Handbook of Forensic Medicine, First Edition. Edited by Burkhard Madea.© 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

Gerhard Kernbach-Wighton and L. Aurelio Luna

34.1  Introduction

Postmortem biochemistry or thanatochemistry can be defined as: ‘The study of biochemistry parameters in the biological means of the cadaver, for solving the problems presented by the diagnosis of cause of death and the circumstances surrounding it (data, survival time, etc.)’. We can date the appearance of the term to 1963 when Evans published The Chemistry of Death, even though Naumann had published a paper in 1950 entitled ‘Studies on postmortem chemistry’.

The use of biochemical markers in forensic pathology is limited to a highly reduced number of pathologies and requires special laboratories. Apart from a few rare exceptions, the diag­nostic side of thanatochemistry is still limited mainly to the field of research. Complementary biochemistry analyses gain importance in the following circumstances:1. Metabolic morbid processes that do not leave morphologi­

cal traces in the majority of cases, although histochemical and pathological anatomy examinations are performed.

2. Iatrogenic and allergic processes.3. Violent deaths that do not leave morphological alterations.4. Cases of intoxication where the physiopathology consists

of biochemical alterations.5. Processes with a pathological anatomy alteration requir­

ing a minimum development time for results to be objective.

The circumstances where an additional test may be considered are diverse:

• To confirm a presumed diagnosis.• To exclude a possible diagnosis.• To guide a diagnosis in a confusing situation.• To interpret data that requires additional information.• As a routine test.

Postmortem alterations cause severe interferences to the analysis of parameters such as lactic acid, glucose, pH, and so on. This means limiting the number of elements in line with the death data and establishing a gradation for their use, trying to take the samples as quickly as possible. It is an absolute prior­ity to monitor the samples in accordance with the postmortem interval. In such a small time interval it is very difficult to give a full and systematic presentation of something so complex and variable as biochemistry studies for complementary diagnosis at autopsy.

The problems presented by biochemical techniques used on cadavers are as follows:1. The time the sample is taken. Establishing normal ranges

for different parameters to be evaluated. Many compounds undergo significant biochemical, qualitative and quantita­tive transformations due to the processes that initially begin during the agonal phase and then during autolysis and putrefaction.

2. Selecting where to take the sample from. It is important to establish a protocol for how to proceed with the collection of biological samples, so there are no significant differences.

3. The condition of the sample. The sample must be well­protected biological material, with no suspended cellular elements (reduction of the effects of autolysis).

34 Postmortem Biochemistry as an Aid in Determining the Cause of Death

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postmortem processes given that, at first the integrity of mem­branes may be affected in a lesioned area, but there is clearance of elements caused by the circulation influencing the surround­ing areas; whilst, secondly, in the cadaver this clearance process stops and, moreover, the release kinetics fit a very complex model. A classic accepted example of postmortem release is the kinetics complying with Fick’s law of simple diffusion. This model does not correspond to the biological reality where there is a series of processes in which membranes are affected by autolytic processes together with oncotic and osmotic pressure on either side of it. This means a constantly changing physical–chemical environment where the classic mathematical models are obviously inadequate and demand new models to be defined to reflect biological reality.

What information can biochemical analysis provide? From a practical perspective it gives information about the following:• Early processes of cellular suffering.• Established processes of cell necrosis.• Autolytic processes.

The basic fluids for postmortem biochemical and toxico­logical complementary studies are as follows:• Vitreous humour.• Cerebrospinal fluid (CSF).• Pericardiac fluid.• Femoral blood/serum.• Blood/serum from a jugular vein.• Blood/serum from the right ventricle.

The efficiency criteria for the selection of a biological fluid are as follows:• Accessibility.• Precautions when taking the sample.• Handling requirements of the sample (centrifugation, etc.).• Normal volume of the fluid.• Possibility of interference from surrounding tissues.• Possible interference from suspended cells.• The general information provided.• The locoregional information provided.Table 34.1 shows the different fluids and their characteristics in relation to the above criteria.

4. Artefacts following autopsy and sample collection.5. The biochemical composition of any organic fluid located

in a sealed compartment depends as much on the previous pathology as the alterations that originate from the imme­diate cause of death.

When choosing a specific parameter as a diagnostic element, there are two matters to be considered:1. The modifications caused by the postmortem interval may

interfere with the evaluation.2. That the specific marker exactly reflects tissue damage.

The ideal biochemical marker should meet all of the follow­ing conditions:1. It should be specific and uniformly distributed in the fluid

to be examined.2. It should be released only as a response to a cellular lesion

in which an alteration in the permeability of the cellular membrane is produced.

3. Its release kinetics should be conditioned by the nature of the mechanism responsible for damaging the cellular membrane.

4. It should provide a real estimate of the organic lesion.5. It should provide a real base for the diagnosis and quantita­

tive evaluation of the lesion.The biochemical determination of a marker will be closely

linked to its release kinetics, in which many factors will be involved. In this way, the speed at which it appears in circula­tion appears to depend on blood flow and the circulation pattern of the tissue in question. Damaged areas with poor blood perfusion will release products more slowly. Areas with better perfusion release biochemical elements more quickly.

In a cadaver, the postmortem diffusion of molecules gener­ally fits an exponential curve that is not defined by a simple diffusion model. The rupture membranes by the autolysis, the size of the molecules, differing concentrations and a relative stagnation of fluids located in the interstitial space will all be determining factors. Previous work has demonstrated the exist­ence of a postmortem circulation in a corpse, even causing renal filtration that can influence the determination of different enzymes. Firstly, there is a qualitative and quantitative differ­ence in the diffusion dynamics between vital processes and

Table 34.1  Efficiency criteria for choosing a biological fluid.

Vitreous humour Pericardial fluid Cerebrospinal fluid Femoral blood Blood right ventricle

Accessibility ***** ** * ** **

Sampling precautions ***** *** ** *** ***

Sample handling ***** **** *** *** ***

Volume available * *** *** **** ****

Possibility of interference **** *** * * *

General information ** ** ** *** ***

Locoregional information * ***** ***** * *

632  PART IV    SUDDEN AND UNEXPECTED DEATH FROM NATURAL CAUSES

The advantages of postmortem analyses of vitreous humour can be divided into the following characteristics:• It is easy to obtain a sample.• Isolated anatomical position (protected from trauma, burns,

autolysis, putrefaction, etc.).• It is a fluid with good (bio)chemical stability.• It is easy to analyse.• It is more resistant to bacterial contamination than blood.

34.1.2  Cerebrospinal fluid

Cerebrospinal fluid is very useful for studying both anoxic and traumatic brain injury. The importance and relevance of hypoxic brain injury evaluation through the study of enzymes in CSF has been described repeatedly. The marker enzymes in CSF, such as neuron­specific enolase and creatine kinase BB, can be used to confirm the extent of brain damage and its postmortem diagnosis in the absence of evident pathological anatomy and morphological findings, such as for example in diffuse brain injury and prolonged cerebral hypoxia.

