP + ADP + AMP + Ado + Ino + Hyp + Xan + Ura PM1 (postmortem index, %) = AMP + Ado + Ino + Hyp + Xan + Ura x 100 ATP + ADP + AMP + Ado + Ino + Hyp + Xan + Ura These indices were defined on the basis that antemortem bloodstains were rich in ATP, ADP and AMP, whereas postmortem bloodstains were rich in AMP, Ado, Ino, Hyp, Xan and Ura.
3. Results 3.1. HPLC profile of blooa%tainextract ATP, ADP, AMP, Ado, Ino, Hyp, Xan, UA, and Ura in the extract were separated by HPLC (Fig. 1). Each peak was certified by the enzymatic peak shift method. For example, the ATP peak was certified with ATPase or myokinase. The peaks of ADP, AMP, Ado and Ino were certified with alkaline phosphatase or nucleosidase,those of Hyp and Xan with xanthine oxidase. The Ura peak was certified by pH peak shift method using pH 3.0 and pH 4.5 ammonium phosphate buffer as HPLC eluents. 3.2. Change of the contents of bloocistainsduring storage 3.2.1. Antemortem bloodstains (bloodstains from volunteers). The antemortem bloodstains on filter papers were analysed after storage of a few hours, 4 months and 12 months at 4” or 23OC(Fig. 2). The metabolic rate of dried bloodstains was very slow in comparison with that of liquid blood. At 23°C ATP decreasedlinearly, and ADP increased gradually during the 12 months. At 4°C ATP decreasedslowly, and ADP decreasedat the beginning and then increased. Concentrations of AMP and Hyp did not change during the 12 months storage at either 4” or 23°C. Ado, Ino, Xan, and Ura were never detected during the 12 months storage at either temperature. 3.2.2. Postmortem bloodstains. Concentrations of metabolites in the postmortem bloodstains were plotted against the storage period of up to 20 years (Fig. 3). ATP was not detected in any specimen. ADP was detected in specimensstored for l-2 years but not in those stored for longer than 5 years. AMP decreasedyear by year.
126
H. Sugie et al. /Forensic Sci. Int. 71 (1995) 123-130
Antemortem
bloodstain
c;
Postmortem bloodstain u) Lo vi 6
I-
IIIIIII,,.,, ,
,
,
I
m
Time ( rni: )
I
I
I U-J
% c-3
,,, Lo
2
2
Time ( min )
Fig. 1. Chromatogram of the extract from bloodstain. Left: Antemortem bloodstain stored for 1 month at 23°C after blood collection. Right: Postmortem bloodstain stored for 1 year at 23°C after collection at medicolegal autopsy. Each numbered peak was assignedto the following compound: 1, ATP; 2, ADP; 3, Uracil (Ura); 4, AMP; 5, uric acid (UA); 6, hypoxanthine (Hyp); 7, xanthine (Xan); 8, inosine (Ino); 9, adenosine (Ado). The compound of the peak (X) was not identified.
(nmol/mg) 10
ATP
AMP
(nmolimg) 10
HYP
0
4
s
12 (Month)
Fig. 2. Monthly change of concentrations in the antemortem bloodstains of 42 volunteers. The bloodstains were stored at 4Oor 23°C. Each plot shows the average. The standard errors are indicated by vertical bars; most were smaller than 0.2 nmol/mg and too small to be shown except for that of ADP concentration at 12 months.
H. Sugie ei al. /Forensic Sci. ht. 71 (1995) 123-130
127
HYP I,
Fig. 3. Concentrations average and standard error in the postmortem bloodstains after storage of various periods at 23°C. The dotted zone representsthe range of concentrations in antemortem bloodstains when stored at 23°C for 12 months.
