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Aminopeptidase and cathepsin A activity in vitreous humor in relation to causes of death
Forensic Science International, 29 (1985) Elsevier Scientific Publishers Ireland Ltd.
171
171-178
AMINOPEPTIDASE AND CATHEPSIN A ACTIVITY HUMOR IN RELATION TO CAUSES OF DEATH
IN VITREOUS
A. LUNA, G. JIMENEZ-RIOS and E. VILLANUEVA Departumento de Medicino Granada (Spain)
Legal, Fucultud
de Medicina,
Universidad
de Granada, 18071
(Received October 10, 1984) (Revision received April 29, 1985) (Accepted July 5,1985)
Summary Brain autolysis happens rapidly, especially when environmental temperatures are high, and poses serious limitations for evaluating damage using morphologic methods. In the present study we have measured total proteins, cathepsin A and aminopeptidase activity in the vitreous humor in relation to cause of death and survival time. We have studied vitreous humor samples from 106 cadavers autopsied in the I.A.F. of Granada. The samples were classified according to causes of death as follows: myocardial infarction; hanging; other mechanical asphyxias; multiple trauma; craniocerebral trauma; other violent death; pulmonary functions; and other natural deaths. Total protein was measured by Lowry’s method, and cathepsin A and aminopeptidase activity by the Bowen and Davison (1973) and Greenberg (1962) methods, respectively. Results are expressed in IU/l and in mIU/mg of protein. We found higher values of aminopeptidase and cathepsin A activity in groups with severe brain damage (craniocerebral trauma, multiple trauma, etc.) and lower values in groups of natural deaths. We believe, therefore, that aminopeptidase activity in vitreous humor may be a useful parameter for evaluating brain damage. Key words: Cathepsin A; Aminopeptidase;
Vitreous humor; Thanatochemistry
Introduction Morphological methods for the postmortem study of brain injury are usually adequate, however, in certain circumstances (brain autolysis, fixation artifacts, etc.) it is difficult to establish and the other main problem during the autopsy may be to establish the vital character of brain injuries. The hypothesis of the present paper is that brain damage can be studied by the aminopeptidase and cathepsin A activity in the vitreous humor. The value of postmortem biochemical determinations in vitreous humor has been well demonstrated by many authors [l-6]. Vitreous humor is anatomically well protected, resists putrefaction better than other body fluids, postmortem contamination is limited by the absence of cellular constituents in suspension, and finally, vitreous humor is easly accessible for sampling immediately 0379-0738/85/$03.30
o 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
172
following death. Lactic acid concentrations in vitreous humor are related to variations in agonal time differences and may be a reflection of different causes of death [7]. It has been found [8] that hypoxanthine levels in vitreous humor may reflect the induced effect of prolonged tissue hypoxia caused by respiratory depression. Materials and methods One hundred six vitreous humor samples from 106 cadavers autopsied in I.A.F. of Granada were studied. Samples were obtained with a sterile syringe in all cases. Vitreous humor was aspirated from the posterior chamber of the eye via the external lateral angle. In ten cadavers two samples from each eye were taken at different postmortem intervals. The samples were frozen at -30°C until the moment of assay. Proteins were measured by Lowry’s method using a Spectrophotometer Beckman mod. 25. Aminopeptidase activity was measured by Greenberg’s method [9] using a Perkin-Elmer mod. MPF 25 Spectrofluorimeter (Ext. 345 nm; Em 412 nm). The final concentration in the reaction mixture was 2 mM leucine+naphthylamide in 0.1 M phosphate buffer at pH 7.4 (containing 2 mM dithiothreitol and 0.1% bovine albumin (v/v), 1 ml and 50 ~1 vitreous humor). Incubation time was 30 min