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Post-mortem hypoxanthine levels in the vitreous humour an introductory report
Forensic Science International, 12 (1978) 33 - 36 @ Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
POST-MORTEM MOUR
HYPOXANTHINE
AN INTRODUCTORY
LEVELS
IN THE
33
VITREOUS
HU-
REPORT
0. D. SAUGSTAD and B. OLAISEN Institute for Surgical (Norway)
Research
(Received November 23,1977;
and Institute
of Forensic
Medicine,
University
of Oslo
accepted July 18, 1978)
Summary Post-mortem hypoxanthine levels in vitreous humour were determined in 86 consecutive legal autopsy cases. In cases of sudden death caused by trauma or by myocardial infarction, levels ranging from 0 to 540 ymol/l were found. The mean value was about ten times higher than normal in vivo plasma levels. The hypoxanthine levels seem to be independent of time post-mortem, at least during the first 48 hours. It is known that augmentation of the hypoxanthine plasma, cerebrospinal fluid, and urine levels reflects tissue hypoxia. In the present material no elevation of hypoxanthine levels in the vitreous humour was found in cases of strangulation or suspension, while statistically significant elevation was found in cases of drug intoxication. It is concluded that this may reflect the effect of drug-induced prolonged tissue hypoxia caused by respiratory depression.
Introduction In the last few years it has been extensively documented that during tissue hypoxia hypoxanthine levels in plasma [ 11, cerebrospinal fluid [2] and urine [3] are raised. It is therefore reasonable to suggest that elevation of hypoxanthine concentrations during tissue hypoxia occurs in other body fluids as well. The vitreous humour of the eye mediates transport to and from the retina and, apart from changes caused by this process (for instance low glucose and high lactic acid levels), its composition is quite similar to that of aqueous fluid, cerebrospinal fluid and serum. Vitreous humour is easy to sample post mortem, and is relatively free from contamination by blood, bacteria and products of post-mortem autolysis. Changes in the concentration of several substances in the vitreous humour are known to occur postmortem, however. The aim of the present study was to investigate post-mortem hypoxanthine levels in vitreous humour and in particular to determine whether these levels might reflect the degree of tissue hypoxia preceding death.
34
Materials
and methods
Eighty-six consecutive legal autopsy cases (3 months to 80 years of age) comprise the material for this study. The cases were divided into five groups. Group 1: severe trauma leading to immediate death (23 cases). Group 2: short-lasting hypoxia by strangulation or suspension (10 cases). Group 3: sudden death from myocardial infarction (19 cases). Group 4: fatal drug intoxication (13 cases). Group 5: others, including cases of bronchopneumonia, gastric haemorrhage, brain catastrophies, drowning and carbon monoxide poisoning (21 cases). Toxicological analyses were performed at the National Institute of Forensic Toxicology. Vitreous humour was obtained 0.5 - 192 hours post mortem using 4 ml vacutainers. The samples were then immediately stored at -20 “C until analysed for hypoxanthine within a few weeks [4]. Since vitreous humour hypoxanthine levels were considerably higher than those of normal plasma 141, the samples were diluted 20 times before analysis. In six cases samples of vitreous humour from both eyes were examined, and in some cases several analyses were made of the same samples after different storage times.
Results The post-mortem hypoxanthine concentrations in vitreous humour samples from groups 1 - 4 are given as histograms in Fig. 1. In group 1 (severe trauma) the hypoxanthine concentrations varied between 0 and 480 pmol/l with a mean of 136 pmol/l. In group 2 (suspension or strangulation) the values ranged between 0 and 300 pmol/l (mean 129 pmol/l). In group 3 (myocardial infarction) hypoxanthine levels between 0 and 540 I*mol/l (mean 143 pmol/l) were found. There were no statistically significant differences between the hypoxanthine levels of these three groups. In group 4 (intoxication) the hypoxanthine concentrations in the vitreous humour ranged between 60 and 1260 pmol/l (mean 363 pmol/l). Compared to the results of groups 1 - 3 these values were significantly higher (P < 0.01, with Wilcoxon rank test for two samples). In group 5 (others) the hypoxanthine values ranged between 0 and 840 pmol/l with a mean of 156 pmol/l. The correlation between the time post mortem when the samples were obtained and hypoxanthine values for all cases are plotted in Fig. 2. By linear regression analyses a correlation coefficient of +0.09 (N.S.) between hypoxanthine concentrations and post-mortem time during the first 48 hours was found. When post-mortem times from 0 to 90 hours were considered, the correlation coefficient increased to +0.28 (P < 0.05), and was further increased when post-mortem times from 0 to 192 hours were considered.