34.1.3  Pericardiac fluid

Pericardiac fluid is the substrate of choice to study the bio­chemical expression of myocardial injury. The markers to choose are creatine kinase–muscle brain (CK­MB) and tro­ponin I.

34.1.4  Diagnostic value

There are many causes of death which, although they have an organic expression, are diagnosed clinically and on the body exclusively through biochemical tests: diabetes, hypoglycaemia, hypothermia, uraemic coma, hepatic coma, acute pancreatitis, electrolyte alterations, anaphylactic shock, acute myocardial infarction evolving quickly to death and infectious processes. In other cases, thanatochemistry is the test that complements the pathological anatomy diagnosis: myocardial infarction, death by fat embolism and wound vitality.

It is evident that functional mechanisms of death are often characterised by only sparse postmortem morphological changes. Therefore, differential diagnosis has to be based particularly on postmortem biochemical alterations which frequently originate from illnesses with internal causes and subsequent metabolic dysregulations such as diabetes mellitus, alterations of kidney and liver function, and imbalances of water and electrolytes. It is not rare that combinations of such disturbances with problematic overlappings are seen due to close physiological and biochemical links.

Postmortem biochemical analyses may represent the main clue to the diagnosis of functional mechanisms or causes of

34.1.1  Vitreous humour

Biochemical studies of the vitreous humour have proven to be extremely useful for postmortem diagnostics. Postmortem diffusion processes following the loss of the selective permea­bility of the cellular membranes are very quick and erratic in other bodily fluids. Also, any alterations present in the serum of living subjects will be reflected in vitreous humour.

Vitreous humour is anatomically protected and will suffer the phenomena associated with autolysis later. It is also located far from the large organs and blood vessels within the abdomi­nal cavity. It is most useful in the study of postmortem interval, with the concentration of potassium being the most used deter­mining factor. There is a notable and steady rise in the concen­tration of potassium after death.

Questions to be considered in biochemical analyses of the vitreous humour are as follows:• What are the ‘normal values’ in vitreous humour in com­

parison with the values in blood and serum?• Are alterations in serum reflected in vitreous humour? If so,

how soon?• Do the values in vitreous humour remain stable in the

corpse?It is very difficult to answer these questions, as ‘normal values’ in the vitreous humour do not exist due to them being extrapo­lations of postmortem values. The only source of in vivo infor­mation is enucleated eyes and they, obviously, are all pathological specimens. Furthermore, values in animals vary from one species to another. Studies to establish normal values for differ­ent biochemical parameters began with Naumann and were continued, in particular, by Coe and Madea. These studies analyse the values of different biochemical parameters relating to age, gender, cause of death, postmortem interval, previous state of health and antemortem serum values. To solve this problem, many studies have to be carried out using random samples. In addition, the values obtained are used as a mirror of the serum values, as postmortem serum concentrations may not reflect those existing at the time of death due to autolysis. Can the values in vitreous humour be taken as a reference for those in serum when the individual died? It seems this can be applied in cases where the concentrations obtained are particu­larly high.

The term ‘reference values’ has been introduced to resolve many of these difficulties. The reference values for a diagnostic test in clinical chemistry or forensic medicine must include five main categories of specifications:1. The reference population and how it was chosen.2. The atmospheric and psychological conditions the samples

were collected under.3. The techniques used and time spent collecting, transport­

ing, preparing and storing the samples.4. The analytical methods used with data on accuracy, preci­

sion and quality controls.5. The data observed and reference intervals derived.

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on the market, which can be used for such screening purposes. These ‘near­table’ methods are useful to support or exclude certain differential diagnoses at the time of the postmortem examination (for further information see http://www.roche­diagnostics.com (last accessed 15 April 2013)).

34.2  Glucose metabolism and diabetes mellitus

34.2.1  Clinical aspects of diabetic coma

Diabetic coma is a life­threatening complication of diabetes mellitus. Owing to a relative or an absolute insulin deficit, there is a typical rise of the blood sugar level with the possibility of further severe acute disturbances or serious damage to blood vessels and nerves after longer duration. Depending on the age group, the incidence of diabetes mellitus varies between approximately 2% and 5%. Causes of the development of coma episodes may include onset of unknown diabetes, missed insulin injections, increased requirement of insulin due to acute infections, poor diet, operations, gastrointestinal diseases or even myocardial infarctions. Twenty­five per cent of all diabetic comas are so­called manifestation comas with previous undi­agnosed diabetes mellitus. Infections are considered the most frequent triggers for coma onset (c. 40% of cases). The fre­quency of fatal comas among known diabetics ranges between 0.5% and 1.5% in the age group 40–60 years. The overall lethality from coma varies from 5% to 25% and rises to 70% with coma of longer duration. Lethality of diabetic coma is 10­fold in 70­year­old individuals compared to 30­year­old patients. Furthermore, the risk for coma in juveniles is 4–7­fold higher than in adults (Table 34.2).

death. One of the main problems is to be in a position to apply clinical biochemical values on postmortem conditions. On the one hand, there are considerable unpredictabilities regarding general postmortem changes in body fluids. On the other, bio­chemical values in postmortem specimens may well represent more or less the results of changes taking place during the agonal or the early postmortem period. In contrast to clinical biochemical estimations, values obtained postmortem do not necessarily allow conclusions regarding the mechanism of death. Postmortem diagnostic procedures therefore require a critical way of looking at them.

Bodily fluids are usually obtained during the postmortem examination. In cases with a limited external examination appropriate samples of, for example CSF, vitreous humour, blood and urine can also be taken by cannulation (e.g. suboc­cipital access, puncture of the eyeball or dissection of the femoral vein and puncture of the urinary bladder). The cranial cavity and the eyeball provide rather good protection of the enclosed bodily fluids against the effects of decomposition. After obtaining vitreous humour, the eyeball should be refilled with water due to piety and cosmetic reasons. The volume of CSF to be found varies from c. 50 mL (baby) to c. 135 mL (adult). A few millilitres are sufficient for postmortem bio­chemical analysis and there is usually no problem obtaining blood­free CSF. Approximately 1–2 mL of vitreous humour can be obtained by the puncture of both eyeballs. Possible aspira­tion of small parts of the retina is of no further relevance. Postmortem blood should be taken from the heart and a (peripheral) femoral vein, and urine from the bladder imme­diately after dissection (a few millilitres per specimen).

During the postmortem examination, several screening tests with diagnostic strips and tablets can be carried out focused on the levels of for example glucose, bilirubin or ketone bodies. Furthermore, there are a number of electronic devices

Table 34.2  Postmortem biochemical values in case of alterations in glucose metabolism.

