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Hypothermia markers: serum, urine and adrenal gland catecholamines in hypothermic rats given ethanol
Forensic Science International 72 (1995) 125-133
ELSEVIER
Hypothermia markers: serum, urine and adrenal gland catecholamines in hypothermic rats given ethanol J. Hirvonen*, P. Huttunen Department
of Forensic
Medicine,
University
of Oh
90220-Oulu.
Finland
Received 18 April 1994; accepted 22 December 1994
Abstract
The effect of ethanol (2 g/kg) on body temperatureand catecholamine(CA) secretionin the cold (-20%) wasinvestigatedin adult maleand femalerats. The temperaturedropped morerapidly in the females,being - 10°Cafter 3 h ascomparedwith 18°Cin the males.Controls receivedthe samedoseof ethanol but werekept at +2O”C.Increasedconcentrationsof adrenaline(A) and noradrenaline(NA) werealreadyobservedin the serumand urine of the femalesafter 0.5 h of exposure,but at 1 h in the males.Serumvalueswerelow in the females after 2 and3 h andurine valueshigh in connectionwith the deephypothermicstate.The urine valuesof the maleswerealsohigh at the end of exposure,whenthey, too, werehypothermic. Depletionof amineswasobservedin the adrenalsduring the hypothermiaphase,while CA concentrationstendedto risein the serumof the ratskept in the warm.The CA index (A:NA) wasgenerally> 1 in both the serumand urine of the cold-exposedrats. The resultsshowthat femalerats arelessresistantto hypothermiathan males,asindicatedby their morerapid drop in body temperatureand exhaustionof CA secretion.It is alsoapparentthat urine CAs are worth measuringin casesof accidentalhypothermiaand possiblyalsoother types of stress. An elevatedCA index (> 1)seems to be an additionalmarkerof hypothermia,showinga proportionally greaterincreasein the secretionof A than NA during cold stress. KeyworrLF:Hypothermiamarkers;Catecholamines; Adrenaline; Noradrenaline
* Corresponding author. 0379-0738/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(95)01694-E
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1. Introduction We have shown in previous experiments that guinea-pigs had high concentrations of adrenaline (A) and noradrenaline (NA) in their urine after cold exposure; and also postmortem [ 11.These results confirmed similar observations made earlier in human victims of hypothermia [2]. Similar activation of the adrenergic nerve system is also seen in other stress situations. In many cases of accidental hypothermia heavy consumption of alcohol is a contributory factor, and frequently it is the only one. Alcohol is known to hinder thermoregulation, rendering the individual poikilothermic and increasing the risk of accidents in both cold and hot environments [3]. One of the aims of our cold exposure research has been to find markers of cold stress which could be used in difficult cases of accidental hypothermia for diagnosis and timing of the events. One ‘marker’ was found to be the 24-fold increase in the A: NA ratio in the urine of guinea-pigs after a period of hypothermia, a ratio which increased further to 40-fold after rewarming [ 11. In the light of these observations we decided to compare the effect of ethanol and hypothermia on such markers. The experiments confirmed that ethanol (2 g/kg) makes animals susceptible to cold, and that exposure increases the excretion of catecholamines (CA) into the urine. Moreover, it was considered that this experiment would provide basic information on differences in resistance to cold stress and the excretion of CAs between female and male rats and some clues regarding the timing of the responses. The victims of accidental hypothermia can be of either sex, however, and thus the individual reconstruction of events can be different in this respect.