at 37°C. Cathepsin A activity was measured by the Bowen and Davison method [lo] in the same spectrofluorimeter (Exe 390 nm, Em 480 nm). The final concentration in the reaction mixture was 0.002 M 0.2 ml N-carbobenzoxy-L-glutamyl-L-tyrosine, 0.75 M (pH 5) 0.2 ml sodium acetate buffer, and 100 ~1 vitreous humor. Incubation time was 1 h at 37°C. Samples were classified according to the cause of death as follows: Group 1, myocardial infarction; Group 2, pulmonary function; Group 3, other natural deaths; Group 4, craniocerebral traumas; Group 5, multiple trauma; Group 6, hanging; Group 7, other violent asphyxias; and Group 8, other violent deaths. The third group (other natural deaths) included: Cirrhosis of the liver Lung cancer Myocarditis Renal failure Pancreatitis Septic shock
2 2 1 2 1 1
cases cases case cases case case
The seventh group (other violent asphyxias) consisted of: Carbon monoxide poisoning Homicidal strangulation
6 cases 2 cases
173
Crush asphyxias Drowning
2 cases 4 cases
The eighth group (other violent deaths)
included:
Acute hemorrhage caused by bullet wound to the heart (gunshot homicide) 1 case Acute hemorrhage from stab wounds 5 cases Multiple burns 2 cases Post-traumatic fat embolism 1 case Renal failure following injury 1 case Septic shock secondary to peritoneal injury 1 case Intoxications:
Lye
2 1 1 1 1
Salicylates Reserprine Ethyl alcohol Barbiturates
cases case case case case
Statistical analysis was carried out using one way ANOVA. Table 1 shows the postmortem interval and the age characteristics of the total sample. Results Results
for aminopeptidase
activity
are expressed
in IU/l and mIU/mg
TABLE 1 TOTAL SAMPLE CHARACTERISTICS DATA N
Myocardial infarction Pulmonary functions Other natural deaths Craniocerebral trauma Multiple trauma Hanging Other violent asphyxias Other violent deaths a Expressed in hours. b Expressed in years.
12 10 9 14 23 10 12 17
IN RELATION
Postmortem
TO AGE AND POSTMORTEM
intervala
Ageb
Mean
SD.
Mean
S.D.
13.41 15.50 15.00 14.25 13.19 18.20 29.04 17.88
8.071 6.096 9.192 5.075 7.554 9.028 31.543 14.401
54.50 56.30 44.27 57.14 46.26 59.10 45.42 39.94
24.56 29.531 25.27 25.839 20.033 10.159 20.951 22.827
174
protein (protein specific activity) (see Table 3). Cathepsin A activity (see Table 2) is expressed in fluorescence units/liter and mg protein. We define 1 unit of fluorescence as the amount of enzyme capable of producing a l-cm rise on the graph paper, with the fluorimeter at sensitivity 3 (PerkinElmer recorder, model 561, potential out signal of 1 mV) in 1 min. Discussion We found in our study a positive correlation between vitreous humor activity of aminopeptidase and cathepsin A (r = 0.249, 94 d.f.; T exp = 2.494; Sig P < 0.05) with the highest values noted in the craniocerebral trauma and multiple trauma groups, followed by other violent asphyxias (group 6). Lower levels were noted in pulmonary functions (group 2) and other natural deaths (group 3) (Figs. 1 and 2). There were no statistically significant differences in the cathepsin A values. The most accurate vitreous humor enzymatic activity expression is referred to as protein content (protein specific activity). We have found significant differences in the aminopeptidase activity of the groups noted in Table 4. The large standard variations found can be explained by the different factor that plays a role in enzymatic release aside from the cause of death: time evolved since injuries, intensity of biological stress and basal situation. The time between death and postmortem examination does not cause QJF.U./l VITREOUS
25
HUMOR
CATHEPSIN
A ACTIVITY
~mF.U./mg
proten
I
F.U./l
25
20
15
10
5
0 GROUP
1
GROUP
2
GROUP
3
GROUP4
GR
5
GROUP6
GROUP
7
GROUP8
Fig. 1. The vitreous humor cathepsin A activity in different causes of death is shown (myocardial infarction; pulmonary functions; other natural deaths; craniocerebral trauma; multiple trauma; hanging; other violent asphyxias; and other violent deaths).