Dysfunction Parameter Compartment Results

Coma (in general) Sum valuea Cerebrospinal fluid Σ > 415 mg/dLb

Vitreous humour Σ > 410 mg/dLc

HbAlc Blood >12.1%d

Glucose Urine >25 mg/dLe

Ketotic coma Acetone Blood, cerebrospinal fluid >21 mg/dLf

Vitreous humour >5 mg/Lf

Urine

Hypoglycaemia Sum value Cerebrospinal fluid Σ < 50–80 mg/dLVitreous humour Σ < 100–160 mg/dL

a According to Traub: concentrations of glucose and lactate.b Mean value = 500–600 mg/dL.c Mean value glucose = 300–950 mg/dL.d Mean value = 13–15%, non-diabetics = 9.15%.e Most coma cases >50 mg/dL, partly 2000–4000 mg/dL.f Coma: mean value = 100–150 mg/L.

634  PART IV    SUDDEN AND UNEXPECTED DEATH FROM NATURAL CAUSES

asphyxia, pneumonia and pancreatitis. This aspect has also to be taken into account regarding other body fluids.

Postmortem diagnosis of diabetes appears therefore an area where thanatochemistry provides a number of possibilities based on studies of glucose, lactate, fructosamine and β­hydroxylbutyrate. Normal levels of glucose in vitreous humour are c. 0–100 mg/dL. They have a diagnostic value if they are high, but no value if they are low. A high level of glucose in vitreous humour is quite a reliable indicator of a high level of glycaemia antemortem. Levels of glucose in vitreous humour above 200 mg/dL are considered indicative of diabetes mellitus. Even if a glucose infusion is administered prior to death, glucose in vitreous humour of normal subjects will be below 200 mg/dL. The glycated haemoglobin in cardiac blood is one of the markers for chronic hyperglycaemia that correlates with values obtained from normal subjects. In cases of death by diabetes levels of 427 mg/dL of glucose and 420 mg/dL of lactate in vitreous humour have been observed. Examining glucose in vitreous humour can be equally interesting in cases of death from hypothermia where an increase glucose levels in vitreous humour has been detected.

34.2.4  Lactic acid (lactate)

The product of postmortem glycolysis is lactate (normal level in CSF c. 9 mg/dL). Its concentration increases postmortem with a rate of approximately 10–15 mg/dL per hour up to the tenth hour following death. After this time, the increasing rates may vary considerably. Under differential diagnostic aspects other disorders may also cause hyperlactacidaemia, for example tumours, respiratory insufficiency, severe chronic inflamma­tions, uraemia, particularly inflammations of the central nervous system or alcohol­induced types with lack of thiamine, physical strain and also alimentary factors (e.g. strict fasting).

34.2.5  Sum value according to Traub

This combined calculation method, according to Traub, com­pensates arithmetically the postmortem production of lactate from glucose by using a ‘sum value’. This is based on the fact that 1 mol of glucose produces, via glycolysis, 2 mol of lactic acid so that the concentrations can be added using milligrams per decilitre. If the sum value exceeds 362 mg/dL in CSF, the probability of fatal diabetic coma is about 89%, if other, toxi­cological and morphological alterations can be excluded. In cases of diabetes mellitus, the sum value remains almost stable up to the 200th hour postmortem. If there are non­diabetic causes of death, the sum value increases up to the 30th hour postmortem, but remains nearly stable afterwards. Although the formula, according to Traub, has always to be used under critical view, the sum value may be considered the most impor­tant criterion for the diagnosis of fatal diabetic coma. However, the author’s own research has revealed that it appears to be more realistic to increase the limit sum value in CSF to

34.2.2  Types of diabetic coma

In typical cases, diabetes mellitus type I is associated with ketoaemic coma, whereas hyperosmolar coma normally appears as a consequence of type II diabetes mellitus. A lack of insulin results in a rise of the blood sugar level with subsequent loss of fluids and electrolytes. Additionally, the body uses increased lipolysis to compensate the energy deficit resulting from the inhibition of glucose metabolism leading to increased levels of ketone bodies with metabolic acidosis. The latter may be excessive (c. 500–1000 mg/L acetone or even higher), whereas hyperglycaemia remains mostly moderate (approximately 250–600 mg/dL). Hyperosmolar coma is more rare (around 10–20% of the cases) and associated with relative insulin deficit causing reduced peripheral utilisation of glucose with simulta­neous release of glucose from the liver. Low levels of insulin prevent ketosis due to inhibition of lipolysis. Therefore, it is typical to find excessive hyperglycaemia (often exceeding 1000 mg/dL) with ketosis lacking or being mild.

Diabetic coma may result in fatal outcome via different pathophysiological pathways. Among others, there can be dif­ferentiated a cardiovascular type with predominant oliguria or a renal type with acute kidney failure. Moreover, there exists a pseudo­peritonitis type with the symptoms of an acute abdomen. Typical accompanying diseases of fatal diabetic decompensation may be myocardial infarction, apoplexy, embolism, pneumonia, pancreatitis, pyelonephritis and a pre­disposition for lactic acidosis.

The most important bodily fluids for postmortem diagnos­tic purposes are CSF and vitreous humour using the so­called sum value according to Traub, which provides a combined calculation to compensate postmortem alterations of blood glucose level due to glycolysis, accordingly. Furthermore, blood can be used for estimations of the HbA1c level to assess the long­term stability of the glucose metabolism. Urine analyses can reflect acute acute periods of glucosuria and ketonuria.

34.2.3  Glucose levels

The hourly metabolic decrease of glucose in CSF is approxi­mately 10–15 mg/dL but may vary between c. 5 and 45 mg/dL. The hourly rate is expected to be below 1 mg/dL around 100 hours postmortem. Given normal metabolic conditions, there­fore, zero levels are reached after 10–12 hours. Longer persisting glucose levels are indicative of antemortem hyperglycaemia.

The speed of postmortem glycolysis depends on a number of factors (e.g. the temperature and duration of body storage). Postmortem glycolysis is slower in diabetics compared to non­diabetic individuals, whereas obesity accelerates degradation of glucose. Isolated assessment of elevated glucose levels in CSF requires critical reserve (normal range c. 50–90 mg/dL), because multiple other dysregulations may be accompanied by the same symptom, such as carbon monoxide poisoning, acute cardiac death, brain trauma, strangulation, protracted agonal period,

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reduction of HbA1c because of separation of its unstable com­ponent. Blood sugar also decreases rapidly after death. The stable part of haemoglobin A1c makes up approximately 90% of the whole. For example, hyperglycaemia around 360 mg/dL takes around 12 hours to cause an increase of HbA1c of c. 1.3% absolute. In reverse, a reduction of around 5% needs approxi­mately 7 days.