Concentrations of Ado and Ino were constant during the first several years but decreasedslightly after 18 years of storage. Concentrations of Hyp, Xan and Ura were fairly high and remained constant in all the specimens. 3.2.3. Antemortem index (AMI) and postmortem index (PMI) of the bloodstains. The two indices, AM1 and PMI, were plotted against the storage period (Fig. 4). All the antemortem bloodstains gave AM1 values higher than 90% and PM1 values lower than 30%, indicating the abundant presenceof ATP, ADP or AMP. All the postmortem bloodstains gave AM1 values lower than 50% and PM1 values higher than 80%, indicating the abundant presence of catabolites such as Hyp and Ino. 3.3. Change of the contents in blood during &day storage To investigate the mechanism of these differences in dried bloodstains, the blood specimensof the volunteers were incubated at 4”, 23” or 37°C in a liquid state and analysed. The metabolic rate was faster at the higher temperatures (Fig. 5). ATP was very rich at the beginning and decreasedrapidly to undetectable level within the first day at 37°C. ATP decreasedmore slowly at a lower temperature, reaching the limit of detection on the second day at 23°C and on the fourth day at 4°C. ADP was less than ATP at the beginning and decreasedduring storage. The AMP content reached
H. Sugie et al. /Forensic Sci. ht. 71 (1995) 123-130
128
( pmoln1 so
(Fmdn) “I
( prnOl/, 80
ATP -e-4r
60 E 1
P--
AMP
^^ f
Fig. 4. AMI (0) and PM1 (0) values for antemortem and postmortem bloodstains. Each plot shows the average for antemortem bloodstains (n = 42) and postmortem bloodstains (n = 18-58). The standard errors are indicated by vertical bars.
its maximum level on the first day at 37°C the second day at 23°C and the first day at 4”C, and then decreased.Ino had beenundetectable from the first day when stored at 37”C, but it increasedand then decreasedduring storage at 23” or 4°C. Hyp reached saturation on the second day and maintained that level when stored at 37°C but it increased linearly day by day during the 6-day storage at 23” or 4°C.
Postmortem bloodstains
Antemortem bloodstains 100
e
e
e
r 80
-
5
60
-
z p
40
-
0
Oll-l-7l 0
4
a12
Storage period (Month)
,
,
,
,
,
2
4
6
a 10 12 14 16 18 20
Storage period
,
,
,
Bee , ,
, 0
,
(Year)
Fig. 5. Daily change of the concentrations in whole blood from 42 volunteers. The blood specimenswere stored at 4O,23” or 37°C. Each plot shows the average. The standard errors are indicated by vertical bars.
H. Sugie et al. /Forensic Sri. hr. 71 (1995) 123-130
129
4. Discussion
The difference in the concentration of the metabolites was significant between ante- and post-mortem bloodstains. In antemortem bloodstains, ATP was present in a large amount, AMP and Hyp in lesser amounts, and Ado, Ino, Xan and Ura were not detected. In postmortem bloodstains, ATP was not detected, AMP and Hyp were present in large amounts, and Ado, Ino, Xan and Ura were present in detectable amounts. The following mechanismsare considered to cause these differences.ATP in red blood cells is catabolized along the metabolic pathway (ATP ADP, AMP - Ino, Hyp) as shown in the present results. Without a supply of ATPgenerating glucose, ATP is exhausted in several hours at 37°C. In a corpse, blood cells do not receive glucose and oxygen, but they are exposed to the still warm and respirating tissues.Therefore, ATP in corpse blood cells decreasesvery rapidly with the complementary increase of ADP and the other metabolites. These changesoccur so rapidly that ATP can not be detected in blood only 2 h after death, and Ado, Ino, Hyp and Ura have increased to significantly high levels. In necrosing heart or other tissue cells, the catabolites such as Ado, Ino and Hyp increase and are releasedinto the blood [6,7], hence, these catabolites may originate in tissue cells as well as blood cells. Since Ura is never produced by red blood cells, Ura in bloodstains could originate only in necrosing or damaged tissue cells. Therefore, a bloodstain containing a significant amount of Ura can be regarded as postmortem, unless gross tissue necrosis occurs before death. We included Ura level in the indices definition; however, its level may better be an independent factor for differentiation. The following cutoff values of the indices were proposed from the results: AMP > 90% and PM1 < 30% for antemortem; AM1 c 50% and PM1 > 80% for postmortem. These values were stable during storage becauseenzymatic and nonenzymatic reactions occur very slowly in a dried state, as shown by the present results and the other reports [5,8,9]. ATP was slowly metabolized into ADP and AMP in antemortem bloodstains during storage but hardly metabolized further into Ino, Hyp, and others. Therefore, we selected the sum of ATP, ADP and AMP for the definition of AM1 value which was calculated to be always higher than 90%. Though the antemortem bloodstains were stored for only up to 1 year, the time course study suggeststhat these values will be stable for much longer. There are some other tests detecting compounds such as amino acids and fibrinogen as described before, but they seem to be unreliable after sample storage of weeks or years. The presently described method may be more reliable for the specimensstored for years. It is possible to quantitate ATP and its related compounds by other methods, but HPLC using a silanol-uncapped reversed-phasecolumn seemsto be suitable because rapid and multiple quantitations can be made in one chromatography run [IO]. References [1] K. Satoh, S. Hayakawa and K. Nakanishi, Identification of antemortem and postmortem blood (report 3). Jpn. J. Legal Med., 29 (1975) 283-290. [2] K. Haba, M. Okano and K. Ito, Identification of antemortem and postmortem blood by means of immunodiffusion technique. Nall. Rex Inst. Police Sci., 23 (1970) 238-242 [in Japanese].
