Forensic Science International 229 (2013) 6065Glucocorticosteroids as markers of death from hypothermia

Krzysztof Banka *, Grzegorz Teresinski, Grzegorz Buszewicz, Roman Madro

Chair and Department of Forensic Medicine, Medical University of Lublin, 20-090 Lublin, Jaczewskiego 8, Poland

A R T I C L E I N F O

Article history:

Received 23 August 2012

Received in revised form 28 February 2013

Accepted 3 March 2013

Available online 19 April 2013

Keywords:

Hypothermia markers

Glucocorticosteroids

Postmortem diagnosis

Blood alcohol concentration

A B S T R A C T

In the course of hypothermia, biochemical changes occur that are associated with stimulation of

protective thermogenic mechanisms as well as mobilization of internal energy resources mediated by

the hormone system. The objective of the investigation was the assessment of validity of determinations

of cortisol, cortisone and corticosterone as hypothermia markers in cases of fatal hypothermia combined

with concomitant insobriety of the victims.

The experimental group consisted of blood samples collected in the course of medico-legal autopsies

of 23 hypothermia victims. The controls included blood samples originating from 34 victims of violent

sudden deaths (deaths by hanging and traffic road accidents at the scene) and from ten individuals

deceased after prolonged agony in consequence of post-traumatic subdural hematomas. In both groups,

three subgroups were distinguished that included cases with ethanol levels within the following ranges:

0.00.99, 1.02.99 and 3.0%.The comparison of determination results showed that irrespectively of blood ethanol concentration,

cortisol, cortisone and corticosterone levels seen in hypothermia victims were significantly higher as

compared to the controls (P < 0.001).

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jou r nal h o mep age: w ww.els evier . co m/lo c ate / fo r sc i in t1. Introduction

Classic morphological changes are insufficient for an unam-biguous diagnosis of death from hypothermia [15]. In addition,such symptoms are relatively rare in such fatalities where victimswere under influence of ethanol, since alcohol facilitates promptlowering of body temperature and shortens the time of agony [4,610].

Thus, verification of the suspicion of fatal hypothermia is as arule based on excluding other causes of death and taking intoconsideration conditions and circumstances in which the body wasfound [1,4,5,1113]. Therefore sought other diagnostic criteria of acharacter biochemical, based on which one might conclude with ahigher probability that death occurred in consequence of excessivelowering of body temperature [11,1418].

As it has been suggested by our earlier investigations [9,17,1921] on the validity of determinations of acetone and acetoaceticand b-hydroxybutyric acid in blood, urine and vitreous body innecrochemical diagnostic management of premortem metabolicdisturbances, high concentrations of ketone bodies may beconsidered a marker of premortem hypothermia, but only insober individuals or in persons demonstrating low (

K. Banka et al. / Forensic Science International 229 (2013) 6065 61Therefore, in the present study, the authors have aimed atverification of the data in a more numerous group of cases and atassessing the usefulness of glucocorticosteroids as new, potentialmarkers of death from hypothermia while taking into consider-ation possible insobriety of victims.

2. Materials and methods

2.1. Study material

The study was performed using samples of blood collected from the bodies of

individuals who had been autopsied at the Chair and Department of Forensic

Medicine, Medical University of Lublin, upon commission of regional prosecutor

offices in the years 20062009.

The experimental (E) group consisted of 23 individuals (21 males and 2 females

aged 3280 years, mean age 52 years), in whom the circumstances of death (the site

of body discovery, weather conditions and data from prosecutor investigation) had

indicated hypothermia as the cause of death (Table 1). The controls (C) consisted of

44 individuals (aged 1792 years) who had died of hanging (N = 13) or in traffic road

accidents (N = 21) at the site (short agony). The authors additionally took into

consideration ten victims who had died due to acute subdural hematomas where

the duration of agony was prolonged (for several hours), similarly as in the case of

hypothermia.