There has been found a positive connection between sum value, urine glucose concentration and HbA1c level. This means that there usually is a coincidence of elevated sum value, high urine glucose and elevated HbA1c. Haemoglobin A1c has proven to be relatively stable versus autolysis especially in haemolysed blood and can be measured postmortem in frozen samples and also in samples stored in a normal fridge. It has been revealed that storage at temperatures between +4 and –80°C does not cause any relevant changes to the HbA1c concentrations. The results are independent from the actual total haemoglobin level because HbA1c is mostly measured as a percentage of the current haemoglobin value.

Falsely elevated haemoglobin HbA1c concentrations can be found due to increased fetal haemoglobin (HbF) levels in cases of thalassaemia or advanced renal failure. In principle, HbA1c has proven to be a reliable parameter for basic diagnosis of diabetes mellitus without being too liable for interferences. It is also possible to measure other glycosylated proteins such as fructosamine, but assessment has proved to be rather difficult. The mean levels of HbA1c in cases of diabetes mellitus differ considerably from those in non­diabetic individuals and are around 12.1% in diabetic coma (range c. 13–15%). However, the lower portion of the range in case of diabetes mellitus may overlap with the upper portions of the range in non­diabetic cases as has been shown for the sum value.

34.2.9  Ketone bodies

Ketotic diabetic coma is characterised by an increased level of ketone bodies in blood and other bodily fluids (acetone and acetylacetate c. 25–35%, β­hydroxylbutyrate c. 65–75%; normal values for acetylacetate 0.8–2.4 mg/L, for β­hydroxylbutyrate 2.5–9.8 mg/L). Estimation of acetone may easily be carried out in connection with blood alcohol analysis using headspace chromatography. The normal concentrations or free acetone range from c. 2.3 to 2.5 mg/L in non­diabetic patients and may reach around 23 mg/L in diabetics. The levels are almost inde­pendent from the postmortem interval.

The levels of acetone in CSF with diabetes mellitus differ considerably from those seen with non­diabetic causes of death, especially in cases of diabetic coma, with an obvious association regarding an elevated sum value. If other causes of death can be ruled out, acetone levels exceeding 5 mg/L are suspicious of diabetes mellitus. Ketotic coma may be associated with levels higher than 100 mg/L, but ketonaemia is rarely seen if the blood glucose concentration is only 200 mg/dL and below. According to the authors’ research, acetone levels in ketotic

415 mg/dL (upper limit of the 95% confidence interval in cases of cardiac death), with cases of diabetic coma ranging on average between c. 500 and 600 mg/dL.

34.2.6  Conditions in vitreous humour

The calculation method according to Traub may also be applied on vitreous humour. The glucose level herein is about 50–85% of the serum glucose. Values for postmortem glucose concen­trations vary from 20 mg/dL (non­diabetics) to 90 mg/dL (known diabetics), but wide variation ranges have to be taken into account. Owing to slower glycolysis in vitreous humour compared to CSF, normal glucose values may be found as long as 2 days postmortem. In cases of fatal coma, glucose levels between c. 300 and 950 mg/dL may be found. Lactate values are already around 80–160 mg/dL in the intramortal period and between c. 210 and 260 mg/dL approximately 20 hours post­mortem. The upper limit value is given as around 410 mg/dL and if it is exceeded, it can be taken as a strong indication of fatal diabetic coma, given the condition that other possibly competing mechanisms can be excluded. The procedure is said to be applicable until the 10th postmortem day.

34.2.7  Value of blood glucose estimations

Blood sugar alone are only of low diagnostic relevance, if at all limited to peripheral blood from the femoral veins within the first and second hour postmortem in which the level is c. 40–100 mg/dL. In contrast, glucose levels in central blood (right ventricle of the heart) may easily reach 1000 mg/dL and over due to postmortem hepatic glycogenolysis. Normally, postmor­tem glycolysis (approximately 13 mg/dL per hour) results in complete metabolisation of the blood glucose within 6–8 hours. This leads to a corresponding increase of lactate up to 180 mg/dL after 1 hour and c. 450–680 mg/dL after 12–24 hours. Espe­cially due to postmortem diffusion of serum and its compo­nents from surrounding tissues into blood vessels, the sum value cannot be used on blood.

34.2.8  Value of haemoglobin HbA1c

This glycosylated fraction of haemoglobin represents an impor­tant parameter regarding a basic diagnosis of diabetes mellitus. Owing to the fact that kinetics of its formation is depending on time and glucose concentrations, HbA1c can be used as a long­term indicator of diabetic conditions (so­called blood sugar memory for c. 120 days). Levels of 6–8% (maximum of 10%) are consistent with a normal glucose metabolism, whereas higher concentrations are indicative of inappropriate metabolic conditions (hyperglycaemias in the past). Periods of increased blood sugar have to last for 6–8 hours minimum to cause sig­nificant rises of HbA1c due to its slow reaction kinetics. Further­more, the prefinal and postmortem drop of the pH value in blood, caused by formation of lactate, are likely to result in a

636  PART IV    SUDDEN AND UNEXPECTED DEATH FROM NATURAL CAUSES

of acids and also by an increased loss of bicarbonate resulting from diarrhoea and/or vomiting.

The central causal mechanism is an increased concentration of pyruvate from protein catabolism together with a lack of oxygen, so that energy can still be provided by glycolysis. Accu­mulation of lactate happens more frequently in diabetics than in other patients due to disturbances of oxygen supply and alterations of metabolic activities. The clinical picture is char­acterised by gastrointestinal discomfort, muscular spasms, central nervous disturbances and deep frequent respiration. The severe type of biguanide­induced lactic acidosis shows a lethality rate of over 50%.

Patients suffering from chronic alcoholism represent a special risk group regarding fatal lactic acidosis and ketotic coma. There are often very few and/or non­specific morpho­logical findings. On the one hand, considerable ketoaemia may follow acute alcoholisation (free acetone from c. 74 to 400 mg/L), but on the other hand, high ‘sum values’ may also result in this condition. Their range (c. 294–594 mg/dL) can also be associ­ated with fatal diabetic coma. Given the precondition that dia­betes mellitus and other competing mechanisms can be ruled out, ketotic coma or lactic acidosis have to be considered as a cause of death in such cases. The lower limiting values for the sum value are c. 300–400 mg/dL, for acetone in blood around 90 mg/L and 6% for HbA1c.

34.2.12  Hypoglycaemia (endogenous versus exogenous hyperinsulinism)

Although fatal hypoglycaemia appears to be a rather rare event among forensically examined death cases, they might be the source of serious diagnostic problems. Under clinical condi­tions, hypoglycaemia is diagnosed if the blood glucose level lies below 40 mg/dL or if the so­called Whipple’s triad can be found. It comprises a blood glucose level below 45 mg/dL, symptoms of hypoglycaemia and relief of these symptoms when the glucose level is raised to normal. Multiple circumstances may be responsible for hypoglycaemia in individuals with an empty stomach (e.g. insulinomas and other tumours, severe hepatic disease, uraemia, glycogenoses). The initial manifestation of diabetes mellitus may also be accompanied by reactive hypogly­caemia as well as alterations of gastric mobility, vegetative insta­bility or massive alcohol intake with simultaneous lack of food due to inhibition of gluconeogenesis. The autonomous or glucopenia­associated spectrum of symptoms includes hypero­rexia, nausea, restlessness, sweating, tachycardia, endocrine neuropsychological disorder, primitive automatisms, risk of convulsions and focal signs with apoplectiform symptoms. The final state with somnolence, coma and central nervous altera­tions of respiration and circulation until death has forensic medical relevance.

