Forensic Science International, 46 (19961255-268 255

Elsevier Scientific Publishers Ireland Ltd.

A STUDY ON THE VITAL REACTION IN WOUNDED SKIN:

SIMULTANEOUS DETERMINATION OF HISTAMINE AND
POLYAMINES IN INJURED RAT SKIN BY HIGH-PERFORMANCE
LIQUID CHROMATOGRAPHY

YOSHITAKA MAENO, FUKUTARO TAKABE, HIBOYUKI INOUE and MINE0 IWASA

Department of Legal Medicine, Nagoya City University Medical School, Kawaswmi Mizuho-ku,
Nagoya 467 Napad
(Received November 6th. 19691
(Accepted January 24th. 19961

This paper describes a modified method of simultaneous quantitative determination of his-

tamine and polyamines (putrescine, cadaverine, spermidine and sperminel in incised skin wounds
of rats using high-performance liquid chromatography (HPLCl with a strong-cation exchange
column. shim-pack ISC-05/SO564P, employing a linear gradient of 0.7-2.5 M sodium chloride.
There was scarcely any change in the histamine content of the vital wounds throughout the one
week experimental period, although content decreased slightly in l-h-old wounds. On the other
hand, concentrations of putrescine and spermidine increased suddenly 12 h after wounding, and
spermine content increased slightly in 4-day-old wounds. There was no post-mortem change in
the histamine, spermidine and spermine contents of vital wounds allowed to stand at 25% up to
24 h after death. Based on these results, this HPLC method may be useful for the determination
of wound age during the period of healing.

Key words: High-performance liquid chromatography; Simultaneous analysis; Histamine and

polyamine; Bat skin wounds; Vital reaction

Introduction

In forensic medical practice, numerous methods for the examination of the

vital reaction in wounds have been published. Histamine determination in
wounds is a well known means of examining the vital reaction of short-term
wounds [l - 41.
Other natural substances of interest are polyamines which are widely dis-
tributed in animal tissues, microorganisms and plants. Spermidine and sper-
mine in particular are related to the initiation of tissue growth [5-g]. Their
metabolism is closely interrelated, sequential formation of putrescine, sper-
midine and spermine.
In this study, we developed a new HPLC method to analyze histamine and
polyamines simultaneously. Furthermore, we applied this method to the
determination of the above chemicals in rat skin wounds at various vital
time periods.

0379-0738/90/803.50
0 1996 Eisevier Scientific Publishers Ireland Ltd.
Printed and Published in Ireland
256

Materials and Methods

Chemicals
Histamine, putrescine, cadaverine, spermidine and spermine hydrochloride
salts were obtained from Sigma (St. Louis, MO, U.S.A.). 1,3_Diaminopropane
dihydrochloride, as an internal standard, was purchased from Tokyo Kasei
(Tokyo, Japan). All other chemicals were of analytical grade, Wako Pure
Chemicals (Osaka, Japan).

Treatment of animals
Male Wistar strain rats, weighing 230-260 g, were used throughout all
experiments. Under light ether anesthesia, rats were shaved on the back and
full skindepth incisions, l-cm long, were made with a knife at various peri-
ods ante-mortem. These wounds were inflicted on the right dorsal side at 5,
20, 60 min, 12, 24, 96 and 168 h before death. In all cases, control specimens
of normal skin were taken from the left dorsal side. To study the changes in
histamine and polyamine contents in wounds after death, post-mortem inci-
sions were made on the skin within 2 min after killing and were left on the
body for 5,20, 60 and 720 min after injury. In order to study the post-mortem
changes of these amine contents in vital wounds, the rats with 24-h ante-
mortem wounds were allowed to stand at 25OC for various periods of 1, 3
and 5 days after death. Specimens were then taken and stored at -8OOC
until used.

Preparation of tissue extracts

Each excised skin tissue was serially sliced from the wound edge parallel
to the wound surface to a thickness of 20 w using a cryostat. These slices
were then divided into 7 fractions of 20 slices each.
Each fraction was transferred into 0.5 ml of 0.4 M perchloric acid, contain-
ing 20 4 of 1.0 mM 1,8diaminopropane as an internal standard. After shak-
ing vigorously, these fractions were incubated for 30 min at 4OC and then
were centrifuged at 1500 g for 20 min. The supernatants were filtered
through a 0.45pm pore size filter (Gelman Sciences Japan, Tokyo, Japan) and
the filtrates were used for the analysis of histamine and polyamines.