2. Experimental
procedure and methods
The experiment was carried out with adult Sprague-Dawley rats of both sexes. There were 80 males weighing 300-540 g and 80 females weighing 200-297 g. Permission for the experiments was obtained from the university Animal Experimentation Committee and the provincial administration. The animals were living in an ordinary colony with a 12-h light/dark regime and an ambient temperature of 21°C. They received pelleted food and water ad libitum. Urine was collected in a bottle containing HC124 h before the experiment for measurement of the basic concentrations of CAs. The rats were divided into two groups, those for exposure at -20°C and controls to be kept at +2O”C for the corresponding time. The exposure times were 0.5 h, 1 h, 2 h and 3 h, and each subgroup consisted of 10 males and 10 females, as did the control subgroups. Each rat was given 2 g/kg of ethanol in a 20% solution i.p. 5 min before the experiment. Urine from all the rats was collected in a bottle in a metabolic cage throughout the exposure. After the experiment the rats were anaesthetized with ether and the following samples were taken for analysis:
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1. A blood sample was taken by cardiac puncture into a tube containing Na&05, centrifuged and the plasma was stored at -70°C until analyzed. 2. The remaining urine was released from the bladder by puncture and added to that collected during the experiment. The acidified urine was frozen. 3. The adrenals were removed, frozen in liquid nitrogen and stored at -70°C until analyzed. Colonic temperature was measured before and after exposure. Temperatures before exposure varied between 38°C and 39°C. 2.1. Assay of CAs
CAs were extracted from the serum and urine with aluminum oxide [4] and NA and A concentrations were measured by high pressure liquid chromatography using an electrochemical detector (HPLC) [5]. The adrenals were homogenized in O.lM HClO, and CAs in the homogenate were again measured by HPLC. 2.2. Statistical analyses An analysis of variance (UWM) was used for testing the differences between group means. The CA index (A:NA) was calculated for the serum and urine values. 3. Results
3.1. Drop in colonic temperature Colonic temperature (T,,,) dropped about 2°C in 0.5 h in both the male and female rats kept at +2O”C, but then remained quite stable (Fig. 1). The rats of both sexeskept at -20°C had suffered a drop in colon temperature of - 5°C by 0.5 h, after which the drop became faster in the females than in the males. After 3 h the temperature had fallen to 18’C in the males and about 10°C in the females (Fig. 1). 40
'O
32-
? 2 2
24-
: E .?!
16-
-
!
+20’ \i \ I d-20'
Q-20'
\
before
'
0.5h
l.Oh
2.0h
3.0h
Fig. 1. Colonic temperature (body temperature) during the exposure to cold and normal ambient temperatures. Note the rapid drop in female rats after 1 h in the cold.
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1.5 -
5
1.2-
5 i
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0.6-
P 2
0.3-
V.”
0.5
h
1.0
h
exposure
2.0
with
h
3.0
h
ethanol
Fig. 2. NA concentration (mean l SE) in serum during the exposure. Elevated values were observed in females at 0.5 h and at the final phase in the cold. In male rats the concentration was stable both in the cold and warm. The concentration was higher in females throughout the exposure.
3.2. Serum CAs 3.2.1. NA concentration. NA was higher in the females than in the males
throughout the 3-h period at +2O”C, and had increased 2-fold in the cold-exposed females after 0.5 h (P < 0.05), but levelled off at 1 h and 2 h, to rise again somewhat, but not significantly, at 3 h. The concentration in the cold-exposed males remained
E =I t v)
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i
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-
0.5
-
E 4
0.0
I
I
’ 0.5
h
1.0
I h
2.0
I h
3.0
h
exposure with ethanol Fig. 3. A concentration (mean l SE) in serum during the exposure. Clearly elevated values were already observed in female rats at 0.5 h and at the agonal phase in the cold, but the concentration rose at +2O“C to the end of the experiment. In males the concentration stayed rather stable in both ambient temperatures.
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Fig. 4. NA concentration (mean f SE) in urine during the exposure. Elevated contents were already observed in females at 0.5 h and in males after 1 h in the cold; 24-h urine concentration means normal excretion during the day before the experiment.
quite stable, with only a slight increase recorded at 1 h and a decline at 2 h and 3 h (Fig. 2). X2.2. A concentration. The A concentration tended to increase in both sexes during the 3-h period at +2O”C, but more so in the females. Cold exposure for 0.5 h increased the concentration significantly more in the females than in the males (P < 0.001). After this it declined, and was lowest at 2 h, but increased again > 1Zfold in the females by 3 h, being significantly higher than in the males (P < 0.05) (Fig. 3).
0) .E 5 5 8 =
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36
F a
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exposure with ethanol Fig. 5. A concentration (mean l SE) in urine during the exposure. A rather remarkable elevation was observed in females from 0.5 h onwards in the cold. In males the increase started at 1 h and was more moderate; 24-h value, see the caption for Fig. 4.