130
H. Sugie et al. /Forensic Sci. ht. 71 (1995) 123-130
[3] A. Takatsu, M. Abe, A. Shigeta, K. Fukui, S. Murata, Y. Aragaki, K. Maruyama and M. Ohtsuki, Identification of blood stain from living and cadaver origin by means of immunohistochemical method. Jpn. J. Legal Med., 45 Suppl. (1991) 124 [in Japanese]. [4] S. Muraoka, Studies on the blood stains by scanning electron microscopy. Jpn. J. Legal Med., 34 (1980) 605-617 [in Japanese]. [5] H. Sugie, M. Furukawa, K. Kurihara and T. Funao, Forensic chemical analysis of blood stains. Kifasato Med., 9 (1979) 149-156 [in Japanese]. [6] R.B. Jennings, K.A. Reimer, M.L. Hill and S.E. Mayer, Total ischemia in dog hearts, in vitro. I. Comparison of high energy phosphate production, utilization, and depletion, and of adenine nucleotide catabolism in total &hernia in vitro vs. severeischemia in vivo. Circ. Rex, 49 (1981) 892-900. [7] E.E. Gardiner, R. C. Newberry and J. Keng, Postmortem time and storage temperature affect the concentrations of hypoxanthine, other purines, pyrimidines, and nucleosidesin avian and porcine vitreous humor. Pediatr. Res., 26 (1989) 639-642. [8] L.A. King, The value of biochemical profiling for the discrimination of bloodstains. J. Forensic Sci. Sot., 14 (1974) 323-327. [9] S. Ameno, C. Sato, S. Suguri, T. Mori, N. Iijima, K. Ameno, C. Fuke, T. Kiriu and I. Ijiri, Species
identification of blood and bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-human hemoglobin antibody. Res. Pratt. Forensic Med., 33 (1990) 21-26. [lo] T. Nishikawa, S. Suzuki, H. Ohtani, M. Shirai, S. Nomiyama and H. Kubo, Isocratic separation of adenosineS&phosphate and its metabolites by reversed-phasehigh performance liquid chromatography: end-capped versus uncapped packings. Anal. Sci., 7 (199 I) 241-246.
ELSEVIER
Forensic Science Internihionid
Quantitation of nucleotides, nucleosides and bases in antemortem and postmortem bloodstains by high-performance liquid chromatography H. Sugie* a, T. Nishikawab, T. Funaoa “Department of Legal Medicine, Kitasato University School of Medicine, 1-15-I Kitasato, Sagamihara, Kanagawa 228, Japan bDepartment of Clinical Pathology. Kitasato University School of Medicine, I-15-1 Kitasato, Sagamihara. Kanagawa 228, Japan
Received21 April 1994;accepted28 September1994 Abstract
Ante- and post-mortem bloodstains prepared from the blood of volunteers and corpses were analysed for ATP and its related compounds by reversed-phasehigh-performance liquid chromatography (HPLC). The results showed that (1) ATP was present in a large amount in antemortem bloodstains but not in postmortem stains, (2) AMP, adenosine, inosine, hypoxanthine, xanthine and uracil either were not detected or were detected in smaller amounts in antemortem than in postmortem bloodstains, and (3) ADP was present in both ante- and postmortem bloodstains. These differences suggestthat quantitation of these compounds may be useful in identifying whether bloodstains are ante- or post-mortem. Keywork Bloodstains; Antemortem and postmortem; ATP metabolites; High-performance liquid chromatography
1. Inlmduction It is sometimes necessary to identify bloodstains as ante- or post-mortem. The following tests have been reported for the differentiation of bloodstains:
(1) thin-layer chromatography for quantitative assay of free amino acids [l], (2) immunodiffusion and immunoelectrophoresis using anti-fibrinogen antibody
[219 l
Correspondingauthor.
