Forensic

ELSEVIER

Biochemical

Forensic Science International 68 (1994) 83-89

Science Interaidiid

diagnosis of the intravital origin of skin wounds

P. Fernbdez*, A.M. Bermejo, M. Lbpez-Rivadulla, L. Concheiro Institute of Legal Medicine, Service of Forensic Toxicology. Faculty of Medicine. University of

Santiago de Composrela, Santiago de Compostela. Spain

Received 22 June 1993; revision received 22 March 1994; accepted 23 May 1994

Abstract

The performance of a spectrophotometric and a spectrofluorimetric method in the deter- mination of histamine and serotonin in skin samples was compared. The spectrophotometric method was found to be superior in terms of reproducibility and sample stability, so it was chosen for subsequent use. An overall 30 rabbits were wounded at different times prior to and after death and the histamine and serotonin concentrations in the wounds were compared to those of control specimens. Vital wounds were found to result in increased concentrations of the amines. The spectrophotometric method was also applied to human skin samples from 12 corpses showing body signs of violence.

Keywords: Histamine; Serotonin; Spectrophotometry; Spectrofluorimetry; Skin

1. Introduction

Proper diagnosis of the intravital origin of wounds calls for a biochemical study in addition to macro- and microscopic examinations. While reddening and swelling of wound edges in cases of acute swelling usually appear 12-24 h after wounding, leucocyte margination can be detected 6-8 h post-wounding by routine histological methods or even within l-2 h by histochemical procedures. However, the hour preceding the time of death can only be investigated by biochemical procedures [ 11.

Fazekas and VirBgos-Kis [2] found increased free histamine concentrations in

* Corresponding author.

0379-0738/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(94)01552-G

84 P. Fermindez et al. /Forensic Sri. ht. 68 (1994) 83-89

hanging grooves, which was subsequently confirmed by other authors [3-61: vital le- sions result in increased histamine (H) and serotonin (S) levels in the wound relative to control samples.

In this work we propose a spectrophotometric method to determine histamine and serotonin in wounds of rabbits injured at different times prior to and after death, and compare the concentrations obtained with those found in control samples. The method was also applied to 12 real cases of suspected violent death in order to deter- mine whether the wounds were vital or post mortem.

2. Material and methods

2. I. Analytical procedures Spectrophotometric method. The spectrophotometric method was implemented on

a Perkin-Elmer 552 UV-visible spectrophotometer. Histamine was quantified by preparing a stock solution containing 200 &ml in 0.1 N HCl, from which working strength solutions of 2, 5, 10, 15 and 20 pg/ml were subsequently made. The solution absorbance at 210 nm, Y, was found to be linearly related to the histamine concen- tration, X, by the following equation:

Y = 0.050X + 0.015 (correlation coefficient = 0.999; R2 = 99.76%) (1)

A stock solution containing 250 &ml serotonin in 0.1 N HCl was similarly prepared and working strength solutions of 2.5, 5, 10, 15 and 20 &ml were made from it by appropriate dilution. The solution absorbance at 275 nm, Y, was linearly related to the serotonin concentration, X, by the following equation:

Y = 0. 033X + 0.014 (correlation coefficient = 0.999; R2 = 99.75%) (2)

Spectrofluorimetric method. The spectrofluorimetric method was carried out on a Kontron SFM-23 spectrofluorimeter. From a stock solution of 250 &ml histamine in 0.1 N HCl, working strength solutions containing 0.2, 0.5, 1, 1.5 and 2 &ml of the analyte were made by dilution. To a 2.5-ml aliquot of each was added in se- quence 0.5 ml of 1 N NaOH and 0.125 ml of o-phthaldehyde, plus 0.25 ml of 3 N HCl 4 min later. The mixture was then shaken and its fluorescence at 450 nm measured on excitation at 360 nm [7]. The solution fluorescence, Y, was found to be linearly related to the concentration of histamine, X, by the following equation:

Y = 49.01X + 2.07 (correlation coefficient = 0.998; R2 = 99.51%) (3)