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3.3. Urine CAs
There were no differences in 24 h (basic) urinary CAs between the sexes. 3.3.1. NA concentration. The NA concentration had already increased in the urine of the females after 0.5 h of cold exposure (P < O.OOl), and had risen in both sexes by 1 h, more so in the females than in the males (P < 0.01). It continued to be high in both sexes when kept in the cold for 2 h, the increase again being greater in the females (P < 0.05). The highest NA concentration was observed after 3 h in the cold in both sexes, with no significant differences between them. Thus NA was as a rule higher in the females during cold exposure but not in the warm (Fig. 4). 3.3.2. A concentration. The A concentration rose steadily in the cold-exposed females up to 3 h, but the highest concentration was reached in the males at 2 h. The rise was higher in the females at 0.5 h (P < O.OOl), 1 h (P < 0.05) and 3 h (P < 0.01). In a warm environment the A concentration varied in both sexes at 0.5-3 h, but was always higher than the 24 h baseline value (Fig. 5). 3.4. CAs in the adrenal gland 3.4.1. NA concentration. The NA content of the adrenal gland was higher in the
males than in the females after 0.5 h in the cold environment (P < 0.05), being higher than under warm conditions in the males, but lower in the females. The concentration was lower after exposure for 1 h, 2 h and 3 h than in those kept in the warm for both sexes, the decrease being more pronounced in the males than in the females at 2 h (P < 0.05) (Fig. 6). 3.4.2. A concentration. The A content was also markedly higher in the coldexposed males than in the females at 0.5 h (P < 0.01) and 1 h. It was lowest in males at 2 h, where the drop was more pronounced than in the females (P < O.OOl), whereas the steepest drop for the females was seen at 3 h (P < 0.05) (Fig. 7). 250
exposure with ethanol Fig. 6. NA concentration (mean f SE) in the adrenal gland during the exposure. A small decrease was observed in males at 2 h in the cold.
J. Hiwonen, P. Huttunen / Forensic Science International 72 (1995) 125-133
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800 600 400
a
200 0
d 0.5h0
d l.Ot?
d
d 3.0t1°
2.OR
exposure with ethanol Fig. 7. A concentration (mean f SE) in the adrenal during the exposure. No major differences were observed in warm conditions in both genders, but in cold conditions the content was low at the 2 h point of the exposure.
3.5 Changes in the CA index (Table I) 3.5.1. Serum index. The ANA index in both serum and urine was slightly > 1.0 in the males kept in the warm after 0.5 h and < 1.0 in the females. The serum index rose slightly in the females but remained around 1 in the males. In the cold the index was slightly elevated at 0.5 h and further at 3 h in both sexes being very high in the females (8.7). 3.5.2. Urine index. The urine index was quite low (C 1.0) at all time points in the males kept in the warm, but was elevated in the females at 1 h, levelling off after that. In the cold, the female urine index rose continuously from the 0.5 h value and
Table 1 CA index (A:NA) at given time points under warm and cold conditions 0.5 h
Ih
2h
3h
M F M F
1.2 0.6 1.6 1.3
1.4 1.1 1.3 1.1
1.0 1.3 0.9 1.2
1.0 1.6 1.8 8.7
M F M F
1.0 0.8 1.2 0.7
0.9 1.9 1.0 1.2
0.4 0.6 1.7 1.4
0.7 0.9 0.7 1.6
serum +2O”C -20°C Urine +2O”C -20°C
M, male; F, female.