Analytical methods
HPLC was carried out on a strong cation exchanger column, Shim-pack
ISC-05/SO504P (50 x 4 mm I.D., Shimadzu, Kyoto, Japan) and the column
oven temperature was 70°C. Analytical separation of histamine and polyam-
ines was performed by the modified method of Fujita et al. [lo]. Briefly, after
30-50 4 of the filtrate were injected, the column was developed with sol-
vent A (0.2 N sodium citrate and 0.7 N sodium chloride) for 5 min. and then
with a linear gradient of O-100% solvent B (2.5 N sodium chloride and 0.2 N
sodium citrate) for 10 min. Then, 100% solvent B was run for 25 min. The
solvent flow rate was 0.6 ml/min. The elute was mixed with o-phthalaldehyde
 257

(OPA) reagent (0.08 % in 0.3 M boric acid, pH 10.5, containing 0.2% 2-mer-
captoethanol and 0.1 %I Brij 35) at a flow rate of 0.36 mllmin. The chemicals
were detected by fluorometry (an excitation wavelength of 345 nm and an
emission of 430 nm).
Histamine and polyamine contents were determined using a calibration
curve, which was obtained from the ratios of the areas of the histamine and
polyamine peaks to the area of the internal standard peak.

Results

Separation of histamine and polyamines

Figure 1 shows the chromatogram of the authentic standard, the
histamine standard and four polyamine standards, and of the extracts
obtained from a wounded rat skin. The retention times of histamine, putres-
tine, cadaverine, spermidine and spermine were 9.58, 20.13, 24.15, 27.33 and
39.51 min, respectively. The 1,8diaminopropane used as an internal standard

(A) (6)
5

1
3
1
6
2

Ii
4

0 ji 0 I!
r I I 1
0 15 30 45 0;55
TIME (min) TIME (min)
Fig. 1. Chromatogram of an authentic standard mixture of histamine and polyamines (A), and a
tissue extract obtained from a fraction of wound skin (B). Peaks: (1) histamine; 6) internal stand-
ard; (3) putrescine; (4) cadaverine; (5) spermidine; (6) spermine.
258

0 50 100
HISTAMINE AND FOLYAMINES (pmol)
Fig. 2. Standard calibration curve for the assay. Various amounts of a mixture of standard his-
tamine and polyamines in 50 CJ of 20 mM HCl were injected. 0, histamine; 0, putrescine; ? ,?
cadaverine; A, spermidine;m spermine.

had a retention time of 18.00 min. As a result, symmetrical peaks and good
separations could be obtained for histamine and the polyamines.
The calibration curves obtained from the peak area ratios of histamine
and polyamines to the internal standard were linear from 5 to 100 pmol, as
shown in Fig. 2. As shown in Table 1, the percentage recovery was 94-
115% for the known amounts of the histamine and polyamine standards 60
pmol each of histamine and polyamines per extract) added to the skin tissue
extracts.

TABLE 1
RECOVERY OF TISSUE HISTAMINE AND POLYAMINES

Compounds Recovery I%P

Histamine 115.1 f 3.3

Putrescine 93.9 f 5.5
Cadaverine 98.2 + 1.8
Spermidine 102.1 + 12.4
Spermine 109.7 f 16.5

‘The percentage recovery of each amine was calculated as {(extract containing a known amount
of standard amine - extra&a known amount of standard amine} x 100. fn = 3. mean f
S.D.).
 TABLE 2

1 CHANGES IN HISTAMINE LEVELS IN RAT SKIN WOUNDS

Fraction Relative histamine levek with time after woundinf

Ix 400 pm1
b-min 20-min 6O-min 12-h 24-h 96-h 168-h
ln = 181 ln = 12) In = 151 ln = 12) In = 9) ln = 12) In = 12)

1st 0.65 + 0.23* 0.72 r?: 0.25* 0.62 + 0.20+ 0.73 f 0.23* 0.52 f 0.21* 0.60 + 0.15* 1.05 & 0.23

2nd 0.89 f 0.23** 0.82 f 0.22** 0.86 f 0.16’ 0.90 -c 0.25 0.84 f 0.24** 0.89 & 0.21** 1.17 f 0.27

3rd 0.93 f 0.17 0.91 f 0.23 0.88 f 0.12’ 1.03 f 0.23 0.94 f 0.23 1.04 f 0.20 1.13 rt 0.26