175
VITREOUS
HUMOR
AMINOPEPTIDASE
ACTIVITY 1
30
25
20
30
15
20 -
10
10
0' GROUP1
GROUP2
GROUP3
GROUP4
GROUP5
GROUP6
GROUP
7
GROUPB
Fig. 2. The vitreous humor aminopeptidase activity in different causes of death is shown (myocardiai infarction; pulmonary processes; other natural deaths; craniocerebrai trauma; multiple trauma; hanging; other natural asphyxias; other violent deaths).
significant variations in the results in the first 48 h, though a rise in enzymatic activity was noted in the ten cadavers on which two sequential postmortem samplings were taken (Figs. 3 and 4). Intracellular release of cathepsins [lo] from lysosomes has been demonstrated during myocardial ischemia with a release into the extracellular space; the activity of acid and alkaline phosphatase and acid cathepsin in TABLE 2 VITREOUS HUMOR CATHEPSIN A ACTIVITY IN THE DIFFERENT CAUSES OF DEATH EXPRESSED IN FLUORESCENCE UNITS/LITER AND IN PROTEIN ESPECIFIC ACTIVITY N
FU/l Mean
Myocardial infarction Pulmonary functions Other natural deaths Craniocerebral trauma Multiple trauma Hanging Other violent asphyxias Other violent deaths
12 10 8 11 20 9 12 17
9.083
13.3 13.687 17.136 19.825 15.5 20.625 22.47
mFU/mgprotein S. D. 5.927 3.649
5.483 10.032 16.956 18.941 16.29 25.036
Mean
S. D.
7.647 11.6 9.21 16.69 17.096 13.87 19.52 20.845
16.081 4.076 14.781 13.609 18.454 20.506 29.802
4.459
176 TABLE 3 VITREOUS HUMOR AMINOPEPTIDASE ACTIVITY IN THE DIFFERENT CAUSES OF DEATH EXPRESSED IN INTERNATIONAL UNITS/LITER AND IN PROTEIN SPECIFIC ACTIVITY N
Mean -.
Myocardial infarction Pulmonary functions Other natural deaths Craniocerebral trauma Multiple trauma Hanging Other violent asphyxias Other violent deaths
12 10 9 14 23 10 11 17
mIU/mg
W/l
17.91 15.73 10.19 36.28 29.86 17.116 27.65 18.874
protein
SD.
Mean
S. D.
20.98 12.21 10.61 32.036 27.288 12.389 27.82 14.44
12.32 9.55 6.98 23.73 21.72 12.69 20.04 14.94
7.054 7.016 7.536 18.904 15.82 9.51 15.016 9.162
subcellular fractions of gray matter of dog brain rises proportionally to the length of ischemia [12]. Cathepsin A may play a role in the turnover of selected hormonal peptides containing c-terminal neutral amino acids and in the sequential breakdown of protein associated with degenerative conditions such as demyelination [ 131. Cathepsin A (catheptic carboxypeptidase) is present in lysosomal brain fraction [14]. Many enzymes with aminopeptidase activity have been characterized: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminoencephalinase, cytosolic aminoencephalinase, leucineaminopeptidase and angiotensinconverting enzyme in brain and peripheral nerves [15]. The enzymes with aminopeptidase activity are more concentrated in the white matter than
TABLE 4 STATISTICAL ANALYSIS OF THE VITREOUS VITY EXPRESSED IN mIU/mg PROTEIN
HUMOR AMINOPEPTIDASE
T exp
Pulmonary functions Pulmonary functions Pulmonary functions Pulmonary functions Other natural deaths Other natural deaths Other natural deaths
-
craniocerebral trauma multiple trauma other violent asphyxias other violent deaths craniocerebral trauma multiple trauma other natural asphyxias
3.066 2.679 2.269 2.086 2.93 2.51 2.065
ACTI-
P
< 0.01 < < < < < <
0.01 0.05 0.05 0.01 0.05 0.05
Number = 106; F exp = 2.789; d.f. = 7.98; In variance = 163.368; 455.757.
Between variance =
AMINOPEPTIDASE 60
1
MULTIPLE
ACTIVITY
TRAUMA
177
0
mI.U./mg proten
. 0
e
0 . D
I ..
. .
..I.............
I...
5
0 POSTMORTEM
10
15
-
I....,.