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that for the males was also high at 2 h. The index at 3 h was twice as high in the females as in the males. Concerning the use of this index as a marker of hypothermia, the conclusion could be reached that an elevation was seen in the serum after 3 h cold exposure in both sexes, and in the urine in the females. An elevation in the urine index was found in the males at 2 h. 4. Discussion The fact that the colonic temperature dropped markedly faster in the females than in the males indicates a weaker resistance to cold stress and the onset of a lifethreatening situation in an earlier stage. The males benefit in this respect from their greater body size, which prevents loss of heat, from their stronger musculature, which is capable of producing more heat by shivering, and perhaps from their better endocrinological and enzymatic ability to resist the effects of ethanol. The authors are unaware of any information or experimental data on possible differences in body temperature drop between human females and males exposed to dry cold. Body size, musculature, enzymes and hormones are factors that can also be applied to humans, but the greater fat content of females can have a beneficial effect regarding heat loss, compensating for the differences in metabolism and musculature. CA excretion into the blood and urine was greatly stimulated by cold stress. The sources of CA are the adrenal medulla and the peripheral sympathicus nerve endings. The ethanol dose of 2 g/kg as such seemed to prevent the release of A and NA from the adrenal medulla during the moderate stress of immobilization in water at 25°C for 15 min [6]. Our experiments show that the release of CA into the blood and its ovefflow into the urine were consequences of severe cold stress, since the difference relative to the rats kept in the warm which also received ethanol was an obvious one. The results also showed that the adrenal NA content tended to increase by 1 h in the warm (+2O”C), which supports the idea that a moderate ethanol dose inhibits the release of CA. A mild rise in the A content of the serum was found in both sexes at +2O”C, however, which points to a slight stress reaction causing increased synthesis of A in the adrenal gland, since no depletion was seen there. The higher serum NA in the males than in the females at +2O”C is probably a normal situation. Serum NA content was high in the females after 3 h in the cold, however, but not in the males, probably reflecting the more rapid exhaustion of the females under cold conditions and a final agonal reaction. A similar rise was also observed in serum A in the female rats after 3 h, when they were either in a deep coma or dead. The coma was not as deep in the males. Increased excretion of A and NA also occurs at maximal exercise stress in humans, but not during submaximal exercise [7]. The greater stress reaction of the females to the cold was also seen in the increase in A and NA in their urine after only 0.5 h, when the values were still normal in that of the males. The NA increase in the males was observed after 1 h in the cold, beyond which time the urine concentrations rose along with cold exposure. The increased consumption of CAs towards the end of exposure was also seen in
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the adrenals, where the A content decreased in the exposed females. A decrease in A had also occurred after the 2 h phase of exposure in the males, but it had increased again at 3 h. The NA content had fallen in the males after 1 h in the cold, probably due to diminishing synthesis as hypothermia started to develop. When the serum CA index (A:NA) was calculated for each time point it was seen to increase steadily in the females kept in the warm but not in the males. In the cold, it was higher in both sexes at 0.5 h, and especially so at the end of the exposure (3 h). The urine index had a tendency to fall during the experiment in both sexes in the warm, but higher values in the cold were obtained at 2 h for both sexes and still higher at 3 h for the females. The high indices indicate A excretion being activated proportionally more than NA, an observation which can be explained as suggesting either a more important role for A in cold survival or a side-reaction, e.g. to fear during cold stress. The urine and serum indices behaved in inverse manners in the cold-exposed females, pointing to an apparent sex difference regarding this parameter as well. When considering the value of the CA index as an extra diagnostic sign or vital reaction with respect to hypothermia death, we think that some support can be obtained by calculating the index from the original values. In practice the urine index would be more reliable, since the postmortem leaking from the adrenals tends to disturb the information. Since the urine index was well above 1 in the females, it could be regarded as elevated in the present experiments, which argues in favour of stress. It was more clearly elevated in guinea-pigs, however [ 11,which indicates a different balance between the CAs in the two species. The value of the urine index needs to be tested in real human cases, both of hypothermia stress and of other stress lasting a few hours. References [l] J. Hirvonen and T. Lapinlampi, Plasma and urine catecholamines and cerebrospinal fluid amine metabolites as hypothermia markers in guinea-pigs. Med. Sci. Law, 29 (1989) 130-135. [2] J. Hirvonen and P. Huttunen, Increased urinary concentration of CAs in hypothermia deaths. J. Forensic
Sci., 21 (1982)
264-271.
[3] M.-L. Kortelainen, Drugs and alcohol in hypothermia and hyperthermia related deaths. A retrospective study. J. Forensic Sci., 32 (1987) 1704-1712. [4] B.M. Eriksson and B.A. Persson, Determination of catecholamines in rat heart tissue and plasma samples by liquid chromatography with electrochemical detection. J. Chromatogr., 228 (1982) 143-154. [5] R.B. Taylor, R. Reid, K.E. Kendle, C. Geddes and P.F. Curie, Assay procedures for the detemtination of biogenic amines and their metabolites in rat hypothalamus using ion-pairing reversed phase high performance liquid chromatography. J. Chromatogr., 277 (1983) 101-114. [6] K. Kuriyama, K. Kanmori and Y. Yoneda, Preventive effect of alcohol against stress-induced alteration in content of monoamines in brain and adrenal gland. Neuropharmacol., 23 (1984) 649-654. [7]
J. Harris, G.S. Krahenbuhl, R.D. Malchow and J.R. Stem, Neurochemistry of stress: Urinary biogenie amine/metabolic excretion rates in exercise. Biogenic Amines., 2 (1985) 261-267.