0379-0738/95/$09.50 0 1995 Elsevier Science Ireland SSDI 0379-0738(94)01650-T
Ltd. All rights reserved
124
H. Sugie et al. /Forensic Sci. ht. 71 (1995) 123-130
(3) enzyme antibody technique using anti-myoglobin antibody or anti-fibrinogen antibody [3], (4) scanning electronic microscopy for the detection of fibrin [4], and (5) enzymatic assay of 2,3-diphosphoglycerate and lactate [5]. The analytes were amino acids, fibrinogen, fibrin, myoglobin or the metabolites of the glycolytic pathway. We have found that amounts of nucleotides, nucleosides and basesdiffer in anteand post-mortem bloodstains, suggesting that quantitation of these compounds will be useful and more reliable than other tests for differentiation. 2. Materials and methods 2.1. Reagents
ATP, ADP, AMP, adenosine (Ado), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA) and uracil (Ura) were obtained from Wako Pure Chemicals (Tokyo, Japan). ATPase, myokinase, alkaline phosphatase, nucleosidase, xanthine oxidase and uricase were obtained from Boehringer Mannheim Co. (Tokyo, Japan). 2.2. Preparation of antemortem blood and bloodstains
Blood specimens from 42 healthy volunteers were collected in heparin-coated tubes. A portion of the blood was dropped onto filter paper immediately after collection to prepare antemortem bloodstains, and the remaining blood was incubated at various temperatures for analysis. 2.3. Preparation of postmortem bloodstains
During medicolegal autopsy (2-240 h after death), blood from the heart was dropped onto filter paper to prepare postmortem bloodstains. These filter papers (n = 554) were stored at room temperature (about 23°C) until extraction. 2.4. Procedure of extraction from bloodstains and blood
About 10 mg of dried mass of the bloodstain was scraped off the paper, weighed on a Shimazu AEG-200 scale,mixed with 0.5 ml of cooled 0.6 M perchloric acid solution and ground in a tube with a glass pestle for 10 min. The suspension was centrifuged at 10 000 rev./min for 10 min at 4°C to obtain supernatant for HPLC analysis. Blood (0.1 ml) from volunteers was mixed with 0.5 ml of the cooled perchloric acid solution and extracted in the same way. 2.5. HPLC conditions
A Waters chromatograph system (a 6000A pump, U6K injector, 441 absorbance detector at 254 nm) with a Hitachi D-2500 integrator was used. A 20-~1aliquot of the supernatant extract was injected into a Waters Radial Cl8 Resolve Cartridge column (100 x 8 mm, 10 pm) and eluted with a 1:99 mixture of ethanol/O.1 M NH4H2P04 (pH 3.0) at a flow rate of 2.0 ml/min.
H. Sugie et al. /Forensic &i. ht. 71 (1995) 123-130
125
2.6. Definition of antemortem index and postmortem index In order to differentiate clearly between ante- and post-mortem bloodstains from the ratio of these metabolites, we defined indices as follows: AM1 (antemortem index, %) = ATP + ADP + AMP x 100 ATP + ADP + AMP + Ado + Ino + Hyp + Xan + Ura PM1 (postmortem index, %) = AMP + Ado + Ino + Hyp + Xan + Ura x 100 ATP + ADP + AMP + Ado + Ino + Hyp + Xan + Ura These indices were defined on the basis that antemortem bloodstains were rich in ATP, ADP and AMP, whereas postmortem bloodstains were rich in AMP, Ado, Ino, Hyp, Xan and Ura.