4th 1.00 f 0.17 0.94 * 0.18 0.90 2 0.13* 0.98 + 0.16 0.97 2 0.23 1.00 2 0.18 1.12 f 0.23

5th 1.02 f. 0.15 0.96 f 0.23 0.93 f 0.15** 1.00 it 0.18 1.02 f 0.23 1.01 + 0.16 1.10 + 0.17

6th 1.03 f 0.17 0.91 + 0.19 0.94 f 0.13** 1.01 f 0.18 1.06 f 0.25 1.02 f 0.18 1.12 f 0.12

7th 1.03 f. 0.23 0.90 & 0.17 0.89 f 0.11* 1.00 f 0.16 1.02 * 0.18 1.04 f 0.20 1.08 f 0.19

‘The histamine level is the ratio of its content in vital wounds to that in intact skin (mean f S.D.).
*Mean < 1 (P < 0.06); **Mean < 1 P < 0.1).
TABLE 3

CHANGES IN PUTRESCINE LEVELS IN RAT SKIN WOUNDS

Fraction Relative putrescine kveLs with time after woulading

1x400 mj
dmin 20&n 60-m& 12-h 24-h 96-h 168-h
In = 181 In = 12) In = 15) In = 121 hc = 9) In = 12) (n = 121

1st 0.74 f 0.29* 0.88 2 0.37 0.73 f 0.27* 2.82 + 2.06’ 4.37 f 2.64* 2.70 2 3.07 2.13 f 0.96’

2nd 0.95 * 0.29 1.22 + 0.49 0.98 -c 0.38 2.76 + 1.99’ 4.2’7 + 2.79* 2.33 +. 1.88’ 1.81 r+ 0.67’

3rd 1.03 f 0.28 1.38 2 0.53’ 1.02 f 0.30 2.53 f 1.93’ 3.51 f 2.57’ 2.06 -c 1.30’ 1.53 * 0.54’

4th 1.01 f 0.25 1.41 2 0.51’ 1.00 f 0.36 2.23 2 1.79’ 2.95 f 2.61 1.69 f 1.09’ 1.63 k 0.61’

5th 1.01 2 0.30 1.48 -c 0.67’ 1.08 + 0.46 1.89 2 1.40’ 2.72 + 2.73 1.62 f 0.98’ 1.54 f 0.54’

6th 0.95 f 0.30 1.43 2 0.61’ 1.00 2 0.31 1.69 -c 1.12 2.88 f 3.20 1.70 & 0.81’ 1.50 f 0.66’

7th 0.96 + 0.32 1.39 -c 0.57’ 1.04 2 0.42 1.59 -c 1.21 2.46 i: 2.84 1.77 f 0.99’ 1.48 f 0.75’

‘The putrescine level is the ratio of its content in vital wounds to that in intact skin (mean 2 S.D.).
*Mean < 1 Lp < 0.05); ‘Mean > 1 (P < 0.05); ‘Mean > 2 (P < 0.05).
TABLE 4

CHANGES IN SPERMIDINE LEVELS IN RAT SKIN WOUNDS

Fraction Relative spermidine kvek with time after wounding”

tx400 rmtl
5-m& 20-min GO-min 12-h 24-h 96-h 168-h
/n = 18) In = 121 In = 151 In = 121 hr = 9) In = 121 ln = 12t

1st 0.75 f 0.15+ 0.83 f 0.21+ 0.77 * 0.12* 1.12 + 0.30 1.65 -c 0.29+ 1.95 f 0.57’ 1.69 f 0.34+

2nd 0.97 f 0.13 1.06 f 0.36 0.93 * 0.14 1.22 f 0.29’ i.79 f 0.39’ 1.82 f 0.71’ 1.46 f 0.23+

3rd 0.98 + 0.14 1.13 2 0.27 0.97 f 0.14 1.21 f 0.21+ 1.61 + 0.29’ 1.64 2 O&J+ 1.30 * 0.13’

4th 0.99 f 0.12 1.14 f 0.28 0.96 f 0.11 1.13 f 0.20+ 1.46 f 0.21’ 1.36 f 0.34’ 1.29 f 0.21’

5th 1.01 + 0.16 1.17 f 0.33 1.00 f 0.18 1.12 f 0.17+ 1.37 2 0.17’ 1.27 f 0.30’ 1.26 + 0.21’