ELSEVIER
Hypothermia markers: serum, urine and adrenal gland catecholamines in hypothermic rats given ethanol J. Hirvonen*, P. Huttunen Department
of Forensic
Medicine,
University
of Oh
90220-Oulu.
Finland
Received 18 April 1994; accepted 22 December 1994
Abstract
The effect of ethanol (2 g/kg) on body temperatureand catecholamine(CA) secretionin the cold (-20%) wasinvestigatedin adult maleand femalerats. The temperaturedropped morerapidly in the females,being - 10°Cafter 3 h ascomparedwith 18°Cin the males.Controls receivedthe samedoseof ethanol but werekept at +2O”C.Increasedconcentrationsof adrenaline(A) and noradrenaline(NA) werealreadyobservedin the serumand urine of the femalesafter 0.5 h of exposure,but at 1 h in the males.Serumvalueswerelow in the females after 2 and3 h andurine valueshigh in connectionwith the deephypothermicstate.The urine valuesof the maleswerealsohigh at the end of exposure,whenthey, too, werehypothermic. Depletionof amineswasobservedin the adrenalsduring the hypothermiaphase,while CA concentrationstendedto risein the serumof the ratskept in the warm.The CA index (A:NA) wasgenerally> 1 in both the serumand urine of the cold-exposedrats. The resultsshowthat femalerats arelessresistantto hypothermiathan males,asindicatedby their morerapid drop in body temperatureand exhaustionof CA secretion.It is alsoapparentthat urine CAs are worth measuringin casesof accidentalhypothermiaand possiblyalsoother types of stress. An elevatedCA index (> 1)seems to be an additionalmarkerof hypothermia,showinga proportionally greaterincreasein the secretionof A than NA during cold stress. KeyworrLF:Hypothermiamarkers;Catecholamines; Adrenaline; Noradrenaline
* Corresponding author. 0379-0738/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(95)01694-E
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1. Introduction We have shown in previous experiments that guinea-pigs had high concentrations of adrenaline (A) and noradrenaline (NA) in their urine after cold exposure; and also postmortem [ 11.These results confirmed similar observations made earlier in human victims of hypothermia [2]. Similar activation of the adrenergic nerve system is also seen in other stress situations. In many cases of accidental hypothermia heavy consumption of alcohol is a contributory factor, and frequently it is the only one. Alcohol is known to hinder thermoregulation, rendering the individual poikilothermic and increasing the risk of accidents in both cold and hot environments [3]. One of the aims of our cold exposure research has been to find markers of cold stress which could be used in difficult cases of accidental hypothermia for diagnosis and timing of the events. One ‘marker’ was found to be the 24-fold increase in the A: NA ratio in the urine of guinea-pigs after a period of hypothermia, a ratio which increased further to 40-fold after rewarming [ 11. In the light of these observations we decided to compare the effect of ethanol and hypothermia on such markers. The experiments confirmed that ethanol (2 g/kg) makes animals susceptible to cold, and that exposure increases the excretion of catecholamines (CA) into the urine. Moreover, it was considered that this experiment would provide basic information on differences in resistance to cold stress and the excretion of CAs between female and male rats and some clues regarding the timing of the responses. The victims of accidental hypothermia can be of either sex, however, and thus the individual reconstruction of events can be different in this respect.