Serotonin was assayed similarly from a stock solution containing 250 &ml in 0.1 N HCl, which was used to prepare working strength solutions of 0.1, 0.2, 0.4, 0.8 and 1.0 &ml by appropriate dilution. A 3-ml aliquot of each dilute solution was adjusted to pH 2.5 with anhydrous sodium carbonate and its fluorescence at 345 nm measured on excitation at 295 nm [8]. The solution fluorescence, Y, and serotonin concentration, X, were found to be related by the following equation:

P. Fernhdez et al. /Forensic Sri. In:. 68 (1994) 83-89 85

Y = 99.87X-0.29 (correlation coefficient = 0.999; R* = 99.74%)

2.2. Extraction procedure Histamine and serotonin in the skin samples were extracted by the Fazekas [2] and

Udenfriend [8] methods, respectively. These methods were tested by extracting in parallel and assaying 10 replicate tirode solutions [2] spiked with 10 pg/ml H and 10 replicate 0.1 N HCl solutions [8] spiked with 10 pg/ml S. The final concentrations were calculated spectrophotometrically on Eqs. 1 and 2, respectively, and compared with the initial concentrations (10 &ml) to determine the mean extraction recoveries (78.4% for H and 92.9% for S). The within-run statistical analysis showed mean coefficients of variation which varied from 1.11% for H to 1.38% for S. The spectrofluorimetric method resulted in higher sensitivity but lower solution stability and hence decreased reproducibility.

2.3. Animal experiments An overall 30 rabbits were classified into six groups of five animals each that were

wounded by cutting in different parts of their bodies (rear left thigh, front left thigh, back, head, front right thigh and abdomen) at different times prior to (15, 10, 5 and 2 min) and after death (5 and 20 mm). The wounds were inflicted under ether anesthesia, without apparent effect on the H and S contents [9]. On sacrificing, two specimens were collected from wound edges and another two from a neighboring zone (- 10 cm apart) in order to determine their histamine and serotonin concentra- tions by the above-described spectrophotometric method.

2.4. Real cases Histamine and serotonin were also assayed in skin samples from 12 corpses with

apparent body signs of violence. The 12 cases included six hangings, two suicides with guns, two accidental deaths (one of thoracic traumatism and another of cardio- vascular collapse) and two murders (one by stabbing and the other by cranioen- cephalic traumatism).

In the hangings (cases 1, 2, 3, 7, 9 and lo), the victims were founded suspended at their homes. In the suicides (cases 5 and 6), the individuals used hunting rifles and were in the surroundings of their houses. In the accidental deaths, case 8 was found- ed dead on the bed with frontal ecchymosis and case 12 was a thoracic traumatism produced by a fall at home, the death happening 12 h later in a hospital. Finally, the two murders were due to injuries inflicted in fights, and the deaths took place 10 h (case 4) and 6 h (case 11) later in a hospital. In all cases the specimens were taken and analyzed inside 24 h after death, and we never had proof of previous medi- cation in the people studied.

3. Results and discussion

A comparison of the spectrofluorimetric and spectrophotometric methods initially assayed for the determination of histamine and serotonin revealed the former to be substantially more sensitive, with detection limits of 0.1 &ml S and 0.2 &ml H,

86

Table 1

P. Ferminde: et al. /Forensic Sci. ht. 68 (1994) 83-89

Histamine (H) and serotonin (S) concentrations in rabbit skin samples

Wound Time (min)

Sample source Average concentration Average concentration (H)

(S) (pplg) (PglP)

Absol. Relat. Absol. Relat.

Antemortem

IS

IO

5

2

Postmortem

5

20

Rear left thigh

Control Front left thigh

Control Back

2.01 6.38 2.76 1.73

0.73 3.68 1.58 4.70

2.08 1.84 0.76 2.55 0.99 5.78

1.87 1.70

Control 0.53 3.40 Head 1.13 7.42

1.80 Control 0.63 3.65

2.03

Front right thigh 2.40 7.32

Control Abdomen

Control

1.46 1.64 1.92

1.36 I.41

1.33 5.50 9.12

1.27 7.65

I I c 15 10 5 D 5 10 15 20

WOUND TIME (MIN)

Fig. I. Variation of the relative concentrations of histamine (m) and serotonin (0) as a function of the time of death (D) in rabbits.