6th 1.00 f 0.10 1.13 f 0.29 0.98 f 0.13 1.14 f 0.19’ 1.36 f 0.23+ 1.22 2 0.22’ 1.26 f 0.23’

7th 0.99 f 0.11 1.12 f 0.28 0.98 + 0.13 1.10 f 0.18+ 1.27 f 0.21’ 1.24 f 0.21’ 1.17 f 0.22+

#The spermidine level is the ratio of its content in vital wounds to that in intact skin (mean + S.D.).
*Mean < 1 (p < 0.05); +Mean > 1 (P < 0.05).
TABLE 5

CHANGES IN SPERMINE LEVELS IN RAT SKIN WOUNDS

Fractian Relative sperm&e levels with time after wounding’

1x400 /.nnl
5-m& 20-m& 60-m& 12-h 24-h 96-h 168-h
In = 181 In = 121 (n = 151 In = 12) ln = 9) (n = 12t ln = 121

1st 0.67 2 0.13* 0.74 f 0.14* 0.71 2 0.12f 0.87 -c 0.21 0.89 + 0.14 1.34 5 0.15’ 1.51 f 0.31’

2nd 0.89 f 0.15* 0.89 f 0.19 0.91 + 0.12* 1.01 2 0.24 1.09 f 0.17 1.29 + 0.19’ 1.35 f 0.23’

3rd 0.95 f 0.15 0.99 + 0.14 0.96 + 0.14 1.04 * 0.22 1.12 & 0.12 1.27 it 0.24’ 1.24 2 0.12’

4th 0.99 f 0.13 1.04 & 0.14 0.95 f 0.10 0.98 f 0.16 1.09 f 0.09 1.14 * 0.13’ 1.23 + 0.17’

5th 1.01 -t 0.13 1.07 f 0.15 1.00 -c 0.15 1.02 z? 0.18 1.09 * 0.11 1.11 -+ 0.15’ 1.21 + 0.10’

6th 1.02 f 0.11 1.03 2 0.16 1.00 f 0.11 1.06 * 0.20 1.14 f 0.14 1.08 & 0.10’ 1.23 2 0.13’

7th 1.01 -c 0.13 1.05 + 0.17 0.98 2 0.11 1.03 f 0.18 1.12 f 0.12 1.12 * 0.14’ 1.16 -c 0.14’

‘The spermine level is the ratio of its content in vital wounds to that in intact skin (mean -C S.D.).
*Mean < 1 (P < 0.05); ‘Mean > 1 (P < 0.05).
 263

TABLE 6
POST-MORTEM CHANGES IN HISTAMINE CONCENTRATION IN WOUNDS INFLICTED
WITHIN 2 min AFTER DEATH

Fmtion Relative histamine contents”

(x400 jtml
5-min 2O-min 6O-min 720-m&
In = 4) (n = S1 In = 9) (n = 4)

1st 0.70 * 0.11’ 0.90 f 0.13 0.62 f 0.12* 0.69 f 0.08*

2nd 0.88 * 0.05’ 0.95 f 0.015 0.86 2 0.30 0.81 f 0.12
3rd 0.94 i. 0.14 1.01 * 0.10 1.11 f 0.21 0.93 * 0.08
4th 0.90 f 0.09 1.01 + 0.19 0.90 * 0.20 0.94 +: 0.17
5th 1.00 1.00 1.00 1.00

‘The histamine content is the ratio of the concentration of each fraction to that of the 5th frac-
tion (mean f S.D.).
*Mean < 1 (P < 0.05): wean < 1 (P < 0.1).

Changes of histamine and polyamine levels in wounded rat skin

There are individual and regional differences in the contents of histamine,
putrescine, spermidine and spermine in rat skin (approx. lOO- 900, 10-300,
300-2500 and 150-750 nmol/g dry wt tissue, respectively). Thus, the
changes in content of these biogenic amines in the vital skin wounds were
indicated by the ratio of their concentration in ante-mortem wounds relative
to that of intact skin taken from the same animal.
As shown in Table 2, in all fractions of 60-min ante-mortem wounds, the
histamine content was lower than that of the intact skin. In the other

TABLE 7
POST-MORTEM CHANGES IN PUTRESCINE CONCENTRATION IN WOUNDS INFLICTED
WITHIN 2 min AFTER DEATH

Fraction Relative putrescine contents”