2. Experimental
procedure and methods
The experiment was carried out with adult Sprague-Dawley rats of both sexes. There were 80 males weighing 300-540 g and 80 females weighing 200-297 g. Permission for the experiments was obtained from the university Animal Experimentation Committee and the provincial administration. The animals were living in an ordinary colony with a 12-h light/dark regime and an ambient temperature of 21°C. They received pelleted food and water ad libitum. Urine was collected in a bottle containing HC124 h before the experiment for measurement of the basic concentrations of CAs. The rats were divided into two groups, those for exposure at -20°C and controls to be kept at +2O”C for the corresponding time. The exposure times were 0.5 h, 1 h, 2 h and 3 h, and each subgroup consisted of 10 males and 10 females, as did the control subgroups. Each rat was given 2 g/kg of ethanol in a 20% solution i.p. 5 min before the experiment. Urine from all the rats was collected in a bottle in a metabolic cage throughout the exposure. After the experiment the rats were anaesthetized with ether and the following samples were taken for analysis:
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1. A blood sample was taken by cardiac puncture into a tube containing Na&05, centrifuged and the plasma was stored at -70°C until analyzed. 2. The remaining urine was released from the bladder by puncture and added to that collected during the experiment. The acidified urine was frozen. 3. The adrenals were removed, frozen in liquid nitrogen and stored at -70°C until analyzed. Colonic temperature was measured before and after exposure. Temperatures before exposure varied between 38°C and 39°C. 2.1. Assay of CAs
CAs were extracted from the serum and urine with aluminum oxide [4] and NA and A concentrations were measured by high pressure liquid chromatography using an electrochemical detector (HPLC) [5]. The adrenals were homogenized in O.lM HClO, and CAs in the homogenate were again measured by HPLC. 2.2. Statistical analyses An analysis of variance (UWM) was used for testing the differences between group means. The CA index (A:NA) was calculated for the serum and urine values. 3. Results
3.1. Drop in colonic temperature Colonic temperature (T,,,) dropped about 2°C in 0.5 h in both the male and female rats kept at +2O”C, but then remained quite stable (Fig. 1). The rats of both sexeskept at -20°C had suffered a drop in colon temperature of - 5°C by 0.5 h, after which the drop became faster in the females than in the males. After 3 h the temperature had fallen to 18’C in the males and about 10°C in the females (Fig. 1). 40
'O
32-
? 2 2
24-
: E .?!
16-
-
!
+20’ \i \ I d-20'
Q-20'
\
before
'
0.5h
l.Oh
2.0h
3.0h
Fig. 1. Colonic temperature (body temperature) during the exposure to cold and normal ambient temperatures. Note the rapid drop in female rats after 1 h in the cold.
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1.5 -
5
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0.6-
P 2
0.3-
V.”
0.5
h
1.0
h
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2.0
with
h
3.0
h
ethanol
Fig. 2. NA concentration (mean l SE) in serum during the exposure. Elevated values were observed in females at 0.5 h and at the final phase in the cold. In male rats the concentration was stable both in the cold and warm. The concentration was higher in females throughout the exposure.
3.2. Serum CAs 3.2.1. NA concentration. NA was higher in the females than in the males
throughout the 3-h period at +2O”C, and had increased 2-fold in the cold-exposed females after 0.5 h (P < 0.05), but levelled off at 1 h and 2 h, to rise again somewhat, but not significantly, at 3 h. The concentration in the cold-exposed males remained
E =I t v)
2.5
-
2.0
-
i
1.5 -
z z
1.0
-
0.5
-
E 4
0.0
I
I
’ 0.5
h
1.0
I h
2.0
I h
3.0
h
exposure with ethanol Fig. 3. A concentration (mean l SE) in serum during the exposure. Clearly elevated values were already observed in female rats at 0.5 h and at the agonal phase in the cold, but the concentration rose at +2O“C to the end of the experiment. In males the concentration stayed rather stable in both ambient temperatures.
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Fig. 4. NA concentration (mean f SE) in urine during the exposure. Elevated contents were already observed in females at 0.5 h and in males after 1 h in the cold; 24-h urine concentration means normal excretion during the day before the experiment.
quite stable, with only a slight increase recorded at 1 h and a decline at 2 h and 3 h (Fig. 2). X2.2. A concentration. The A concentration tended to increase in both sexes during the 3-h period at +2O”C, but more so in the females. Cold exposure for 0.5 h increased the concentration significantly more in the females than in the males (P < 0.001). After this it declined, and was lowest at 2 h, but increased again > 1Zfold in the females by 3 h, being significantly higher than in the males (P < 0.05) (Fig. 3).
0) .E 5 5 8 =
72
54
36
F a
16
exposure with ethanol Fig. 5. A concentration (mean l SE) in urine during the exposure. A rather remarkable elevation was observed in females from 0.5 h onwards in the cold. In males the increase started at 1 h and was more moderate; 24-h value, see the caption for Fig. 4.