Hypoglycaemias due to exogenous causes are mostly seen with an existing diabetes mellitus. Important mechanisms are accidental or intentional overdosage of insulin or sulphonylu­

coma exceed 21 mg/L in most of the cases, with mean values in this group of 100–150 mg/L. Single cases may show levels of more than 1000 mg/L. Non­diabetic factors that might cause elevated ketone levels include chronic hepatic and renal disease, pancreatitis, shock, chronic alcoholism and isopropanol poi­soning (levels up to 160 mg/L) as well as protracted fasting (acetone levels may exceed 5000 mg/L).

34.2.10  Urine

As the fourth column of postmortem diabetes mellitus diag­nostics, an examination of urine can reveal important clues. Urine glucose levels higher than 25 mg/dL (maximum in healthy individuals) may be indicative of diabetes. Diabetic coma is sometimes associated with urine glucose concentra­tions above a few 1000 mg/dL, but usually higher than 500 mg/dL. These excessively high values only show very small overlap with other causes of death, although positive findings for glucose in urine are only of lower diagnostic value. Glucosuria is a rather frequent non­specific symptom (e.g. due to brain trauma, myocardial infarction, intoxication, apoplexia and leu­kaemia). Likewise, glucosuria may be absent even in cases of manifest diabetes mellitus caused by diabetic glomerulosclero­sis itself or postmortem degradation. Ketone bodies are likely to be found in urine more than 24 hours postmortem. Concen­trations exceeding 0.5 mg/dL (=5 mg/L) of free acetone may be indicative of ketotic dysregulation. However, a positive test for ketonuria is not a proof for ketonaemia, because the kidneys have a relatively high clearance rate for ketone bodies. Further­more, there are multiple conditions that might cause consider­able ketonaemia (see Sections 34.2.1 and 34.2.2). Hyperosmolar coma is typically characterised by a lack of ketonaemia (approx­imately 30% of diabetic comas).

34.2.11  Lactic acidosis

There are some secondary effects of lactic acidosis that might gain special forensic relevance. For example, moving potassium to the extracellular space may cause hyperkalaemia. Acidosis decreases the reactivity versus catecholamines with a negative­inotrope effect on the heart. Severe acidosis may result in massive reduction of the kidney blood circulation leading to acute renal failure. Diabetic coma can also cause acidosis by production of β­hydroxylbutyrate and acetylacetate (see Sec­tions 34.2.1 and 34.2.2). Lactic acidosis plays an important role, particularly regarding overlap with postmortem diagnosis of diabetes mellitus. Considerable amounts of lactic acid are being released during shock and hypoxia, due to poor perfusion caused by diabetes mellitus, following renal failure, hepatic disease and ethanol/methanol intake, rarely as a complication of treatment with biguanides or due to severe lack of thiamine with chronic increased alcohol intake. The conditions can be exacerbated by chronic renal failure due to reduced excretion

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because diffusion of insulin from the pancreas via the portal vein might take place postmortem. The sum value calculated from glucose and lactate levels is of special importance (see Section 34.2.5).

34.3  Alterations of liver function

In cases of advanced stage hepatic cirrhosis from different causes, it is not rare that there develops an alteration of liver metabolism, often resulting in potentially reversible complica­tions, due to retention of neurotoxic substances in blood with decompensation and final hepatic failure. Suspicion may arise from the previous medical history, desolate housing conditions, known alcohol abuse and sometimes the presence of jaundice. Acute deterioration of hepatic insufficiency with a danger of hepatic coma originates from an increased production of ammonia due to a high proportion of proteins in the intestinal contents that may be caused by gastrointestinal haemorrhages (especially oesophageal varicosis due to alcoholism), protein­rich nutrition, febrile infections with increased protein catabo­lism and drugs (e.g. benzodiazepines, analgesics). Clinically, the advanced stage is characterised by permanent drowsiness but patients can be woken up, later on hepatic smell, and electro­encephalogram (EEG) alterations. This picture leads to coma with unmistakable fetor hepaticus and massive EEG changes until fatal outcome with total hepatic failure.

The terms acute hepatic insufficiency or endogenous hepatic coma describe a failure of liver function without previously existing chronic liver disease. Contrary to chronic hepatic failure, decompensation can occur suddenly without any indi­cations from the medical history. Important morphological findings are dermal and scleral jaundice and, clinically, distur­bances of blood coagulation and consciousness (such as som­nolence, coma). This is especially of the fulminant type with duration of less than 7 days, which may gain forensic medical relevance. Important causes are viral hepatitis (65%) and hepa­totoxic substances (30%) such as medication (acetaminophen), drugs, chemicals (carbon tetrachloride) or poisons from mush­rooms (Amanita phalloides). This elucidates the importance of accompanying toxicological analyses. Potentially fatal compli­cations may be brain oedema (80%, most frequent cause of death), gastrointestinal haemorrhages (50%) as well as hypogly­caemia and renal failure with electrolyte imbalances.

The typical enzymes of liver metabolism represent impor­tant parameters, which can also be examined postmortem, as does bilirubin measurement. Daily bilirubin production comes to approximately 510 μmol/L (30 mg/dL; normal value up to 1.1 mg/dL). Hepatic failure is typically associated with an increased level of serum bilirubin causing jaundice if it exceeds c. 34 μmol/L (2 mg/dL). Differentiation between direct bilirubin bound to biglucoronide and non­direct bilirubin bound to albumin is only useful under clinical circumstances. Postmortem bilirubin levels may compare with those obtained

rea derivatives with subsequent reactive hypoglycaemia. Such a situation may arise from lack of regular alimentation due to intercurrent diseases without changing the doses of antidiabetic drugs. Other possibilities for hypoglycaemias can be interfer­ences with drugs which decrease the blood sugar level indirectly or unusual physical strains. However, types of hypoglycaemia with a forensic medical impact are those caused by overdose of antidiabetics.

The so­called factitious hypoglycaemia needs special atten­tion. It is caused by (unnecessary) administration of insulin or sulphonylurea derivatives and can be seen in connection with psychic alterations (e.g. borderline personality disorder) or sui­cidal intention. It is rare to find a primary criminal background (e.g. cases of homicide). The most important diagnostic crite­rion of this type of hypoglycaemia is that it happens independ­ently from alimentation. Affected persons often have relations to professional health care or are relatives of known diabetics.

