Mello de Oliveira & Santos-Martin: Enzyme Histochemistry ofthe Liver 201
Enzyme Histochemistry of the Liver in Autopsy Material at
Different Post-mortem Times
JOSE ALBERTO MELLO DE OLIVEIRA, MD, Laboratory of Histoenzymology
CARMEN CINIRA SANTOS-MARTIN, MD, Area of Legal Medicine
Department of Pathology, School of Medicine of Ribeirao Pteto, University of Sao Paulo, Brazil
ABSTRACT
The authors report the enzyme histochemistry of the
liver obtained from autopsy material in 22 corpses (2
to 12 hours post-mortem) and performed to evaluate
the sensitivity of enzyme activities to the autolysis
process and the use of enzymes to estimate time in
forensic pathology. The earliest sample was at 2
hours post-mortem; there was five cases up to 5
hours; eight cases up to 8 hours and eight cases up to
12 hours since death. Active phosphorylase (PHYLA
a) and total phosphorylase (PHYLA t) were negative
two hours after death. PHYLA t reaction represents
the activity of PHYLA a increased with the inactive
phosphorylase b which can be activated by the addi-
tion of ATP and Mg 2+ to the incubation medium for
phosphorylase a; this activation proved to be ineffec-
tive in the post-mortem periods of this study.
Glucose-6-phosphatase (G6P-A) also showed a ten-
dency to be sensitive to the autolysis process,
displaying a reaction progressively weaker or nega-
tive in the post-mortem periods of observation. The
results indicate these enzymes as a possible tool to
estimate time in forensic pathology deserving fur-
ther investigation. Lactate dehydrogenase (L-D), a-
glycerophosphate dehydrogenase (a-GP-D) and ~-hy­
droxybutyrate dehydrogenase (~-HOB-D) instead
showed stronger reactions as the autolysis process
evolved.
INTRODUCTION
Raekallio (1961) published the first paper on
the medico-legal application of enzyme histo-
chemistry, followed by reports by Fatteh (1966)
and Raekallio (1966). Goffin (1968) studied
acid phosphatase, alkaline phosphatase and
naphthol-AS-esterase activities in the skin of
cadavers. Goffin and Beeckmans (1973) stud-
ied the same tissue for the activities of glucose-
6-phosphate dehydrogenase, succinate
dehydrogenase and nicotinamideadenine dinu-
cleotide phosphate tetrazolium-reductase. In
this later report the authors tried to identify
the earliest signs of autolytic process through
the enzyme histochemistry in order to deter-
mine the number of hours elapsed since death.
They were unsuccessful in estimating the post-
mortem time by skin histoenzymology because
all systems studied were active for a long time
after death. Pentilla and Ahonen (1976) found
the activities oflactate dehydrogenase, malate
dehydrogenase and succinate dehydrogenase
in the rat myocardium to still be active four
days after death. Probably because of those
unsuccessful attempts to show changes in the
enzyme activity through histochemistry this
method is little referred to in research for post-
mortem time estimate. Knight (1991) in his
treatise on Forensic Pathology refers to its use
in distinguishing ante-mortem and post-mor-
tem wounds but not in the study of the post-
mortem interval. Van den Oever (1976) and
Ravache-Quiriny (1986) reviewing the subject
of post-mortem interval considered its estima-
tion as a question not well answered and de-
serving further investigation. Recently Zharov
and Mel'nikova (1989) reported changes in the
skeletal and cardiac muscle cathepsins within
five days of death and considered that is possi-
ble to use the observed values as one criterion
in determining post-mortem interval.
The present paper is a report of histoenzy-
mological studies undertaken to investigate
enzyme activities in human livers obtained
from autopsy material, in order to identify
those more sensitive to the autolytic process
and evaluate their utilization to estimate time
of death.
202 Med. Sci. Law (1995) Vol. 35, NO.3

Table I. Enzyme histochemistry of the liver: techniques applied to study the autopsy
material.