35
Trauma Mean:
136 gmol/l
Infarctus Mean:
cordis
143 pmol/l
Intoxication N= 13
nb 3 2 \
363 gmol/l 0
120 240 360 480 MI0 )6M)
Hypoxanthine
,,,,,,,,
10
20
30
LO
50
(pmol/l)
Fig. 1. Distribution of the hypoxanthine samples from groups 1 - 4.
concentrations
Fig. 2. Correlation between hypoxanthine concentrations time post mortem when the samples were obtained.
in post-mortem
in vitreous
60
‘+
70 80 9Oz Hours post mortem
vitreous
humour
humour
and the
Hypoxanthine values obtained in samples from both eyes did not show statistically significant deviations, neither were there any changes in hypoxanthine levels as a result of storage at -20 “C (up to 20 weeks).
Discussion The normal hypoxanthine concentration in vitreous humour in the living body is not known. It is reasonable to assume, however, that the values found in cases of sudden death reflect the levels in uiuo. In this study we have therefore regarded the concentrations in the vitreous humour of individuals from group 1 (severe trauma) and group 3 (myocardial infarction) as reference values. The normal hypoxanthine concentration in vitreous humour thus appears to range between 0 and 540 pmol/l. The mean value found is lo- to 20-times higher than that in plasma [4]. In the reference material no statistically significant differences in hypoxanthine concentrations were found in the samples obtained more than 48 hours post mortem when compared to samples obtained 0 - 48 hours post mortem. In the total material a positive correlation was found between
36
hypoxanthine concentrations and the post-mortem sampling time when this exceeded 48 hours. However, this is obviously caused by the fact that the time interval from death to autopsy tended to be high in the individuals who had died of intoxication. In patients of groups 1 and 3 circulatory arrest occurred suddenly, probably with no or only minor preceding tissue hypoxia. Compared to this reference group, no elevated hypoxanthine levels were found in patients from group 2, who had died by suspension or strangulation. Although tissue hypoxia in such patients probably preceded circulatory arrest, this period may have been of too short a duration for hypoxanthine to accumulate in the vitreous fluid. The drugs found in group 4 cases were barbiturates, methadone, ketobemidone, mebrobamate and dextropropoxiphen, either alone, in combination, or together with non-lethal ethanol concentrations. These drugs are known to cause respiratory depression. The significant elevation of hypoxanthine values, compared to the reference group, found in patients dying of intoxication can be explained by respiratory depression preceding death. In these patients tissue hypoxia may have occurred for rather a long period before circulatory arrest. Hence accumulation of hypoxanthine in the vitreous humour could be presumed. Obviously the hypoxanthine levels are a reflection of the degree and duration of tissue hypoxia. The significance of each of these factors is impossible to estimate in this study, however. The present data indicate that determination of the hypoxanthine concentration in vitreous humour post mortem may give information about whether or not tissue hypoxia preceded circulatory arrest.
Acknowledgement During this study 0. D. Saugstad was a research wegian Research Council for Science and the Humanities.
fellow
of the Nor-
References 0. D. Saugstad, Hypoxanthine as a measurement of hypoxia, Pediat. Res., 9 (1975) 158 - 161. 0. D. Saugstad, H. Schrader and A. 0. Aasen, Alteration of the hypoxanthine level in cerebrospinal fluid as an indicator of tissue hypoxia, Brain Res., 112 (1976) 188 189. H. Manzke, K. Orner, J. Unitz and H. Wensky, Increased hypoxanthine, creatinine and alpha-fetoprotein excretion in urine of newborn infants with birth complications, Monatsschr. Kinderheilk., 124 (5) (1976) 492 - 493. 0. D. Saugstad, The determination of hypoxanthine with a pOz-electrode, Pediat. Res., 9 (1975) 575 - 579.