The calculation procedure regarding the sum value can also be used for the diagnosis of hypoglycaemia. Consequently, low ‘sum values’ in CSF and in vitreous humour below c. 50–80 mg/dL or rather 100–160 mg/dL are strongly indicative of fatal hypoglycaemia. This conclusion is particularly supported by simultaneously high insulin levels suggesting that estimation of insulin levels and also of C­peptide postmortem is essential. In cases of endogenous secretion, insulin and C­peptide are both found to be elevated. If there is exogenous hypoglycaemia due to administration of insulin, the level of C­peptide will be noted as much lower than normal. An administration of exog­enous insulin can also be diagnosed by the quotient between the level of insulin (increased) and C­peptide (diminished) in serum. Contrary to this, there are usually increases of insulin and C­peptide concentrations following an intake of sulphony­lurea derivatives, but, in diabetic individuals, often rather high insulin levels can be seen without any indication of hypogly­caemia. The procedure has also proven to be reliable in cases of suspected hypoglycaemia in car drivers.

Postmortem estimations of insulin levels can be carried out by radioimmunoaessay (RIA) and have revealed levels very similar to those of healthy individuals in blood from femoral veins and also from the heart. Nevertheless, postmortem con­centrations of insulin in blood from the right ventricle may be increased about 10­fold of normal values due to release of insulin after death. Putrefaction may cause problems as well. Furthermore, single estimations may have a wide variation and therefore cannot be used as the only criterion for the diagnosis of insulin­based hypoglycaemia. Sometimes it is possible and useful to have a proof of suicidal insulin injection by analyses of the tissues close to the injection site. It is a strict rule that the postmortem diagnosis of hypoglycaemia must be based on a combined assessment of different criteria and can only be made as a diagnosis of exclusion. According to this, especially cardiac disease, cerebral haemorrhages, pulmonary embolism, strangulation/asphyxia, rupture of vessels and intoxication have to be ruled out. Estimations of insulin should always be carried out in peripheral venous blood or CSF/vitreous humour

638  PART IV    SUDDEN AND UNEXPECTED DEATH FROM NATURAL CAUSES

include diabetes mellitus (nephropathy, c. 35%), hypertension (c. 25%), chronic inflammations (c. 15%) and abuse of analget­ics (c. 1%). Chronic reduction of renal function can also show acute decompensation leading to unexpected sudden death, which is not an unusual development during diabetic coma. The compensated chronic phase showing only functional reduction of a low degree and the phase of compensated reten­tion (azotaemia, creatinine levels up to 6 mg/dL) are not associ­ated with symptoms of uraemia. Preterminal renal failure with creatinine levels above 8 mg/dL plus symptoms of uraemia is called decompensated retention. Terminal renal failure (uraemia), showing creatinine levels over 10 mg/dL, is associ­ated with massive symptomatology of uraemia. During the phase of decompensated retention (preterminal phase), there may be oedematous changes, cardiac failure, gastroenteritis due to uraemia and neuropathy. The terminal phase is characterised by acute life­threatening symptoms, such as neuropathy and encephalopathy, overhydration with pulmonary oedema, bleed­ing tendency, coma and death (Table 34.3).

antemortem. Differences only range around 0.1 mg/dL, espe­cially in death showing jaundice. During the postmortem period, a slight but steady increase can be seen (c. 0.2 mg/dL after 2 hours and 0.7 mg/dL after 20 hours). Furthermore, there is an increase of enzymes typical for liver – glutamate pyruvate transaminase (GPT), γ­glutamyl transferase (GGT) and alka­line phosphatise (AP) – as well as of ammonia (>100 mg/dL; normal value below 0.05 mg/dL) primarily but not only in the blood but also in other bodily fluids (e.g. CSF, vitreous humour). However, clinical reference ranges of values can only be used as a basis for assessment. Most of the bilirubin in CSF belongs to the conjugated type, often associated with hypokalaemia and hypoglycaemia.

34.4  Disturbances of kidney function

Chronic renal failure represents the result of a non­reversible reduction of the function of both kidneys. Important causes

Table 34.3  Postmortem biochemical values in cases of renal failure (insufficiency).

Dysfunction Parameter Clinical values Compartment Results

Compensated retention Creatinine ≤6 mg/dL

Preterminal failure >8 mg/dL

Terminal failure >10 mg/dL

CSF/VH Creatinine:

RF ruled out <2.5 mg/dL

RF possible 2.5–4.0 mg/dL

RF primary fatal >4.0 mg/dL

Normal values Blood (heart) Maximum 179((urea–nitrogen)/urea)a (83) mg/dL

(mean value = 102)(47) mg/dL

CSF Maximum 197(92) mg/dL(mean value = 89)(41) mg/dL

Uraemia (First 13 hpm) Blood and CSF >200 mg/dL (93)((urea–nitrogen)/ureaa)dysfunctionb

CSF/blood (heart) CSF Blood (heart)urea: creatinine: creatinine:

RF ruled out <100 mg/dL <2.5 mg/dL <3.5 mg/dL

RF possible 100–200 mg/dL 2.5–4.0 mg/dL 3.0–4.5 mg/dL

RF primary fatal >200 mg/dL >4.0 mg/dL >4.5 mg/dL

CSF, cerebrospinal fluid; hpm, hours postmortem; pm, postmortem; RF, renal failure; VH, vitreous humour.a Urea–nitrogen × 2148 (mg/dL) = urea (mg/dL).b Different method of assessment (see text and references).

  CHAPTER 34    POSTMORTEM BIOCHEMISTRY AS AN AID IN DETERMINING THE CAUSE OF DEATH  639

atinine retention but partial renal function can still occur during uraemia.

34.4.2  Urea

In cases of renal failure, there exists a rather close correlation between the levels of urea in serum and CSF (normal range: 13.8–34.6 mg/dL). The urea level in CSF is approximately three­quarters of the serum value. However, there have been reported reduced levels in CSF and also slight increases in blood from the femoral veins compared to antemortem values and also independent from the cause of death. If renal disease can be excluded, such changes may be due to agonal or postmortem effects. Furthermore, there is a rising difference between the concentrations of urea in liquor and blood with increasing postmortem interval. Often postmortem values are slightly higher compared to intravital estimations. However, this increase is lower if the intravital concentration has been rather high. In cases of manifest renal insufficiency, possibly with uraemia, there are usually considerable differences to the levels found in healthy individuals.