Ix400 PI
5-min 20-min 60-m& 720-m&
ln = 41 ln = Sl In = .Y In = 41

1st 1.00 + 0.38 0.93 r 0.05 1.25 f 0.24 0.91 f 0.51

2nd 1.02 f 0.27 1.05 k 0.04 0.98 -c 0.12 1.01 f 0.46
3rd 1.05 2 0.18 0.95 f 0.07 1.00 f 0.05 0.90 f 0.11
4th 1.22 + 0.31 0.90 f 0.34 1.00 + 0.36 0.88 + 0.13
5th 1.00 1.00 1.00 1.00

*The putrescine content is the ratio of the concentration of each fraction to that of the 5th frac-
tion (mean f S.D.).
 264

TABLE 8
POST-MORTEM CHANGES IN SPERMIDINE CONCENTRATION IN WOUNDS
INFLICTED WITHIN 2 min AFTER DEATH

Fraction Relutive spermidine contentsa

lx400 fd

5min 2O-min 6O-min 72O-min

ln = u ln = Sl ln = S) ln = 41

1st 1.02 f 0.16 0.93 f 0.10 1.08 f 0.10 0.80 + 0.19

2nd 1.05 f 0.14 1.05 f 0.05 0.99 f 0.11 0.84 f 0.09*
3rd 1.03 f 0.20 0.99 f 0.09 0.97 + 0.02 0.88 f. 0.090
4th 0.97 f 0.09 1.01 * 0.09 0.95 + 0.15 0.94 + 0.16
5th 1.00 1.00 1.00 1.00

‘The spermidine content is the ratio of the concentration of each fraction to that of the 5th frac-
tion (mean f S.D.).
*Mean < 1 Cp< 0.05); Mean < 1 (P < 0.1).

wounds, with the exception of 7-day-old vital wounds, histamine levels of

fractions obtained from the vicinity of the wound edge were lower than that
of intact skin. However, no difference was observed between the histamine
contents of the wounds and the intact skin at 7 days before death.
Tables 3, 4 and 5 show changes in putrescine, spermidine and spermine
levels, respectively, in the vital wounds. The putrescine concentration
increased slightly in the fractions at a distance of 1200 to 2800 pm from the

TABLE 9
POST-MORTEM CHANGS IN SPERMINE CONCENTRATION IN WOUNDS INFLICTED
WITHIN 2 min AFTER DEATH

Fraction Relative spermine contents”

lx400 pj
5-min BO-min 6Umin 720-min
In = 41 In = s/ In = s/ In = 4)

1st 0.80 + 0.150 0.87 + O.Ol* 0.88 + 0.12 0.85 + 0.09”

2nd 0.92 f 0.15 1.01 + 0.11 0.93 + 0.05 0.83 f 0.08*
3rd 0.97 f 0.13 0.94 f 0.10 0.93 + 0.02 0.93 zk 0.10
4th 0.96 + 0.09 0.97 -c 0.13 0.95 f 0.16 0.95 f 0.17
5th 1.00 1.00 1.00 1.00

*The spermine content is the ratio of the concentration of each fraction to that of the 5th frac-
tion (mean f S.D.).
*Mean < 1 (P < 0.05); Mean < 1 (P < 0.1).
 265

wound edge in 20-min vital wounds compared with that of intact skin. The
concentration of putrescine, however, did not remarkably change within 60
min. although contents increased significantly from 12 h onwards, and
reached a maximum in 24 h. The maximum levels of putrescine were over
twice that of intact skin, especially in the fractions up to 800 pm from the
wound edge. Afterward, levels decreased and returned to nearly intact skin
levels after 7 days.
The levels of spermidine in the wounds were not higher than those in
intact skin until 12 h after injury. The zone with the highest concentration of
spermidine was up to 800 I.crnfrom the wound edge in 1-_-day-old vital
wounds. The contents of spermidine were about 1.4 times that of the intact
skin. Thereafter, levels returned gradually to nearly that of the normal skin,
increasing with distance from the wound edge.
Spermine content, however, increased only in the 4- and ‘I-day-old (o-2800
pm) vital wounds. Cadaverine concentrations were also less than 10 nmol/g
dry tissue and changes were not consistent in these experiments. Contents
of these amines in post-mortem wounds were almost the same as those of
the intact skin.