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3.3. Urine CAs
There were no differences in 24 h (basic) urinary CAs between the sexes. 3.3.1. NA concentration. The NA concentration had already increased in the urine of the females after 0.5 h of cold exposure (P < O.OOl), and had risen in both sexes by 1 h, more so in the females than in the males (P < 0.01). It continued to be high in both sexes when kept in the cold for 2 h, the increase again being greater in the females (P < 0.05). The highest NA concentration was observed after 3 h in the cold in both sexes, with no significant differences between them. Thus NA was as a rule higher in the females during cold exposure but not in the warm (Fig. 4). 3.3.2. A concentration. The A concentration rose steadily in the cold-exposed females up to 3 h, but the highest concentration was reached in the males at 2 h. The rise was higher in the females at 0.5 h (P < O.OOl), 1 h (P < 0.05) and 3 h (P < 0.01). In a warm environment the A concentration varied in both sexes at 0.5-3 h, but was always higher than the 24 h baseline value (Fig. 5). 3.4. CAs in the adrenal gland 3.4.1. NA concentration. The NA content of the adrenal gland was higher in the
males than in the females after 0.5 h in the cold environment (P < 0.05), being higher than under warm conditions in the males, but lower in the females. The concentration was lower after exposure for 1 h, 2 h and 3 h than in those kept in the warm for both sexes, the decrease being more pronounced in the males than in the females at 2 h (P < 0.05) (Fig. 6). 3.4.2. A concentration. The A content was also markedly higher in the coldexposed males than in the females at 0.5 h (P < 0.01) and 1 h. It was lowest in males at 2 h, where the drop was more pronounced than in the females (P < O.OOl), whereas the steepest drop for the females was seen at 3 h (P < 0.05) (Fig. 7). 250
exposure with ethanol Fig. 6. NA concentration (mean f SE) in the adrenal gland during the exposure. A small decrease was observed in males at 2 h in the cold.
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800 600 400
a
200 0
d 0.5h0
d l.Ot?
d
d 3.0t1°
2.OR
exposure with ethanol Fig. 7. A concentration (mean f SE) in the adrenal during the exposure. No major differences were observed in warm conditions in both genders, but in cold conditions the content was low at the 2 h point of the exposure.
3.5 Changes in the CA index (Table I) 3.5.1. Serum index. The ANA index in both serum and urine was slightly > 1.0 in the males kept in the warm after 0.5 h and < 1.0 in the females. The serum index rose slightly in the females but remained around 1 in the males. In the cold the index was slightly elevated at 0.5 h and further at 3 h in both sexes being very high in the females (8.7). 3.5.2. Urine index. The urine index was quite low (C 1.0) at all time points in the males kept in the warm, but was elevated in the females at 1 h, levelling off after that. In the cold, the female urine index rose continuously from the 0.5 h value and
Table 1 CA index (A:NA) at given time points under warm and cold conditions 0.5 h
Ih
2h
3h
M F M F
1.2 0.6 1.6 1.3
1.4 1.1 1.3 1.1
1.0 1.3 0.9 1.2
1.0 1.6 1.8 8.7
M F M F
1.0 0.8 1.2 0.7
0.9 1.9 1.0 1.2
0.4 0.6 1.7 1.4
0.7 0.9 0.7 1.6
serum +2O”C -20°C Urine +2O”C -20°C
M, male; F, female.