There is an arithmetical connection between urea–nitrogen and urea as follows: urea–nitrogen × 2.148 (mg/dL) = urea (mg/dL). Urea levels in CSF above 20 mg/dL (9.3 mg/dL urea–nitrogen) are indicative of renal disease, whereas the postmor­tem ‘normal values’ for blood from the heart is 179 mg/dL maximum (83 mg/dL urea–nitrogen), with a mean value of 102 mg/dL (47 mg/dL urea–nitrogen). The corresponding con­centrations in CSF are 197 mg/dL (92 mg/dL urea–nitrogen) with a mean value of 89 mg/dL (41 mg/dL urea–nitrogen). Con­trary to this, urea levels in CSF and blood from the heart do usually exceed 200 mg/dL (93 mg/dL urea–nitrogen) during the first 13 hours postmortem in the case of uraemia from all imaginable causes.

34.4.3  Diagnosis

Postmortem estimation of creatinine and urea levels in blood from the heart (left ventricle preferred) and CSF have impor­tant relevance regarding the postmortem diagnosis of renal failure. The following ranges of values can be differentiated for a practicable combined diagnostic procedure:1. Urea below 100 mg/dL in CSF/blood, creatinine below

2.5 mg/dL in liquor and below 3.5 mg/dL in blood: renal failure can be excluded.

2. Urea 100–200 mg/dL in CSF/blood: renal failure possible if there is an additional creatinine level of 2.5–4.0 mg/dL in liquor and of 3.0–4.5 mg/dL in blood from the heart.

3. Urea above 200 mg/dL in CSF or blood: renal failure rep­resents the primary cause of death if creatinine levels in liquor simultaneously exceed 4.0 and 4.5 mg/dL in blood from the heart.

Acute renal failure or acute renal insufficiency represent a mostly reversible reduction of renal function with loss of urine production and increasing retention parameters (urea, creati­nine). Fifteen per cent of cases with acute renal failure show polyuria or normuria with an increase of retention values being the only symptom. Without sufficient therapy (e.g. dialysis), acute renal failure mostly has a fatal outcome. Sometimes bilat­eral necroses of the renal cortex can be seen. There are multiple possible causes for acute renal failure, such as alterations of the blood circulation, toxins, medication (antirheumatics, cytostat­ics and antibiotics), chemicals (glycols) and inflammatory or vascular processes.

The most critical clinical phase is the third one with polyuria and extensive loss of water/electrolytes and simultaneous increase of urea and creatinine. Fatal complications may occur associated with other organs, for example shock lung, cardiac failure and arrhythmia, and cerebral oedema with further central nervous system complications. The most significant biochemical changes of acute and chronic renal failure are increased levels of urea and creatinine, electrolyte imbalances (often decreased with acute renal failure) and reduced concen­tration of urine. In summary, urea and creatinine are com­pounds that appear relatively stable postmortem. Postmortem results reflect well the antemortem figures and are therefore rather valid for diagnosing pathologies accompanied by an increase of urea and creatinine such as kidney failure, hyper­thermia and hypothermia, as well as methamphetamine toxicity.

34.4.1  Creatinine

Under postmortem conditions, an increased level of creatinine in CSF and vitreous humour can be indicative of renal failure (normal range 0.6–1.4 mg/dL). During the early postmortem interval, the creatinine concentration is rather stable. In healthy individuals, the mean values are 1.6 mg/dL (8 hours postmor­tem), 1–2 mg/dL (12 hours postmortem) and 3–4 mg/dL (24 hours postmortem). Therefore, reliable assessment is possible for pathological levels if the specimens are obtained during the early postmortem period.

Renal failure can be ruled out if the creatinine level is below 2.5 mg/dL. It is possible if its concentration ranges between 2.5 and 4.0 mg/dL and renal failure is to be considered as the primary cause of death with levels exceeding 4.0 mg/dL, if CSF is obtained within the first few hours postmortem. After death, the normal relation between creatinine levels in serum and CSF remains almost the same.

On the one hand, problems may arise from a connection between renal damage and creatinine level. On the other hand, high creatinine values are seen without any or only slight altera­tions of the kidney. However, there is also the possibility that advanced kidney damage coincides with levels below 4 mg/dL. Disturbances of the circulation and toxicaemia may cause cre­

640  PART IV    SUDDEN AND UNEXPECTED DEATH FROM NATURAL CAUSES

sudden death (acute myocardial failure due to arrhythmias). Particularly, intestinal or renal loss or insulin treatment of dia­betic coma are likely to result in hypokalaemia (<3.6 mmol/L). The main causes of hyperkalaemia (>5.0 mmol/L) are acute renal failure, chronic renal insufficiency or extensive tissue damage. The main possible complications are disturbances of conduction, ventricular flutter and fibrillation, which may lead to asystolia (acute danger to life with potassium levels >6.5 mmol/L).

Estimation of potassium in blood and serum specimens obtained postmortem have proved not to be reliable due to extremely fast and intense potassium release from cytolysis. In CSF, the potassium value can reach up to seven­fold of the normal level within the first 10 hours postmortem, but the range of variation is rather wide. The potassium content of liquor is largely independent from serum level and in infants lower than in adults (normal range: c. 2.1–4.6 mmol/L). Con­trary to this, increase of potassium concentration in vitreous humour has been reported to be regular. This can provide certain conclusions regarding the time of death within the first 12 hours postmortem. There seem to be no other relevant dis­turbances from other diseases on the potassium content of vitreous humour except hepatic failure. Furthermore, there are no comprehensible associations between concentration differ­ences of sodium and potassium that allow further reliable conclusions.

34.5.2  Sodium and chloride

There is an extracellular decrease in sodium parallel to an increase of potassium postmortem (see Section 34.2.2). Gener­ally, variation in sodium level within CSF mostly corresponds to serum concentration (c. 128–157 mmol/L), except in situa­tions of severe infection of the central nervous system.

Without differentiation regarding the mechanism of death, sodium levels in CSF and serum are usually found within the normal range, but the variation range differs considerably from intravital values (c. 123–205 mmol/L). Although there is a dis­tinct decrease of sodium in CSF and serum after death, its concentration in vitreous humour remains rather stable up to 30 hours postmortem, followed by an almost linear decrease in the following 50 hours. Sodium levels above 155 mmol/L and below 130 mmol/L in adults and larger differences outside the normal range in children can be indicative of hypernatraemia or hyponatraemia antemortem. Sodium levels in fluid from the pericardial sac show distinct correlation to postmortem inter­val, namely, a decrease of approximately 0.4 mmol/L during the first 85 hours after death, but there is also a wide range of variation.

The level of chloride in CSF is approximately 20% higher compared to serum and shows a range of c. 110–129 mmol/L in healthy individuals. The postmortem changes of chloride are comparable to those of sodium, so that there is also a typical decrease of chloride concentration in plasma and CSF. The

34.5  Water and electrolyte imbalances

Regulation of water and electrolyte balance aims to maintain isotonia and isovolumia within the intravasal space. Sodium, chloride and bicarbonate show the highest extracellular con­centrations, whereas potassium and phosphoric esters predom­inate in the intracellular space. Because the relation between extracellular fluid volume and water exchange is much lower in infants than in adults, water imbalances may develop much earlier and be life­threatening.