20
25
30
hours
INTERVAL
Fig. 3. Different values of vitreous humor aminopeptidase activity in the multiple trauma group classified in order of the postmortem interval. AMINOPEPTIDASE mI.U./my
ACTIVITY
protein *
5:
5c
25
2
p
e---
*
_*--
*’
I
,’
I’
1 5-
,9
I’
,’ 1, 3-
dr
, ,’ ,’
9
,’
I I
I’
, X0
,,’
,*
,
,,’
/’
*’
._*’
,’
,’
,’
,
**
/’
,’
?--*
,*’ ,’
_.**
,’
,‘
.-’
i’ .”
,’ _*
I*
5’
, 5 POSTMORTEM
-
10
15
70
25
30 hrs.
INTERVAL
Fig. 4. Vitreous humor aminopeptidase activity in two sequential,samples at different postmortem intervals in ten cadavers. Each sample was obtained from both eyes.
178
in the grey, and play an important role in the metabolic processes of retinal membranes. Though the oscure physiopathology of vitreous humor does not permit an accurate analysis of the results, the retinal membrane-like constituents of the central nervous system could explain, through the release of the different substances in the vitreous humor, the brain damage intensity, with an increased enzyme liberation. We believe that the aminopeptidase activity measured in vitreous humor may be a useful parameter for the postmortem study of brain damage. References 1 J.L. Coe, Postmortem chemistries on human vitreous humor. Am. J Clin. Pathol., 51 (1969) 741-750. 2 J.L. Coe, Postmortem chemistry of blood cerebrospinal fluid and vitreous humor. In C.G. Tedeschi, W.G. Eckert and L.G. Tedeschi feds.), Forensic Medicine, Vol. II, W.B. Saunders Company, Philadelphia, 1977, pp. 1033-1060. 3 W.Q. Sturner, A.B.C. Dowdey, R.S. Putnam and J.L. Dempsey, Osmolality and other chemical determinations on postmortem human vitreous humor. J. Forensic Sci., 17 * (1972) 387-393. 4 W.Q. Sturner and J. Dempsey, Sudden infant death: Chemical analysis of vitreous humor. J. Forensic Sci., 18 (1973) 12-19. 5 W.Q. Sturner, Postmortem vitreous humor analysis: A review of forensic applications. Forensic Sci. Gaszete, 3 (1972) l-4. 6 E. Villanueva, Progresos Tecnicos de la autopsia medico-legal y de SUBexamenes complementarios: TCnicas bioqufmicas. Acta J. Mediterrbneas Med. Legal, 1 (1977) 4570 (VaIencia). 7 W.Q. Sturner et al., Lactic acid concentration in vitreous humor: their use in asphyxiaI deaths in children. J. Forensic Sci., 28 (1983) 222-230. 8 O.D. Sangstad and B. Olassen, Postmortem hypoxanthine levels in the vitreous humor. An introductory report. Nor. Forensic Sci., 12 (1978) 33-36. 9 L.J. Greenberg, Fluorimetric measurement of alkaline phosphatase and aminopeptidase activities in the order of 10-l’ moles. Biochem. Biophys. Res. Commun., 9 (1962) 430-435. 10 D.M. Bowen and A.N. Davison, Cathepsin A in human brain and splee. Biochem. J., 131 (1973) 417-419. 11 A.R. Poole and J.S. Mort, Biochemical and inmundogical studies of lysosomal and related proteinases in health and disease. J. Histochem. Cytochem., 23 (1981) 494500. 12 L.V. Molchanova and V.L. Kozhura, Changes in phosphatase and acid cathepsin activity in the cerebral cortex of dogs in terminal states. Bull. Esp. Biol. Med., 80( 12) (1976) 1419-1421. 13 A. Grynbaum and N. Marks, Characterization of a rat brain cathepsin carboxypeptidase (Cathepsin A) inactivating angiotensin II. J. Neurochem., 26 (1976) 313-318. 14 C.W.M. Adams and A.N. Davison, The myelin seath. In C.W.M. Adams (ed.), NeuroElsevier Publishing Company, Amsterdam, London, New York, histochemistry, 1965, p. 367. 15 Hui Koon-Sea, M. Hui and A. Lajtha, Properties of a brain membrane aminoenkephalinase: Inhibition studies. In G.Y. Sun, N. Bazan, Wu Jang-Yen, G. Porcellati and A.Y. Sun, Neural Membranes, Human Press, Clifton, New Jersey, 1983, pp. 375-393.