POST-MORTEM MOUR
HYPOXANTHINE
AN INTRODUCTORY
LEVELS
IN THE
33
VITREOUS
HU-
REPORT
0. D. SAUGSTAD and B. OLAISEN Institute for Surgical (Norway)
Research
(Received November 23,1977;
and Institute
of Forensic
Medicine,
University
of Oslo
accepted July 18, 1978)
Summary Post-mortem hypoxanthine levels in vitreous humour were determined in 86 consecutive legal autopsy cases. In cases of sudden death caused by trauma or by myocardial infarction, levels ranging from 0 to 540 ymol/l were found. The mean value was about ten times higher than normal in vivo plasma levels. The hypoxanthine levels seem to be independent of time post-mortem, at least during the first 48 hours. It is known that augmentation of the hypoxanthine plasma, cerebrospinal fluid, and urine levels reflects tissue hypoxia. In the present material no elevation of hypoxanthine levels in the vitreous humour was found in cases of strangulation or suspension, while statistically significant elevation was found in cases of drug intoxication. It is concluded that this may reflect the effect of drug-induced prolonged tissue hypoxia caused by respiratory depression.
Introduction In the last few years it has been extensively documented that during tissue hypoxia hypoxanthine levels in plasma [ 11, cerebrospinal fluid [2] and urine [3] are raised. It is therefore reasonable to suggest that elevation of hypoxanthine concentrations during tissue hypoxia occurs in other body fluids as well. The vitreous humour of the eye mediates transport to and from the retina and, apart from changes caused by this process (for instance low glucose and high lactic acid levels), its composition is quite similar to that of aqueous fluid, cerebrospinal fluid and serum. Vitreous humour is easy to sample post mortem, and is relatively free from contamination by blood, bacteria and products of post-mortem autolysis. Changes in the concentration of several substances in the vitreous humour are known to occur postmortem, however. The aim of the present study was to investigate post-mortem hypoxanthine levels in vitreous humour and in particular to determine whether these levels might reflect the degree of tissue hypoxia preceding death.
34
Materials
and methods
Eighty-six consecutive legal autopsy cases (3 months to 80 years of age) comprise the material for this study. The cases were divided into five groups. Group 1: severe trauma leading to immediate death (23 cases). Group 2: short-lasting hypoxia by strangulation or suspension (10 cases). Group 3: sudden death from myocardial infarction (19 cases). Group 4: fatal drug intoxication (13 cases). Group 5: others, including cases of bronchopneumonia, gastric haemorrhage, brain catastrophies, drowning and carbon monoxide poisoning (21 cases). Toxicological analyses were performed at the National Institute of Forensic Toxicology. Vitreous humour was obtained 0.5 - 192 hours post mortem using 4 ml vacutainers. The samples were then immediately stored at -20 “C until analysed for hypoxanthine within a few weeks [4]. Since vitreous humour hypoxanthine levels were considerably higher than those of normal plasma 141, the samples were diluted 20 times before analysis. In six cases samples of vitreous humour from both eyes were examined, and in some cases several analyses were made of the same samples after different storage times.
Results The post-mortem hypoxanthine concentrations in vitreous humour samples from groups 1 - 4 are given as histograms in Fig. 1. In group 1 (severe trauma) the hypoxanthine concentrations varied between 0 and 480 pmol/l with a mean of 136 pmol/l. In group 2 (suspension or strangulation) the values ranged between 0 and 300 pmol/l (mean 129 pmol/l). In group 3 (myocardial infarction) hypoxanthine levels between 0 and 540 I*mol/l (mean 143 pmol/l) were found. There were no statistically significant differences between the hypoxanthine levels of these three groups. In group 4 (intoxication) the hypoxanthine concentrations in the vitreous humour ranged between 60 and 1260 pmol/l (mean 363 pmol/l). Compared to the results of groups 1 - 3 these values were significantly higher (P < 0.01, with Wilcoxon rank test for two samples). In group 5 (others) the hypoxanthine values ranged between 0 and 840 pmol/l with a mean of 156 pmol/l. The correlation between the time post mortem when the samples were obtained and hypoxanthine values for all cases are plotted in Fig. 2. By linear regression analyses a correlation coefficient of +0.09 (N.S.) between hypoxanthine concentrations and post-mortem time during the first 48 hours was found. When post-mortem times from 0 to 90 hours were considered, the correlation coefficient increased to +0.28 (P < 0.05), and was further increased when post-mortem times from 0 to 192 hours were considered.