Changes in amine contents in skin wounds inflicted within 2 min after death
Histamine, putrescine, spermidine and spermine levels in post-mortem
wounds inflicted within 2 min after death did not increase within 720 min
after injury, as shown in Tables 6, 7, 8 and 9. However, histamine and sper-
mine contents in the vicinity of the wound edge, within O-400 ccni, were

!I ,
F
E h

d . .
M .
0 1 3 5
DAYS FLAPSED
Fig. 3. Post-mortem changes of histamine and polyamine contents in 24-h-old vital wounds at
2fi°C. The relative amine content is the ratio of amine contents in vital wounds allowed to stand
for various periods after death to that in vital wounds taken immediately after death. ??, histam-
ine; A, spermidine; ??, spermine.
266

significantly lower than those of the 5th fraction used as a control’(Tables 6

and 9).

Post-mortem changes in amine contents in wounds

As shown in Fig. 3, there were no post-mortem changes in histamine,
spermidine and spermine contents in 24-h-old vital wounds at 25OC up to 1
day after death, as compared with those in the vital wounds which were
taken immediately after death. However, histamine and spermidine contents
decreased with time after death, although there was no change in spermine
content. Putrescine and cadaverine rapidly increased in concentration 1 and
3 days after death, respectively.

Discussion

In the field of forensic medicine, this is the first report of estimation of

wound age from changes in levels of histamine and polyamines in wound
inflicted skin tissues, using HPLC methods, although some investigators
have used histamine levels to estimate the vital reaction in wounds [l-4].
Different methods have been reported of simultaneous analysis of histamine
and polyamines in biological material using a HPLC system with cation
exchange resin columns [ll-141. Those methods, however, have in common
the problem of separation and symmetry of the amine peaks and/or the time
required for a complete separation. The gradient elution system used here
was designed to meet the requirements for adequate separation of histamine
and polyamines. As a result, a good separation of these amines was obtained
within 45 min and none of the additional peaks coincided with the symmetri-
cal histamine and polyamine peaks.
It is well known that determination of the free histamine concentration in
injured skin is important in estimating wound age and differentiating
between vital and post-mortem wounds [l-4]. Raekallio and Makinen [2]
reported that free histamine content in guinea pig skin increased immedi-
ately and reached a maximum within 20-30 min after injury, but there was
no such acute increase in the skin of rats. According to Miles [15], this
discrepancy might be related to a difference in the vascular permeability of
guinea pig and rat skin. In the present study, an increase in histamine con-
tent was not observed in rat skin wounds throughout the week-long experi-
mental period, however, in the 60-min vital wounds, histamine levels were
lower than that in intact skin. A similar decrease in histamine from damaged
rat skin has been reported [16-B]. At a distance of up to 800 pm from the
wound edge of vital wounds, the level of histamine decreased up to 4 days
after injury, and this phenomenon was also observed at the same location in
the post-mortem wounds. In the post-mortem wounds, however, no decrease
in histamine content was observed in any fraction.
With respect to polyamine levels, changes were more pronounced at a
later stage, compared with that of histamine. Our present findings concern-
 267

ing polyamine changes were approximately in agreement with changes of

spermidine and spermine in wounded skin as described by Mizutani et al
WI.
Viziam et al. [20] reported that within 6 h after wounding, the major event
appeared to be migration toward the wound of many intra- and perivascular
polymorphonuclear neutrophilic leukocytes (polysl as well as some lympho-
cytes, and the full development of the zone of polys between 12 and 24 h. In
the present study, the concentrations of putrescine, spermidine and
spermine in vital wounds increased in the fractions up to 800 pm from the
wound edge. These facts suggest a very close relationship between the
increase of polyamine contents and cell migration such as in the above zone
of polys, and also designate a possible marker for determinating the later
vital reaction in wounds.
There was no post-mortem change in the histamine, spermidine or
spermine contents in vital wounds at 25OC within 1 day after death. Further-
more, spermine levels showed no change for up to 5 days post-mortem. In
contrast, putrescine and cadaverine contents increased rapidly 1 and 3 days
after death, respectively. It is possible that increases of both amines
resulted from tissue putrefaction. In other words, when a high concentration
of cadaverine is found in skin, it may be inferred that death had occurred at
least 3 days earlier.
In conclusion, the simultaneous analytical method of determining these
biogenic amines using HPLC is very rapid and sensitive, and is very useful
for differentiating between ante-mortem and post-mortem wounds as well as
for the estimation of wound age. Although this study was conducted using
rat skin, we believe that this HPLC method may also be applied to wounded
human skin.

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