The study included only cases in which not taken any action rescue, death

occurred at the site where the body was discovered and the body itself showed no

signs of advanced putrefaction. Additionally, based on routine chemico-toxicologi-

cal tests and determinations of glycated hemoglobin (HbA1c) concentration levels

performed in the experimental group and the controls, any possibility of the

subjects being poisoned or suffering from diabetes had been excluded. Post-

mortem examinations of the subjects did not reveal injuries or macro- or

microscopic pathological lesions that might be considered the cause of death,

although in some victims, diseases of the circulatory or respiratory tract were seen

concomitantly with evident morphological changes characteristic of hypothermia

(Table 2).

The bodies were stored in a cold store at approximately +4 8C (25 days, onaverage 3 days), while material collected for biochemical tests was stored at 70 8Cuntil analysis. The effect of time lapse between death and autopsy was disregarded,

since the available literature did not indicate glucocorticosteroids concentration

values to undergo any significant changes in the course of postmortem

transformations [32].

2.2. Analytical methods

In all the cases, blood ethanol level was determined by headspace gas

chromatographyflame ionization detector (GCFID) [38]; we noted that at the

time of death, 15 individuals from the experimental group and 29 individuals from

the control group were under influence of alcohol. Using the previously developed

method [39] we also determined the level of glycated hemoglobin (HbA1c), or in

other words the index of long-term diabetes control, which in all cases did notTable 1Data on the circumstances of finding the body (N = 23), N/A no data available.

No. Sex and age Site of body discovery Air temperature (8C)

Max. Min.

1 < 38 Asphalt road shoulder +14.8 2.0 2 < 45 Field 2.7 9.0 3 < 41 Yard 2.4 8.9 4 < 45 Snow-covered ground 5.6 9.1 5 < 36 Forest +2.5 3.2 6 , 51 Unheated house +1.6 9.4 7 < 46 Field 4.0 9.4 8 < 50 Field 5.6 17.0 9 < 51 Field road +4.1 2.6

10 < 74 Yard 8.6 12.6 11 < 56 Field +8.8 5.4 12 < 44 Unheated house 16.1 25.8 13 < 62 Field +3.5 0.9 14 < 32 Field +7.2 +1.2 15 < 52 Snow-covered ground 4.6 11.1 16 < 54 Asphalt road +7.5 +5.0 17 , 80 Asphalt road shoulder 3.2 9.0 18 < 40 Unheated house +1.5 4.9 19 < 50 Railway station +2.2 2.8 20 < 63 Forest complex +3.1 +1.1 21 < 73 Unheated house 7.5 13.8 22 < 72 Field +7.2 +1.2 23 < 47 Yard +0.8 3.4 exceed the intravital level observed in healthy individuals, i.e. approximately 6%,

what indicated normal mean glycemia within approximately 120 days prior to

death.

Routine blood toxicology (including determinations of the activity of acetyl-

cholinesterase, carboxyhemoglobin, methemoglobin, volatile toxic substances

derivatives of hydrocarbons, as well as screening for the presence of pharmaceu-

ticals and substances of abuse), was negative in all the 67 cases.

Glucocorticosteroids were determined using the previously developed method

[40]. We employed an ultra-high pressure Accela liquid chromatograph (Thermo

Finningan, USA) coupled with the LCQ Advantage Max ion trap mass spectrometer

(Thermo Finningan, USA). The mass detector operated in the atmospheric pressure

positive chemical ionization mode and data were collected using the Selected Ion

Monitoring option. Chromatographic separation was performed using an Adsorbo-

sphere HS-C18 (5 mm, 250 4.6 mm) column, from which glucocorticosteroidswere eluted by means of a two-component mobile phase consisting of

Chromasolv1 LC-MS acetonitrile (Riedel de Haen, Germany) and deionized water

obtained employing the Milli-Q system (Millipore, USA). Isolation of glucocorti-

costeroids from 0.5 ml of blood using dichloromethane. Prior to chromatographic

analysis, the dry residue was dissolved in 30 ml of methanol.