171
171-178
AMINOPEPTIDASE AND CATHEPSIN A ACTIVITY HUMOR IN RELATION TO CAUSES OF DEATH
IN VITREOUS
A. LUNA, G. JIMENEZ-RIOS and E. VILLANUEVA Departumento de Medicino Granada (Spain)
Legal, Fucultud
de Medicina,
Universidad
de Granada, 18071
(Received October 10, 1984) (Revision received April 29, 1985) (Accepted July 5,1985)
Summary Brain autolysis happens rapidly, especially when environmental temperatures are high, and poses serious limitations for evaluating damage using morphologic methods. In the present study we have measured total proteins, cathepsin A and aminopeptidase activity in the vitreous humor in relation to cause of death and survival time. We have studied vitreous humor samples from 106 cadavers autopsied in the I.A.F. of Granada. The samples were classified according to causes of death as follows: myocardial infarction; hanging; other mechanical asphyxias; multiple trauma; craniocerebral trauma; other violent death; pulmonary functions; and other natural deaths. Total protein was measured by Lowry’s method, and cathepsin A and aminopeptidase activity by the Bowen and Davison (1973) and Greenberg (1962) methods, respectively. Results are expressed in IU/l and in mIU/mg of protein. We found higher values of aminopeptidase and cathepsin A activity in groups with severe brain damage (craniocerebral trauma, multiple trauma, etc.) and lower values in groups of natural deaths. We believe, therefore, that aminopeptidase activity in vitreous humor may be a useful parameter for evaluating brain damage. Key words: Cathepsin A; Aminopeptidase;
Vitreous humor; Thanatochemistry
Introduction Morphological methods for the postmortem study of brain injury are usually adequate, however, in certain circumstances (brain autolysis, fixation artifacts, etc.) it is difficult to establish and the other main problem during the autopsy may be to establish the vital character of brain injuries. The hypothesis of the present paper is that brain damage can be studied by the aminopeptidase and cathepsin A activity in the vitreous humor. The value of postmortem biochemical determinations in vitreous humor has been well demonstrated by many authors [l-6]. Vitreous humor is anatomically well protected, resists putrefaction better than other body fluids, postmortem contamination is limited by the absence of cellular constituents in suspension, and finally, vitreous humor is easly accessible for sampling immediately 0379-0738/85/$03.30
o 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
172
following death. Lactic acid concentrations in vitreous humor are related to variations in agonal time differences and may be a reflection of different causes of death [7]. It has been found [8] that hypoxanthine levels in vitreous humor may reflect the induced effect of prolonged tissue hypoxia caused by respiratory depression. Materials and methods One hundred six vitreous humor samples from 106 cadavers autopsied in I.A.F. of Granada were studied. Samples were obtained with a sterile syringe in all cases. Vitreous humor was aspirated from the posterior chamber of the eye via the external lateral angle. In ten cadavers two samples from each eye were taken at different postmortem intervals. The samples were frozen at -30°C until the moment of assay. Proteins were measured by Lowry’s method using a Spectrophotometer Beckman mod. 25. Aminopeptidase activity was measured by Greenberg’s method [9] using a Perkin-Elmer mod. MPF 25 Spectrofluorimeter (Ext. 345 nm; Em 412 nm). The final concentration in the reaction mixture was 2 mM leucine+naphthylamide in 0.1 M phosphate buffer at pH 7.4 (containing 2 mM dithiothreitol and 0.1% bovine albumin (v/v), 1 ml and 50 ~1 vitreous humor). Incubation time was 30 min at 37°C. Cathepsin A activity was measured by the Bowen and Davison method [lo] in the same spectrofluorimeter (Exe 390 nm, Em 480 nm). The final concentration in the reaction mixture was 0.002 M 0.2 ml N-carbobenzoxy-L-glutamyl-L-tyrosine, 0.75 M (pH 5) 0.2 ml sodium acetate buffer, and 100 ~1 vitreous humor. Incubation time was 1 h at 37°C. Samples were classified according to the cause of death as follows: Group 1, myocardial infarction; Group 2, pulmonary function; Group 3, other natural deaths; Group 4, craniocerebral traumas; Group 5, multiple trauma; Group 6, hanging; Group 7, other violent asphyxias; and Group 8, other violent deaths. The third group (other natural deaths) included: Cirrhosis of the liver Lung cancer Myocarditis Renal failure Pancreatitis Septic shock
2 2 1 2 1 1
cases cases case cases case case
The seventh group (other violent asphyxias) consisted of: Carbon monoxide poisoning Homicidal strangulation
6 cases 2 cases
173
Crush asphyxias Drowning
2 cases 4 cases
The eighth group (other violent deaths)
included:
Acute hemorrhage caused by bullet wound to the heart (gunshot homicide) 1 case Acute hemorrhage from stab wounds 5 cases Multiple burns 2 cases Post-traumatic fat embolism 1 case Renal failure following injury 1 case Septic shock secondary to peritoneal injury 1 case Intoxications:
Lye
2 1 1 1 1
Salicylates Reserprine Ethyl alcohol Barbiturates
cases case case case case
Statistical analysis was carried out using one way ANOVA. Table 1 shows the postmortem interval and the age characteristics of the total sample. Results Results
for aminopeptidase
activity
are expressed
in IU/l and mIU/mg
TABLE 1 TOTAL SAMPLE CHARACTERISTICS DATA N
Myocardial infarction Pulmonary functions Other natural deaths Craniocerebral trauma Multiple trauma Hanging Other violent asphyxias Other violent deaths a Expressed in hours. b Expressed in years.
12 10 9 14 23 10 12 17
IN RELATION
Postmortem
TO AGE AND POSTMORTEM
intervala
Ageb
Mean
SD.
Mean
S.D.
13.41 15.50 15.00 14.25 13.19 18.20 29.04 17.88
8.071 6.096 9.192 5.075 7.554 9.028 31.543 14.401
54.50 56.30 44.27 57.14 46.26 59.10 45.42 39.94
24.56 29.531 25.27 25.839 20.033 10.159 20.951 22.827
174
protein (protein specific activity) (see Table 3). Cathepsin A activity (see Table 2) is expressed in fluorescence units/liter and mg protein. We define 1 unit of fluorescence as the amount of enzyme capable of producing a l-cm rise on the graph paper, with the fluorimeter at sensitivity 3 (PerkinElmer recorder, model 561, potential out signal of 1 mV) in 1 min. Discussion We found in our study a positive correlation between vitreous humor activity of aminopeptidase and cathepsin A (r = 0.249, 94 d.f.; T exp = 2.494; Sig P < 0.05) with the highest values noted in the craniocerebral trauma and multiple trauma groups, followed by other violent asphyxias (group 6). Lower levels were noted in pulmonary functions (group 2) and other natural deaths (group 3) (Figs. 1 and 2). There were no statistically significant differences in the cathepsin A values. The most accurate vitreous humor enzymatic activity expression is referred to as protein content (protein specific activity). We have found significant differences in the aminopeptidase activity of the groups noted in Table 4. The large standard variations found can be explained by the different factor that plays a role in enzymatic release aside from the cause of death: time evolved since injuries, intensity of biological stress and basal situation. The time between death and postmortem examination does not cause QJF.U./l VITREOUS
25
HUMOR
CATHEPSIN
A ACTIVITY
~mF.U./mg
proten
I
F.U./l
25
20
15
10
5
0 GROUP
1
GROUP
2
GROUP
3
GROUP4
GR
5
GROUP6
GROUP
7
GROUP8
Fig. 1. The vitreous humor cathepsin A activity in different causes of death is shown (myocardial infarction; pulmonary functions; other natural deaths; craniocerebral trauma; multiple trauma; hanging; other violent asphyxias; and other violent deaths).