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that for the males was also high at 2 h. The index at 3 h was twice as high in the females as in the males. Concerning the use of this index as a marker of hypothermia, the conclusion could be reached that an elevation was seen in the serum after 3 h cold exposure in both sexes, and in the urine in the females. An elevation in the urine index was found in the males at 2 h. 4. Discussion The fact that the colonic temperature dropped markedly faster in the females than in the males indicates a weaker resistance to cold stress and the onset of a lifethreatening situation in an earlier stage. The males benefit in this respect from their greater body size, which prevents loss of heat, from their stronger musculature, which is capable of producing more heat by shivering, and perhaps from their better endocrinological and enzymatic ability to resist the effects of ethanol. The authors are unaware of any information or experimental data on possible differences in body temperature drop between human females and males exposed to dry cold. Body size, musculature, enzymes and hormones are factors that can also be applied to humans, but the greater fat content of females can have a beneficial effect regarding heat loss, compensating for the differences in metabolism and musculature. CA excretion into the blood and urine was greatly stimulated by cold stress. The sources of CA are the adrenal medulla and the peripheral sympathicus nerve endings. The ethanol dose of 2 g/kg as such seemed to prevent the release of A and NA from the adrenal medulla during the moderate stress of immobilization in water at 25°C for 15 min [6]. Our experiments show that the release of CA into the blood and its ovefflow into the urine were consequences of severe cold stress, since the difference relative to the rats kept in the warm which also received ethanol was an obvious one. The results also showed that the adrenal NA content tended to increase by 1 h in the warm (+2O”C), which supports the idea that a moderate ethanol dose inhibits the release of CA. A mild rise in the A content of the serum was found in both sexes at +2O”C, however, which points to a slight stress reaction causing increased synthesis of A in the adrenal gland, since no depletion was seen there. The higher serum NA in the males than in the females at +2O”C is probably a normal situation. Serum NA content was high in the females after 3 h in the cold, however, but not in the males, probably reflecting the more rapid exhaustion of the females under cold conditions and a final agonal reaction. A similar rise was also observed in serum A in the female rats after 3 h, when they were either in a deep coma or dead. The coma was not as deep in the males. Increased excretion of A and NA also occurs at maximal exercise stress in humans, but not during submaximal exercise [7]. The greater stress reaction of the females to the cold was also seen in the increase in A and NA in their urine after only 0.5 h, when the values were still normal in that of the males. The NA increase in the males was observed after 1 h in the cold, beyond which time the urine concentrations rose along with cold exposure. The increased consumption of CAs towards the end of exposure was also seen in
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the adrenals, where the A content decreased in the exposed females. A decrease in A had also occurred after the 2 h phase of exposure in the males, but it had increased again at 3 h. The NA content had fallen in the males after 1 h in the cold, probably due to diminishing synthesis as hypothermia started to develop. When the serum CA index (A:NA) was calculated for each time point it was seen to increase steadily in the females kept in the warm but not in the males. In the cold, it was higher in both sexes at 0.5 h, and especially so at the end of the exposure (3 h). The urine index had a tendency to fall during the experiment in both sexes in the warm, but higher values in the cold were obtained at 2 h for both sexes and still higher at 3 h for the females. The high indices indicate A excretion being activated proportionally more than NA, an observation which can be explained as suggesting either a more important role for A in cold survival or a side-reaction, e.g. to fear during cold stress. The urine and serum indices behaved in inverse manners in the cold-exposed females, pointing to an apparent sex difference regarding this parameter as well. When considering the value of the CA index as an extra diagnostic sign or vital reaction with respect to hypothermia death, we think that some support can be obtained by calculating the index from the original values. In practice the urine index would be more reliable, since the postmortem leaking from the adrenals tends to disturb the information. Since the urine index was well above 1 in the females, it could be regarded as elevated in the present experiments, which argues in favour of stress. It was more clearly elevated in guinea-pigs, however [ 11,which indicates a different balance between the CAs in the two species. The value of the urine index needs to be tested in real human cases, both of hypothermia stress and of other stress lasting a few hours. References [l] J. Hirvonen and T. Lapinlampi, Plasma and urine catecholamines and cerebrospinal fluid amine metabolites as hypothermia markers in guinea-pigs. Med. Sci. Law, 29 (1989) 130-135. [2] J. Hirvonen and P. Huttunen, Increased urinary concentration of CAs in hypothermia deaths. J. Forensic
Sci., 21 (1982)
264-271.
[3] M.-L. Kortelainen, Drugs and alcohol in hypothermia and hyperthermia related deaths. A retrospective study. J. Forensic Sci., 32 (1987) 1704-1712. [4] B.M. Eriksson and B.A. Persson, Determination of catecholamines in rat heart tissue and plasma samples by liquid chromatography with electrochemical detection. J. Chromatogr., 228 (1982) 143-154. [5] R.B. Taylor, R. Reid, K.E. Kendle, C. Geddes and P.F. Curie, Assay procedures for the detemtination of biogenic amines and their metabolites in rat hypothalamus using ion-pairing reversed phase high performance liquid chromatography. J. Chromatogr., 277 (1983) 101-114. [6] K. Kuriyama, K. Kanmori and Y. Yoneda, Preventive effect of alcohol against stress-induced alteration in content of monoamines in brain and adrenal gland. Neuropharmacol., 23 (1984) 649-654. [7]
J. Harris, G.S. Krahenbuhl, R.D. Malchow and J.R. Stem, Neurochemistry of stress: Urinary biogenie amine/metabolic excretion rates in exercise. Biogenic Amines., 2 (1985) 261-267.