Enzyme reaction Abbrev. ECnumber* References

Active phosphorylase PHYLA a EC 2.4.1.1 Guha & Wegmann, 1959

Total phosphorylase PHYLAt See text Guha & Wegmann, 1959
Glucose-6-phosphatase G6P-A EC 3.1.3.9 Chiquoine, 1953
Acid phosphatase a.-GP-AII EC 3.1.3.2 Gomori, 1942
Glucose-6-phosphate dehydrogenase G6P-D EC 1.1.1.49 Wegmann & Gerzelli, 1961
NADH2-tetrazolium-reduetase NADH2-TR EC 1.6.99.1 Scarpelli et al., 1958
NADPH2-Tetrazolium-reductase NADPH2-TR EC 1.6.99.3 Scarpelli et al., 1958
Lactate dehydrogenase L-D EC 1.1.1.27 Wegmann & Sotelo, 1962
Malate dehydrogenase M-D EC 1.1.1.37 Wegmann & Sotelo, 1962
Alcohol dehydrogenase A-D EC 1.1.1.1 Hardonk, 1965
Glutamate dehydrogenase GL-D EC 1.4.1.3 Diculesco & Wegmann, 1964
Succinate dehydrogenase S-D EC 1.3.99.1 Wegmann & Tordet-Caridroit, 1960
Monoamine oxidase MAO EC 1.4.3.4 Glenner et al., 1957
a-Glycerophosphate dehydrogenase a.-GP-D EC 1.1.1.8 Wegmann et al., 1964
[3-Hydroxybutyrate dehydrogenase [3.-HOB-D EC 1.1.1.30 Wegmann et al., 1964
(*) Reference 11.

MATERIAL AND METHODS a control for the effectiveness of every incuba-

There was no refrigeration after death of the
bodies selected for this study. The access to the
liver was made soon after opening the abdomi-
nal cavity, and the fragments were sampled
and frozen avoiding contact with tap water. We
sampled the right lobe of the liver during 22
necropsies and studied them in the Laboratory
of Histoenzymology (Department of Pathology;
School of Medicine of Ribeirao Prete). Frag-
ments measuring 5 x 5 x 3mm were frozen in
liquid nitrogen (-196°C), cut with a Harris
cryotome model CTD of the International
Equipment Company, and incubated in solu-
tions prepared through the techniques listed in
Table I. In every technique a neighbouring sec-
tion incubated in substrate-free media served
as control of enzymatic action on that sub-
strate.
We scored the intensity of the reactions as
negative (N), weak (W), moderate (M) or strong
(S). As a reference for the intensity of the reac-
tions we used liver biopsies with normal
results from our archives whose process
and techniques were the same as for the
autopsy material. Liver and striated muscle
from Wistar rats prepared and incubated si-
multaneously as for the autopsy material gave
tion medium in developing the enzymatic
reactions. We used the periodic acid Schiff
(PAS) reaction (McManus, 1946) to detect gly-
cogen and the reaction of Chevremont and
Frederic (1943) to evaluate the SH-radicals in
the hepatocytes.
RESULTS AND COMMENTS
Table II shows data on post-mortem time, room
temperature, sex, age, colour, major diseases
and hepatic histopathology in 22 autopsies.
Post-mortem time varied from 2 to 12 hours.
Room temperature at the beginning of the
autopsies ranged from 18°C to 29°C. The cases
included 17 males and 5 females, aged 20 to 82
years. There are 10 cases in which no lesions
were reported in liver microscopic examina-
tion. Two cases showed acute congestion; 5
cases chronic congestion; 2 cases fat degenera-
tion and 3 cases steatosis. The present results
will refer only to the enzyme activities in the
hepatocytes.
The intensities of the enzyme reactions in
the hepatocytes of liver biopsies were as fol-
lows: moderate to strong activity for PHYLA a
and PHYLA t in 4 cases; strong activity for
G6P-Ain 9 cases; weak activity for u-GP-AII in
 Mello de Oliveira & Santos-Martin: Enzyme Histochemistry of the Liver 203