Table 2

P. Fernbndez et al. /Forensic Sci. ht. 68 (1994) 83-89 87

Histamine (H) and serotonin (S) concentrations found in real death cases

Case Age Sample source Cont. (S) Wg) Cont. (H) Wg) Cause of death

Absol. Relat. Absol. Relat.

I 13

2 21

3 21

4 30

5 38

6 42

7 52

8 51

61

66

72

83

Groove

Control Groove

Control Groove

Control Back

Control Right cheek

Control Precordial region

Control Groove

Control Frontal ecchymosis

Control Groove

Control Groove

Control Forehead

Control Chest

Control

5.58 6.2-l 2.21 I.64

2.46 3.82 7.51 7.20

I .98 1.88 3.19 3.83

14.38 21.46 2.82 1.84

5.10 II.66 43.00 32.91

1.85 1.33 23.22 21.55 82.21 73.89

1.99 1.94 41.31 38.09 40. I I 36.96

3.00 1.60 13.39 23.03 2.16 4.18

3.18 2.47 0.68 1.69

210.68 233.61

4.84 43.50 27.91

2.85 9.16

21.22 1.87

10.77 4.38

2.86 1.53

63.15 2.45

26.01

4.59 50.92 23.60

1.89 12.49 24.30

I.28 18.98 28.46

1.58 18.01

Hanging

Hanging

Hanging

Stabbing

Shooting

Shooting

Hanging

Cardiovascular collapse

Hanging

Hanging

Cranioencephalic traumatism

Thoracic traumatism

than the latter (detection limits, 2.5 &ml S and 2 &ml H). However, the spec- trophotometric method surpassed the spectrofluorimetric method in three respects that justified choosing it, namely: it provided good reproducibility, with average coefficients of variation of 1.38% for S and 1.11% for H; it resulted in higher solution stability; and it was insensitive to potentially interfering substances present in the skin.

By application of Eqs. 1 and 2 and the corresponding extraction yields, the abso- lute and relative concentrations of H and S in the rabbit skin samples from both vital

P. Ferntinde: et al. /Forensic Sei. ht. 68 (1994) 83-89

11 10 20 30 40 50 60 70 80

SUBJECT AGE (YEARS)

Fig. 2. Variation of the relative concentrations of histamine (m and serotonin (0) as a function of subject age in real death cases.

and postmortem wounds were obtained (Table 1). As can be seen, the H and S con- tents of skin varied over wide ranges; on the other hand, the relative concentrations of the two amines was quite consistent with values > 2 for vital wounds and < 1.5 for postmortem wounds. However, it should be noted that the serotonin levels ex- ceeded those of histamine at all the antemortem times studied, with the exception of the wound made immediately prior to death, where the relative concentration of H was somewhat higher than of S. No clearcut relationship, though, was found be- tween these results and the wounding time, as can be seen from Fig. 1.

Table 2 shows the results obtained in the 12 real cases of violent death. The cause of death and the absolute and relative concentration of H and S found in the wounds are stated. The results were similar to those obtained for the rabbits, with relative S concentrations of 1 - 2, andrelative H concentrations of - 1.5 in most cases. This is seemingly consistent with previous findings [ 10,111. The fact that the relative con- centrations of H in cases 4 and 10 were < 1.5 does not necessarily imply that the wounds were made post mortem, even though a positive reaction would be indicative of the antemortem origin of the wound; also, the relative concentrations of S were < 2. This can be ascribed to the high alcohol concentrations detected ( > 2 g/l), which caused a decreased release of both amines, results in concordance with earlier in- vestigations 112). In view of the results, all the wounds were of vital origin and the H and S concentrations varied markedly between individuals and body zones.

P. Ferncinde-_ et al. /Forensic Sci. Int. 68 (1994) 83-89 89

Fig. 2 shows the variation of the relative concentrations of the two ammes as a function of casualty age. As can be seen, the concentration of S increased more markedly than did that of H.

Finally, it should be noted that, while the vita1 origin of wounds can be ascertained by using some individual procedures, estimation of wound time is more accurate if several methods (histological, biochemical, histochemical) are used in conjunction. This in turn provides more reliable evidence for clarifying cases of violent or sudden death.

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