2.3. Statistical procedures

The data were characterized by the mean values, median, minimum and

maximum values, as well as lower (25%) and upper (75%) quartiles. In view of the

fact that the distributions of concentrations of glucocorticosteroids markedly

differed from the normal distribution, the statistical analysis was performed using

the non-parametric tests of KruskalWallis and U MannWhitney ANOVA in order

to verify the significance of differences between the mean values of the quantitative

variable in unrelated groups. To compare classic hypothermia signs between the

groups, the Fishers exact test was employed. Statistical significance was assumed

at P < 0.05. The calculations were performed using Statistica 6.0 PL software.

3. Results

The experimental group and the controls demonstratedstatistically significant differences in prevalence of only someclassic hypothermia markers, i.e. skin discoloration in the kneeand elbow region and the so-called paradoxical undressing(Table 2). No cases of acute pancreatitis or blood extravasation intothe iliopsoas muscle [41] were noted.

Distribution of concentration values of particular glucocorti-costeroids is presented as boxplots (Fig. 1), which illustratedifferences in the range of concentrations observed in theexperimental group and the controls taking into considerationblood alcohol concentration.Mean wind velocity (km/h)

Min. at ground level Mean daily temp.

N/A +5.0 18

8.6 6.2 1411.0 5.6 413.2 7.3 115.7 +0.1 1718.0 2.5 08.9 7.8 1021.4 9.5 54.2 +0.4 1112.2 10.5 226.2 +0.7 928.3 23.5 00.2 +0.2 26+1.5 +3.8 11

11.5 7.9 7+3.9 +5.9 11

7.7 6.7 8N/A 2.4 04.4 +0.3 15+1.5 +2.0 6

18.7 11.5 0+1.5 +3.8 11

3.1 0.5 19

Table 2Results of postmortem assessment of subjects died from hypothermia (NS non-significant differences).

Case no. Hypothermia signs Other pathologies

Red livores Skin discoloration

in the knee and

elbow region

Gastric mucous

hemorrhages

Paradoxical

undressing

1 + Liver steatosis (low grade); chronic gastritis

2 + Liver steatosis (moderate)

3 +

4 +

5 +

6 + + + + Liver steatosis (high grade)

7 Gallstone

8 + + Old brain contusions

9 +

10 + + Small myocardial infarction scar (without coronary

atherosclerosis); Spondylosis; gallstone; old brain

contusions

11 + + + Liver steatosis (low grade); Small left ventricular

hypertrophy; Cardiac fibrosis

12 + Liver steatosis (high grade); Chronic gastritis

13 + + Bronchiectasis; aortic atherosclerosis; valvular heart

calcification; cardiac fibrosis; liver steatosis (moderate)

14 + + +

15 + Cardiac fibrosis; liver steatosis (moderate)

16 Liver steatosis (moderate)

17 + + + Coronary artery atherosclerosis (moderate); left

ventricular hypertrophy (moderate); cardiac fibrosis

18 Aortic atherosclerosis and moderate coarctation;

chronic gastritis

19 + Tuberculous lesions of the left lung; cardiac fibrosis;

liver steatosis (moderate); chronic gastritis

20 + + Old brain contusions

21 + + Atherosclerosis

22 + + Atherosclerosis; small myocardial infarction scar;

tuberculous lesions of the right lung

23 + + Liver steatosis (moderate); old brain contusions

Frequency

Examined 65% 52% 26% 13%

Controls 41% 3% 30% 0%

Fishers exact test NS P < 0.001 NS P < 0.03

Fig. 1. Comparison of concentration levels of cortisol (A), cortisone (B) and corticosterone (C) in the examined group and controls and taking into consideration concentrationof blood alcohol (A1, B1, C1).

K. Banka et al. / Forensic Science International 229 (2013) 606562

Table 3Results of statistical analysis of cortisol, cortisone and corticosterone concentrations values with respect to alcoholemia levels.