35
Trauma Mean:
136 gmol/l
Infarctus Mean:
cordis
143 pmol/l
Intoxication N= 13
nb 3 2 \
363 gmol/l 0
120 240 360 480 MI0 )6M)
Hypoxanthine
,,,,,,,,
10
20
30
LO
50
(pmol/l)
Fig. 1. Distribution of the hypoxanthine samples from groups 1 - 4.
concentrations
Fig. 2. Correlation between hypoxanthine concentrations time post mortem when the samples were obtained.
in post-mortem
in vitreous
60
‘+
70 80 9Oz Hours post mortem
vitreous
humour
humour
and the
Hypoxanthine values obtained in samples from both eyes did not show statistically significant deviations, neither were there any changes in hypoxanthine levels as a result of storage at -20 “C (up to 20 weeks).
Discussion The normal hypoxanthine concentration in vitreous humour in the living body is not known. It is reasonable to assume, however, that the values found in cases of sudden death reflect the levels in uiuo. In this study we have therefore regarded the concentrations in the vitreous humour of individuals from group 1 (severe trauma) and group 3 (myocardial infarction) as reference values. The normal hypoxanthine concentration in vitreous humour thus appears to range between 0 and 540 pmol/l. The mean value found is lo- to 20-times higher than that in plasma [4]. In the reference material no statistically significant differences in hypoxanthine concentrations were found in the samples obtained more than 48 hours post mortem when compared to samples obtained 0 - 48 hours post mortem. In the total material a positive correlation was found between
36
hypoxanthine concentrations and the post-mortem sampling time when this exceeded 48 hours. However, this is obviously caused by the fact that the time interval from death to autopsy tended to be high in the individuals who had died of intoxication. In patients of groups 1 and 3 circulatory arrest occurred suddenly, probably with no or only minor preceding tissue hypoxia. Compared to this reference group, no elevated hypoxanthine levels were found in patients from group 2, who had died by suspension or strangulation. Although tissue hypoxia in such patients probably preceded circulatory arrest, this period may have been of too short a duration for hypoxanthine to accumulate in the vitreous fluid. The drugs found in group 4 cases were barbiturates, methadone, ketobemidone, mebrobamate and dextropropoxiphen, either alone, in combination, or together with non-lethal ethanol concentrations. These drugs are known to cause respiratory depression. The significant elevation of hypoxanthine values, compared to the reference group, found in patients dying of intoxication can be explained by respiratory depression preceding death. In these patients tissue hypoxia may have occurred for rather a long period before circulatory arrest. Hence accumulation of hypoxanthine in the vitreous humour could be presumed. Obviously the hypoxanthine levels are a reflection of the degree and duration of tissue hypoxia. The significance of each of these factors is impossible to estimate in this study, however. The present data indicate that determination of the hypoxanthine concentration in vitreous humour post mortem may give information about whether or not tissue hypoxia preceded circulatory arrest.
Acknowledgement During this study 0. D. Saugstad was a research wegian Research Council for Science and the Humanities.
fellow
of the Nor-
References 0. D. Saugstad, Hypoxanthine as a measurement of hypoxia, Pediat. Res., 9 (1975) 158 - 161. 0. D. Saugstad, H. Schrader and A. 0. Aasen, Alteration of the hypoxanthine level in cerebrospinal fluid as an indicator of tissue hypoxia, Brain Res., 112 (1976) 188 189. H. Manzke, K. Orner, J. Unitz and H. Wensky, Increased hypoxanthine, creatinine and alpha-fetoprotein excretion in urine of newborn infants with birth complications, Monatsschr. Kinderheilk., 124 (5) (1976) 492 - 493. 0. D. Saugstad, The determination of hypoxanthine with a pOz-electrode, Pediat. Res., 9 (1975) 575 - 579.