3. Results 3.1. HPLC profile of blooa%tainextract ATP, ADP, AMP, Ado, Ino, Hyp, Xan, UA, and Ura in the extract were separated by HPLC (Fig. 1). Each peak was certified by the enzymatic peak shift method. For example, the ATP peak was certified with ATPase or myokinase. The peaks of ADP, AMP, Ado and Ino were certified with alkaline phosphatase or nucleosidase,those of Hyp and Xan with xanthine oxidase. The Ura peak was certified by pH peak shift method using pH 3.0 and pH 4.5 ammonium phosphate buffer as HPLC eluents. 3.2. Change of the contents of bloocistainsduring storage 3.2.1. Antemortem bloodstains (bloodstains from volunteers). The antemortem bloodstains on filter papers were analysed after storage of a few hours, 4 months and 12 months at 4” or 23OC(Fig. 2). The metabolic rate of dried bloodstains was very slow in comparison with that of liquid blood. At 23°C ATP decreasedlinearly, and ADP increased gradually during the 12 months. At 4°C ATP decreasedslowly, and ADP decreasedat the beginning and then increased. Concentrations of AMP and Hyp did not change during the 12 months storage at either 4” or 23°C. Ado, Ino, Xan, and Ura were never detected during the 12 months storage at either temperature. 3.2.2. Postmortem bloodstains. Concentrations of metabolites in the postmortem bloodstains were plotted against the storage period of up to 20 years (Fig. 3). ATP was not detected in any specimen. ADP was detected in specimensstored for l-2 years but not in those stored for longer than 5 years. AMP decreasedyear by year.
126
H. Sugie et al. /Forensic Sci. Int. 71 (1995) 123-130
Antemortem
bloodstain
c;
Postmortem bloodstain u) Lo vi 6
I-
IIIIIII,,.,, ,
,
,
I
m
Time ( rni: )
I
I
I U-J
% c-3
,,, Lo
2
2
Time ( min )
Fig. 1. Chromatogram of the extract from bloodstain. Left: Antemortem bloodstain stored for 1 month at 23°C after blood collection. Right: Postmortem bloodstain stored for 1 year at 23°C after collection at medicolegal autopsy. Each numbered peak was assignedto the following compound: 1, ATP; 2, ADP; 3, Uracil (Ura); 4, AMP; 5, uric acid (UA); 6, hypoxanthine (Hyp); 7, xanthine (Xan); 8, inosine (Ino); 9, adenosine (Ado). The compound of the peak (X) was not identified.
(nmol/mg) 10
ATP
AMP
(nmolimg) 10
HYP
0
4
s
12 (Month)
Fig. 2. Monthly change of concentrations in the antemortem bloodstains of 42 volunteers. The bloodstains were stored at 4Oor 23°C. Each plot shows the average. The standard errors are indicated by vertical bars; most were smaller than 0.2 nmol/mg and too small to be shown except for that of ADP concentration at 12 months.
H. Sugie ei al. /Forensic Sci. ht. 71 (1995) 123-130
127
HYP I,
Fig. 3. Concentrations average and standard error in the postmortem bloodstains after storage of various periods at 23°C. The dotted zone representsthe range of concentrations in antemortem bloodstains when stored at 23°C for 12 months.
Concentrations of Ado and Ino were constant during the first several years but decreasedslightly after 18 years of storage. Concentrations of Hyp, Xan and Ura were fairly high and remained constant in all the specimens. 3.2.3. Antemortem index (AMI) and postmortem index (PMI) of the bloodstains. The two indices, AM1 and PMI, were plotted against the storage period (Fig. 4). All the antemortem bloodstains gave AM1 values higher than 90% and PM1 values lower than 30%, indicating the abundant presenceof ATP, ADP or AMP. All the postmortem bloodstains gave AM1 values lower than 50% and PM1 values higher than 80%, indicating the abundant presence of catabolites such as Hyp and Ino. 3.3. Change of the contents in blood during &day storage To investigate the mechanism of these differences in dried bloodstains, the blood specimensof the volunteers were incubated at 4”, 23” or 37°C in a liquid state and analysed. The metabolic rate was faster at the higher temperatures (Fig. 5). ATP was very rich at the beginning and decreasedrapidly to undetectable level within the first day at 37°C. ATP decreasedmore slowly at a lower temperature, reaching the limit of detection on the second day at 23°C and on the fourth day at 4°C. ADP was less than ATP at the beginning and decreasedduring storage. The AMP content reached
H. Sugie et al. /Forensic Sci. ht. 71 (1995) 123-130
128
( pmoln1 so
(Fmdn) “I
( prnOl/, 80
ATP -e-4r
60 E 1
P--
AMP
^^ f
Fig. 4. AMI (0) and PM1 (0) values for antemortem and postmortem bloodstains. Each plot shows the average for antemortem bloodstains (n = 42) and postmortem bloodstains (n = 18-58). The standard errors are indicated by vertical bars.
its maximum level on the first day at 37°C the second day at 23°C and the first day at 4”C, and then decreased.Ino had beenundetectable from the first day when stored at 37”C, but it increasedand then decreasedduring storage at 23” or 4°C. Hyp reached saturation on the second day and maintained that level when stored at 37°C but it increased linearly day by day during the 6-day storage at 23” or 4°C.