Group N Cortisol (ng/ml) Cortisone (ng/ml) Corticosterone (ng/ml)

Median value/quartile (25%75%)

E Examined 23 333.96 14.47 12.13

164.95838.87 7.7221.19 4.4623.16

a 0% ethanol < 1% (0.000.98) 9 550.00 14.69 25.89105.841232.37 9.9826.82 3.3931.21

b 1% ethanol < 3% (1.052.82) 9 385.57 17.42 14.23269.13722.66 10.1321.19 8.2315.19

c Ethanol 3% (3.254.03) 5 193.11 7.72 6.56181.52193.40 6.399.14 4.467.70

C Controls 44 34.16 1.63 2.77

23.5439.90 0.833.21 2.533.53

a 0% Ethanol < 1% (0.000.99) 22 36.15 1.73 2.7229.5139.90 0.893.21 2.643.39

b 1% Ethanol < 3% (1.022.46) 14 35.60 1.66 2.9420.9645.29 0.752.90 2.603.64

c Ethanol 3% (3.043.63) 8 26.57 1.32 2.7118.7029.15 0.772.95 2.473.07

x short agony (hanging and traffic accident victims) 34 29.46 1.59 2.67

19.14 32.34 0.81 2.64 2.50 2.95

y prolonged agony (subdural hematomas) 10 30.27 1.65 2.69

28.6633.11 0.843.04 2.623.29

MannWhitney U-test

E/C P < 0.001 P < 0.001 P < 0.001

Ea/Ca P < 0.001 P < 0.001 P < 0.005

Eb/Cb P < 0.001 P < 0.001 P < 0.001

Ec/Cc P < 0.005 P < 0.005 P < 0.005

K. Banka et al. / Forensic Science International 229 (2013) 6065 63The concentration values of cortisol, cortisone and corticoste-rone determined in blood of hypothermic individuals weremarkedly higher as compared to blood concentration levels ofthe same compounds noted in the control subjects (Table 3). Therelationship proved to be highly significant (P < 0.001).

Additionally, the studies showed significantly higher concen-trations of adrenal cortex hormones in blood of experimentalsubjects divided into subgroups with respect to their concentra-tion of alcohol as compared to blood concentration levels of thesame hormones seen in individuals who died while normothermicand were subdivided in the same manner (P < 0.001). Glucocorti-costeroids secretion was found to be independent of ethanolconcentration both in the experimental group (Ea/Eb/Ec) and thecontrols (Ca/Cb/Cc). No statistical significance was also demon-strated between the control subjects who died instantly (Cx) andthe subjects who died after prolonged agony due to post-traumaticsubdural hematomas (Cy).

In more than 50% of hypothermia victims, the concentrationvalues of the investigated hormones (especially cortisol) weremore than two times higher as compared to the highest levelsdemonstrated in the controls.

4. Discussion

Glucocorticosteroids play a special role in hypothermia in viewof their multidirectional activity [42]. As energy reserves becomeexhausted, they stimulate gluconeogenesis (in the liver), catabolicprocesses (in extrahepatic tissues) and lipolytic processes (in fattytissues) [26,27,43]. In the opinion of Silva [44], glucocorticoster-oids also coordinate heat-generating reactions depending onavailability of energy substrates, while according to Deavers andMusacchia [43] they intensify the activity of other hormones(including catecholamines), which also stimulate carbohydrateand lipid metabolism. An important role of adrenal hormones inthermogenesis is confirmed by experiments in rats, where survivaltime at 4 8C following adrenal medulla excision was shown to bedecreased twofold [45]. Rabbits demonstrated a twofold increaseof corticosterone level at internal temperature of 32 8C [35] and afour-fold increase at 30 8C [46].

Data on the role of adrenal cortex hormones in hypothermia inhumans are ambiguous. Leppaluoto et al. [36] observed astatistically significant decrease in cortisol concentration (by20%) in 20 healthy male volunteers after a 120-min stay in a roomwith a temperature of 10 8C. This observation was confirmed byWittert et al. [37] in six healthy volunteers, in whom a significantdrop in plasma cortisol levels was noted as early as within theinitial 15 min of exposure to cold temperatures (4 8C). Whereas,Wilkerson et al. [47] demonstrated a significant increase of bloodcortisol levels in naked males after 2 h of exposure to low ambienttemperatures (

K. Banka et al. / Forensic Science International 229 (2013) 606564hypothermia increased fourfold, while the increase wassevenfold for cortisone and as high as 14-fold for cortisol ascompared to the mean concentration values in individuals whohad died in normotermic conditions. Thus, the present resultsmay indicate a higher cortisol activity in thermogenesis-associated processes in a hypothermic body, compared to theremaining glucocorticosteroids.