to strong activity in normal tissue except MAO

and P-HOB-Dwhich displayed weak to moder-
ate activity and a-GP-A II which had weak
activity.
Among the enzyme activities of the hepato-
cytes in the autopsy material we observed
reactions sensitive or resistant to the autolysis
process as summarized in Table III. Figure 1
illustrates the histochemical reactions in biop-
sies and autopsies for PHYLA t, G6P-A, A-D,
G6P-D and NADPH2-TR. The changes ob-
served in the enzymatic reactions did not
correlate with the degenerative lesions of the
hepatocytes referred in the histopathologic ex-
amination.
The phosphorylase system in the glyco-
genolytic pathway soon loses its activity with
negative PHYLAa and PHYLA t reactions two
hours after death (Table III). The activity of
phosphorylase in enzyme histochemistry is di-
rectly proportional to the length and number of
glucosyl units added to the pre-existing intra-
cellular glycogen (Guha and Wegmann, 1959).
The negative reactions as observed by us could
be due to the diffusion and loss of glycogen
during autolysis. However, the PHYLA reac-
tions were negative with glycogen well
preserved in the hepatocytes, as detected by
Figure 1. Enzyme histochemistry of the liver in the PAS technique with previous digestion
biopsies (column A) and autopsies (column B): with a-amylase and p-amylase. Guha and Weg-
1: PHYLA t - positive reaction in biopsy and mann (1959) pointed out that addition of ATP
negative in autopsy; and M~+ to the incubation medium for PHYLA
2: G6P-A - positive reaction in biopsy; notice in a is able to demonstrate the active a form of the
B2 a positive reaction 2 hours after death (left) enzyme plus the activated phosphorylase b;
and a negative reaction 6V2 hours after death this reaction thus represents the activity of
(right); total phosphorylase (PHYLA t), In our mate-
3: A-D; rial, the addition of ATP and M~+ always
4: G6P-D; failed to activate phosphorylase b (Figure 1-
5: NADPH2-TR. Bl.) and both the PHYLA a and t reactions
were negative two hours after death and in
all the other periods of observation in our
2 cases; moderate to strong activity for G6P-D
cases.
in 9 cases; moderate to strong activity for
G6P-A in gluconeogenesis is an acid phos-
NADH2-TR in 4 cases; moderate to strong ac- phatase specific for glucose-6-phosphate that
tivity for NADPH2-TR in 10 cases; moderate usually displays strong reaction in the hepato-
activity for L-D, M-D, GLD and SoDin 3 cases; cytes in biopsy fragments (Figure l-A2). In our
strong activity for A-D in 3 cases; weak to material it was shown to be moderately active
moderate activity for a-GP-D and MAO in 3 2 hours after death and negative (Figure I-B2
cases; moderate to strong activities for J3-HOB- right) or weakly reactive with increasing fre-
D in 3 cases. All the enzymes showed moderate quency in the following periods of study.
204 Med. Sci. Law (1995) Vol. 35, NO.3

Table II. Enzyme histochemistry of the liver: data concerning 22 autopsies at different
post-mortem times (PMT)

CasePMT Temp. Sex Age Colour Major disease Liver

No. (h) (oC) (y) histopathology

01 2:00 26.0 m 77 W Necrotizing acute pancreatitis Acute congestion

02 3:00 20.0 m 40 M Bronchopneumonia + Chagas' disease Acute congestion
03 3:00 24.0 m 34 W Chronic alcoholism Steatosis
04 3:20 23.0 f 59 W Hypertensive heart disease (HHD) Chronic congestion
05 3:50 24.0 m 51 W Rattlesnake bite Steatosis
06 4:00 28.0 m 82 M Chagas' disease + HHD Chronic congestion
07 5:00 26.0 m 29 M Perforating wound of the heart No lesion
08 5:00 22.0 m 25 M Accidental polytraumatism No lesion
09 5:00 29.0 f 58 W Chagas' disease No lesion
10 6:00 28.5 m 20 W Rheumatic heart disease No lesion
11 6:30 25.0 m 38 M Chagas' disease No lesion
12 7:00 26.0 f 66 M Chagas' disease No lesion
13 8:00 28.0 m 27 W Chagas' disease Fat degeneration
14 8:00 21.5 m 48 N Hypertensive heart disease Steatosis
15 9:00 21.0 m 47 W Acute tracheobronchitis Fat degeneration
16 9:00 25.0 m 53 W Bronchopneumonia No lesion
17 10:00 18.0 m 45 W Chagas' disease Chronic congestion
18 10:00 19.0 f 57 W Hypertensive heart disease Chronic congestion
19 11:00 18.0 m 48 W Adenocarcinoma of the rectum + Chagas' disease No lesion
20 12:00 22.0 f 21 W Epilepsy No lesion
21 12:00 24.2 m 41 N Chagas' disease No lesion
22 12:00 21.0 m 64 W Acute myocardium infarct Chronic congestion

W=White; NeNegro; M=Mulatto.