175
VITREOUS
HUMOR
AMINOPEPTIDASE
ACTIVITY 1
30
25
20
30
15
20 -
10
10
0' GROUP1
GROUP2
GROUP3
GROUP4
GROUP5
GROUP6
GROUP
7
GROUPB
Fig. 2. The vitreous humor aminopeptidase activity in different causes of death is shown (myocardiai infarction; pulmonary processes; other natural deaths; craniocerebrai trauma; multiple trauma; hanging; other natural asphyxias; other violent deaths).
significant variations in the results in the first 48 h, though a rise in enzymatic activity was noted in the ten cadavers on which two sequential postmortem samplings were taken (Figs. 3 and 4). Intracellular release of cathepsins [lo] from lysosomes has been demonstrated during myocardial ischemia with a release into the extracellular space; the activity of acid and alkaline phosphatase and acid cathepsin in TABLE 2 VITREOUS HUMOR CATHEPSIN A ACTIVITY IN THE DIFFERENT CAUSES OF DEATH EXPRESSED IN FLUORESCENCE UNITS/LITER AND IN PROTEIN ESPECIFIC ACTIVITY N
FU/l Mean
Myocardial infarction Pulmonary functions Other natural deaths Craniocerebral trauma Multiple trauma Hanging Other violent asphyxias Other violent deaths
12 10 8 11 20 9 12 17
9.083
13.3 13.687 17.136 19.825 15.5 20.625 22.47
mFU/mgprotein S. D. 5.927 3.649
5.483 10.032 16.956 18.941 16.29 25.036
Mean
S. D.
7.647 11.6 9.21 16.69 17.096 13.87 19.52 20.845
16.081 4.076 14.781 13.609 18.454 20.506 29.802
4.459
176 TABLE 3 VITREOUS HUMOR AMINOPEPTIDASE ACTIVITY IN THE DIFFERENT CAUSES OF DEATH EXPRESSED IN INTERNATIONAL UNITS/LITER AND IN PROTEIN SPECIFIC ACTIVITY N
Mean -.
Myocardial infarction Pulmonary functions Other natural deaths Craniocerebral trauma Multiple trauma Hanging Other violent asphyxias Other violent deaths
12 10 9 14 23 10 11 17
mIU/mg
W/l
17.91 15.73 10.19 36.28 29.86 17.116 27.65 18.874
protein
SD.
Mean
S. D.
20.98 12.21 10.61 32.036 27.288 12.389 27.82 14.44
12.32 9.55 6.98 23.73 21.72 12.69 20.04 14.94
7.054 7.016 7.536 18.904 15.82 9.51 15.016 9.162
subcellular fractions of gray matter of dog brain rises proportionally to the length of ischemia [12]. Cathepsin A may play a role in the turnover of selected hormonal peptides containing c-terminal neutral amino acids and in the sequential breakdown of protein associated with degenerative conditions such as demyelination [ 131. Cathepsin A (catheptic carboxypeptidase) is present in lysosomal brain fraction [14]. Many enzymes with aminopeptidase activity have been characterized: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminoencephalinase, cytosolic aminoencephalinase, leucineaminopeptidase and angiotensinconverting enzyme in brain and peripheral nerves [15]. The enzymes with aminopeptidase activity are more concentrated in the white matter than
TABLE 4 STATISTICAL ANALYSIS OF THE VITREOUS VITY EXPRESSED IN mIU/mg PROTEIN
HUMOR AMINOPEPTIDASE
T exp
Pulmonary functions Pulmonary functions Pulmonary functions Pulmonary functions Other natural deaths Other natural deaths Other natural deaths
-
craniocerebral trauma multiple trauma other violent asphyxias other violent deaths craniocerebral trauma multiple trauma other natural asphyxias
3.066 2.679 2.269 2.086 2.93 2.51 2.065
ACTI-
P
< 0.01 < < < < < <
0.01 0.05 0.05 0.01 0.05 0.05
Number = 106; F exp = 2.789; d.f. = 7.98; In variance = 163.368; 455.757.
Between variance =
AMINOPEPTIDASE 60
1
MULTIPLE
ACTIVITY
TRAUMA
177
0
mI.U./mg proten
. 0
e
0 . D
I ..
. .
..I.............
I...
5
0 POSTMORTEM
10
15
-
I....,.