On the other hand, no significant differences were notedbetween the control subjects who had died instant deaths (Cx) andindividuals who had died after prolonged agony (Cy). Acutesubdural hematomas were selected as a model of delayed deathafter prolonged agony; since they usually develop in consequenceof traumatic damage of small bridge veins that connect the duramatter with the pia matter of the brain [55]. Subdural hemorrhage even if a large number of veins are ruptured develops relativelyslowly and death occurs only after several hours after the injury.

Blood concentration levels of glucocorticosteroids in alcohol-intoxicated individuals who had died due to hypothermia weresignificantly higher as compared to blood concentration values ofthe hormones noted in intoxicated subjects who had died undernormothermic conditions regardless of the blood alcoholconcentration. Thus, we confirmed our earlier observations [19],that cooling of the body increases secretion of adrenocorticalhormones (especially cortisol), also in alcohol-intoxicated individ-uals and alcohol abusers, what results in the fact that insobrietydoes not diminish the diagnostic value of the markers, as ithappens in the case of ketone bodies [9] and catecholamines [34].Glucocorticosteroids may be thus treated as a potential marker offatal hypothermia, also in alcohol-intoxicated subjects.

For purposes of diagnosis antemortem hypothermia one mightpropose threshold concentration values of the studied compounds,taking into consideration the range of minimum and maximumlevels observed in the examined group and the controls. The cut-offvalue for cortisol might be accepted as >300 ng/ml, for cortisone>10 ng/ml and for corticosterone >20 ng/ml.

5. Limitations of the study

While assessing the usefulness of glucocorticosteroids aspotential hypothermia markers one should take into considerationpossible preexisting hypercorticosolemia being a consequence ofabnormal secretion of adrenal hormones [5658]. The risk of anerroneous interpretation of glucocorticosteroids determinationsbecause of their abnormal secretion seems, however, statisticallylow in view of a low incidence of such syndromes in clinicalpractice and of hypophyseal and adrenal tumors in medico-legalpractice.

In our studies, the diurnal changes in cortisol concentrationwere not taken into account due to the inability to accuratelyestimate the time of death (however, most of the victims left homein the evening hours and probably died due to hypothermia atnight, as their corpses were found in the morning). Glucocorti-costeroid concentrations in the study group were significantlyhigher than the range of physiological concentrations of thesehormones, even taking into account the maximum morningsecretion.

The controls constituted a homogenous group of victims, sincethe objective and assumption of the present study was acomparison of hormone concentration levels in the group ofhypothermia victims and in cases of pure violent deaths withshort and prolonged agony without any other additional factorsthat might affect the hormonal system and interfere withglucocorticosteroids secretion.

It should be additionally emphasized that even very highconcentration levels of glucocorticosteroids in post-mortemblood cannot be regarded the sole reason for diagnosing fatalhypothermia, similarly as a low level of the hormones does notprovide grounds for excluding fatal hypothermia. To diagnosedeath from hypothermia, it is necessary to take into considerationalso other morphologic and biochemical markers, as well ascircumstances of death, following prior exclusion of otherpotential causes of death (injury, disease, poisoning).

6. Conclusions

1. In blood of victims of fatal hypothermia there is observed astatistically significant increase of glucocorticosteroids levels(especially of cortisol).

2. In hypothermic individuals there occurs increased secretion ofcortisol as compared to cortisone and corticosterone, what mayindicate a higher activity of the former in thermogenesisprocesses involving a hypothermic body as opposed to the otherglucocorticosteroids.

3. The degree of alcoholemia does not affect the level ofglucocorticosteroids in cases of fatal hypothermia.

4. Glucocorticosteroids may be treated as a potential marker offatal hypothermia, also in alcohol-intoxicated subjects.

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Glucocorticosteroids as markers of death from hypothermiaIntroductionMaterials and methodsStudy materialAnalytical methodsStatistical procedures

ResultsDiscussionLimitations of the studyConclusionsReferences