Postmortem bloodstains
Antemortem bloodstains 100
e
e
e
r 80
-
5
60
-
z p
40
-
0
Oll-l-7l 0
4
a12
Storage period (Month)
,
,
,
,
,
2
4
6
a 10 12 14 16 18 20
Storage period
,
,
,
Bee , ,
, 0
,
(Year)
Fig. 5. Daily change of the concentrations in whole blood from 42 volunteers. The blood specimenswere stored at 4O,23” or 37°C. Each plot shows the average. The standard errors are indicated by vertical bars.
H. Sugie et al. /Forensic Sri. hr. 71 (1995) 123-130
129
4. Discussion
The difference in the concentration of the metabolites was significant between ante- and post-mortem bloodstains. In antemortem bloodstains, ATP was present in a large amount, AMP and Hyp in lesser amounts, and Ado, Ino, Xan and Ura were not detected. In postmortem bloodstains, ATP was not detected, AMP and Hyp were present in large amounts, and Ado, Ino, Xan and Ura were present in detectable amounts. The following mechanismsare considered to cause these differences.ATP in red blood cells is catabolized along the metabolic pathway (ATP ADP, AMP - Ino, Hyp) as shown in the present results. Without a supply of ATPgenerating glucose, ATP is exhausted in several hours at 37°C. In a corpse, blood cells do not receive glucose and oxygen, but they are exposed to the still warm and respirating tissues.Therefore, ATP in corpse blood cells decreasesvery rapidly with the complementary increase of ADP and the other metabolites. These changesoccur so rapidly that ATP can not be detected in blood only 2 h after death, and Ado, Ino, Hyp and Ura have increased to significantly high levels. In necrosing heart or other tissue cells, the catabolites such as Ado, Ino and Hyp increase and are releasedinto the blood [6,7], hence, these catabolites may originate in tissue cells as well as blood cells. Since Ura is never produced by red blood cells, Ura in bloodstains could originate only in necrosing or damaged tissue cells. Therefore, a bloodstain containing a significant amount of Ura can be regarded as postmortem, unless gross tissue necrosis occurs before death. We included Ura level in the indices definition; however, its level may better be an independent factor for differentiation. The following cutoff values of the indices were proposed from the results: AMP > 90% and PM1 < 30% for antemortem; AM1 c 50% and PM1 > 80% for postmortem. These values were stable during storage becauseenzymatic and nonenzymatic reactions occur very slowly in a dried state, as shown by the present results and the other reports [5,8,9]. ATP was slowly metabolized into ADP and AMP in antemortem bloodstains during storage but hardly metabolized further into Ino, Hyp, and others. Therefore, we selected the sum of ATP, ADP and AMP for the definition of AM1 value which was calculated to be always higher than 90%. Though the antemortem bloodstains were stored for only up to 1 year, the time course study suggeststhat these values will be stable for much longer. There are some other tests detecting compounds such as amino acids and fibrinogen as described before, but they seem to be unreliable after sample storage of weeks or years. The presently described method may be more reliable for the specimensstored for years. It is possible to quantitate ATP and its related compounds by other methods, but HPLC using a silanol-uncapped reversed-phasecolumn seemsto be suitable because rapid and multiple quantitations can be made in one chromatography run [IO]. References [1] K. Satoh, S. Hayakawa and K. Nakanishi, Identification of antemortem and postmortem blood (report 3). Jpn. J. Legal Med., 29 (1975) 283-290. [2] K. Haba, M. Okano and K. Ito, Identification of antemortem and postmortem blood by means of immunodiffusion technique. Nall. Rex Inst. Police Sci., 23 (1970) 238-242 [in Japanese].
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H. Sugie et al. /Forensic Sci. ht. 71 (1995) 123-130
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