20
25
30
hours
INTERVAL
Fig. 3. Different values of vitreous humor aminopeptidase activity in the multiple trauma group classified in order of the postmortem interval. AMINOPEPTIDASE mI.U./my
ACTIVITY
protein *
5:
5c
25
2
p
e---
*
_*--
*’
I
,’
I’
1 5-
,9
I’
,’ 1, 3-
dr
, ,’ ,’
9
,’
I I
I’
, X0
,,’
,*
,
,,’
/’
*’
._*’
,’
,’
,’
,
**
/’
,’
?--*
,*’ ,’
_.**
,’
,‘
.-’
i’ .”
,’ _*
I*
5’
, 5 POSTMORTEM
-
10
15
70
25
30 hrs.
INTERVAL
Fig. 4. Vitreous humor aminopeptidase activity in two sequential,samples at different postmortem intervals in ten cadavers. Each sample was obtained from both eyes.
178
in the grey, and play an important role in the metabolic processes of retinal membranes. Though the oscure physiopathology of vitreous humor does not permit an accurate analysis of the results, the retinal membrane-like constituents of the central nervous system could explain, through the release of the different substances in the vitreous humor, the brain damage intensity, with an increased enzyme liberation. We believe that the aminopeptidase activity measured in vitreous humor may be a useful parameter for the postmortem study of brain damage. References 1 J.L. Coe, Postmortem chemistries on human vitreous humor. Am. J Clin. Pathol., 51 (1969) 741-750. 2 J.L. Coe, Postmortem chemistry of blood cerebrospinal fluid and vitreous humor. In C.G. Tedeschi, W.G. Eckert and L.G. Tedeschi feds.), Forensic Medicine, Vol. II, W.B. Saunders Company, Philadelphia, 1977, pp. 1033-1060. 3 W.Q. Sturner, A.B.C. Dowdey, R.S. Putnam and J.L. Dempsey, Osmolality and other chemical determinations on postmortem human vitreous humor. J. Forensic Sci., 17 * (1972) 387-393. 4 W.Q. Sturner and J. Dempsey, Sudden infant death: Chemical analysis of vitreous humor. J. Forensic Sci., 18 (1973) 12-19. 5 W.Q. Sturner, Postmortem vitreous humor analysis: A review of forensic applications. Forensic Sci. Gaszete, 3 (1972) l-4. 6 E. Villanueva, Progresos Tecnicos de la autopsia medico-legal y de SUBexamenes complementarios: TCnicas bioqufmicas. Acta J. Mediterrbneas Med. Legal, 1 (1977) 4570 (VaIencia). 7 W.Q. Sturner et al., Lactic acid concentration in vitreous humor: their use in asphyxiaI deaths in children. J. Forensic Sci., 28 (1983) 222-230. 8 O.D. Sangstad and B. Olassen, Postmortem hypoxanthine levels in the vitreous humor. An introductory report. Nor. Forensic Sci., 12 (1978) 33-36. 9 L.J. Greenberg, Fluorimetric measurement of alkaline phosphatase and aminopeptidase activities in the order of 10-l’ moles. Biochem. Biophys. Res. Commun., 9 (1962) 430-435. 10 D.M. Bowen and A.N. Davison, Cathepsin A in human brain and splee. Biochem. J., 131 (1973) 417-419. 11 A.R. Poole and J.S. Mort, Biochemical and inmundogical studies of lysosomal and related proteinases in health and disease. J. Histochem. Cytochem., 23 (1981) 494500. 12 L.V. Molchanova and V.L. Kozhura, Changes in phosphatase and acid cathepsin activity in the cerebral cortex of dogs in terminal states. Bull. Esp. Biol. Med., 80( 12) (1976) 1419-1421. 13 A. Grynbaum and N. Marks, Characterization of a rat brain cathepsin carboxypeptidase (Cathepsin A) inactivating angiotensin II. J. Neurochem., 26 (1976) 313-318. 14 C.W.M. Adams and A.N. Davison, The myelin seath. In C.W.M. Adams (ed.), NeuroElsevier Publishing Company, Amsterdam, London, New York, histochemistry, 1965, p. 367. 15 Hui Koon-Sea, M. Hui and A. Lajtha, Properties of a brain membrane aminoenkephalinase: Inhibition studies. In G.Y. Sun, N. Bazan, Wu Jang-Yen, G. Porcellati and A.Y. Sun, Neural Membranes, Human Press, Clifton, New Jersey, 1983, pp. 375-393.