a.GP-A 11 which is usually weak in the normal
hepatocyte showed negative reaction in all the
autopsy cases reported here excluding the in-
terference of a lysosomal non-specific acid
phosphatase in the G6P-A reaction. We ob-
served that the glycolytic enzyme L-D showed
stronger activity in the later post-mortem peri-
ods (Table III) than in the fresh biopsy
specimens. a-GP-D and P-HOB-D respectively
displaying weak and moderate activities in the
hepatocytes of biopsy specimens, also showed
cases with a tendency to increasing activity in
the autopsy material, especially in the later
periods (Table III). The other enzyme reactions
studied in the autopsy cases displayed the
same ranges of activity as are usually observed
in fresh material or showed minor changes
that could not be inferred as being due to the
autolysis process. These are the oxidoreduc-
tases G6P-D, NADH2-TR, NADPH2-TR, M-D,
A-D, GL-D, S-D and MAO (Table III). To con-
trol the interference ofSH-radicals in the redox
reactions, we applied the reaction of Chevre-
mont and Frederic (1943). This was positive in
the liver but did not appear to interfere with
the enzymatic activities, as demonstrated by
the wide gamut of reaction intensities that
depended exclusively on the presence of the
substrate in the incubation medium.
Pearse (1968) stated that when intra-mito-
chondrial enzyme systems are being studied,
post-mortem tissues are unsuitable for any-
thing except relatively crude studies at
histological levels. Damage to cellular mem-
branes and molecules during the autolysis
process can produce artifacts in the localiza-
tion ofthe final product of the enzyme reaction
or even false negative reactions concerning en-
zyme histochemistry. This could lead to the
misinterpretation of the results when used in
research or diagnosis. However, the loss of the
activity of enzymes could be followed during
 Mello de Oliveira & Santos-Martin: Enzyme Histochemistry of the Liver 205

Table III. Frequency and intensity of reactions in enzyme histochemistry of the liver at
different post-mortem times (PMT) studied in 22 autopsies.
= =
(2:00h I case; -,5:00h 5 cases; @8:00h 8 cases; @12:00h 8 cases) =
PMT(h) 2:00 -Q:OO -8:00 -12:00
Reactions N W M S N W M S N W M S N W M S
PHYLA a 1 5 8 8
PHYLAt 1 5 8 8
G6P-A 1 2 2 1 4 2 2 4 3 1
u.-GP-All 1 5 8 8
G6P-D 1 5 1 7 3 5
NADH2-TR 1 5 2 6 8
NADPH2-TR 1 4 1 6 2 7 1
L-D 1 1 4 1 7 3 5
M-D 1 5 7 1 7 1
A-D 1 3 2 4 4 5 3
GL-D 1 4 1 7 1 3 5
SoD 1 5 8 8
MAO 1 3 2 6 2 8
u-GP-D I 2 3 2 5 I 3 5
~-HOB-D I I 4 I 4 3 5 3

Nenegative: W=weak; Memoderate: Sestrong.

the autolysis process and could be useful in
determining the time elapsed since death. Pre-
vious reports (Goffin, 1968; Goffin and
Beeckmans, 1973; Pentilla and Ahonen, 1976)
refer to enzymes that remain active for a long
time after death, meaning that the active site
of the enzymatic protein is still operative. Gof-
fin (1968) and Goffin and Beeckmans (1973)
were unsuccessful in estimating time of death
by skin histoenzymology because all the enzy-
matic systems studied were active for a long
time in the post-mortem period studied. In-
stead, the liver which is easily accessed by
needle sampling or during autopsy, shows en-
zyme systems very sensitive to the autolytic
process as shown for PHYLA a and t and G6P-
A. Our results accord with those of Zharov and
Mel'nikova (1989) who also found enzymes
(cathepsins) to be sensitive to the autolysis
process, indicating them as criteria to be used
in estimating post-mortem time.
In summary, this work shows that histo-
chemistry can be useful to study enzyme
activities in autopsy material depending on the
choice of the tissue or the enzymatic systems
and opens interesting perspectives to approach
the medico-legal aspects of death through a
morphological method which is easily done.
The PHYLA and G6P-A reactions in the liver
which represent enzymes sensitive to the
autolysis process early in the post-mortem pe-
riod may be promising tools in estimating the
time of death and thus deserve further investi-
gation.
ACKNOWLEDGMENTS
Technical assistance provided by Miss M. P. M. Scan-
dar. This investigation received fmancial support
from: UNDPIWORLD BANKIWHO Special Program
for Research and Training in Tropical Diseases -
Process ID 780460; Conselho Nacional de Desen-
volvimento Cientifico e Tecnol6gico (CNPq), Proc. no
302333 - 82 (Area 1); Bolsa de Dedicaeao Academica
da Coordenaeao de Aperfeireoamento de Pessoal de
Nivel Superior - (CAPES).
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