It is not rare for electrolyte imbalances to occur due to other diseases such as diabetes mellitus, chronic alcoholism and nutritive disturbances. There are some types of dysregulation that can lead to sudden unexpected death and may therefore be of medical forensic relevance. Isotonic dehydration is charac­terised by extracellular loss of sodium and water in isotonic relation, for example during the polyuric phase of acute and chronic renal failure, vomiting and diarrhoea, pancreatitis and peritonitis, and due to dermal loss (following burn injuries). The main mechanism of hypotonic dehydration is salt deple­tion together with an extracellular deficit of water. Delirium and convulsions are typical cerebral symptoms, which have to be considered as causes of sudden death. Hypertonic dehydra­tion (with hypernatraemia) leads to a deficit of free water in the extracellular and also in the intracellular space and is caused by factors such as a lack of water supply, dermal loss (sweating) and loss via the lungs (e.g. hyperventilation from infections and fever), the kidneys (diabetic coma) and the gastrointestinal tract (diarrhoea, vomiting). Typical morphology comprises tightening of the skin, sunken eyes, galea dryness and/or dry cutting area of organs. A biochemical pattern was proposed as a diagnostic tool. The so­called dehydration pattern consists of an elevation of sodium >155 mmol/L, chloride >135 mmol/L and urea >40 mg/dL. Persisting imbalances also result in cor­responding alterations within the CSF (osmotic gradient).

Regarding the postmortem diagnosis of water and electro­lyte imbalances, measurements of pH are of no value. Estima­tions of electrolytes in CSF and vitrous humour can only be of limited meaningfulness. On the one hand, pH strongly depends on the state of the body, and, on the other hand, liquor often becomes sanguinolent when it is obtained so that there may be considerable alterations especially to electrolytes. Centrifuga­tion may help, but cannot remove all components originating from damaged erythrocytes. This is why liquor from the lateral ventricles should be obtained, because after 12–24 hours there are no differences to lumbar liquor.

34.5.1  Potassium

Disturbances of potassium balance can gain forensic medical relevance because they have been described occurring not only in isolation but also in connection with other diseases and

  CHAPTER 34    POSTMORTEM BIOCHEMISTRY AS AN AID IN DETERMINING THE CAUSE OF DEATH  641

Particularly increased noradrenaline levels in CSF and vitre­ous humour are indicative of a protracted stress reaction. The authors’ research has revealed massively increased catecho­lamine concentrations, partly exceeding the normal ranges many times—adrenaline values in vitreous humour and CSF 100–8000 ng/L; noradrenaline levels 4000–70 000 ng/L (normal ranges in serum: adrenaline 20–120 ng/L and for noradrenaline 150–170 ng/L). Especially high noradrenaline levels indicate a longer duration of stress.

Hypothermia can also cause a massive release of catecho­lamines during intense stress. The levels are within the ranges of high excitation with noradrenaline concentrations consider­ably higher than those of adrenaline (10­ to 32­fold) compara­ble to cases with prolonged agonal states. Contrary to this, adrenaline levels often exceed those of noradrenaline in death cases with short agonal states. Death due to hypothermia results in mean quotients of adrenaline/noradrenaline considerably less than 1, whereas quotients above 1 are typical for short agonal states (e.g. myocardial infarction, head trauma) being indicative of higher adrenaline levels.

Additional analyses of volatile substances (ethanol, metha­nol, propanol­1, propanol­2 and acetone) usually show elevated acetone concentrations in all compartments being indicative of hypothermia, but basically only in cases that are ethanol­free. Acetone and propanol­2 are then altered equally. If relevant alcoholisation is found, both substances can only be found in very low or physiological ranges indicative of an antilipolytic effect for ethanol (acetone >35 mg/L if the blood alcohol level is <10 mg/dL vs. <5 mg/L if the blood alcohol level is >185 mg/dL).

34.7  Chronic alcoholism

Chronic alcoholism can be diagnosed using biochemical markers (carbohydrate­deficient transferring (CDT) and GGT).

34.8  Anaphylactic shock

One of the specific indications of thanatochemistry is the diag­nosis of anaphylactic shock. The levels of tryptase and specific immunoglobulins in serum are useful markers for the diagnosis of this alteration postmortem.

34.9  Genetic alterations

The need to find helpful markers for genetic alterations means the investigation of the molecular expression of the conse­quences of genetic alterations. The authors’ research group has

levels of chloride and sodium in vitreous humour appear to be almost ‘parallel’ and remain nearly constant for over 30 hours postmortem. However, any close correlations between chloride values and causes of death or time could not be identified postmortem.

34.5.3  Calcium

The homoeostasis of calcium has an important impact on neuromuscular conduction. Hypocalcaemia (total calcium <2.2 mmol/L, ionised calcium <1.1 mmol/L) results in patho­logical reflexes or arrhythmia. Causes of hypocalcaemia (total calcium >2.7 mmol/L, ionised calcium >1.3 mmol/L) are chronic osteolytic or endocrine processes in most of cases, which may be the reason for sudden unexpected deaths via electrolyte imbalance with arrhythmias, somnolence and coma. Under postmortem conditions, the serum calcium concentra­tion is constant for c. 10 hours with a slight increase thereafter (normal range in healthy individuals: 1.96–2.60 mmol/L). The calcium content of CSF reflects approximately the serum level of ionised calcium. In vitreous humour, calcium levels are much more stable and there is less influence of agonal and postmortem effects.

34.5.4  Diagnosis

Postmortem diagnosis of imbalance of electrolyte and water metabolism cannot be based on isolated single parameters. Assessment must always include a synopsis of different values. Furthermore, the postmortem interval has to be taken into account in each case. Postmortem biochemical analyses regard­ing electrolyte imbalance are believed to be most successful in cases characterised by elevation of parameters such as states of dehydration. One main disadvantage is represented by the wide range of variation in single analysis results. This requires a combined interpretation of different values with consideration of all morphological and toxicological findings as well as the possibility of combined dysregulations (e.g. kidney and glucose metabolism).

34.6  High excitation and hypothermia

A state of high excitation is characterised by a massive release of catecholamines, especially in situations with mechanical restraints and also in cases of prolonged agonal period. Such stress situations can be classified by estimation of adrenaline and noradrenaline levels using high­performance liquid chro­matography (HPLC) in serum, CSF and vitreous humour. Analyses in different compartments are useful to achieve semi­quantification of the intensity of stress and its impact on the mechanism of death.

642  PART IV    SUDDEN AND UNEXPECTED DEATH FROM NATURAL CAUSES

34.10  Conclusions

Regarding the postmortem diagnosis of fatal diabetic coma, morphological findings are only of indicative value. Therefore, the diagnosis ‘death due to diabetic coma’ always has to be a synopsis comprising medical history, macromorphology and histology completed by postmortem biochemistry. Specimens (CSF, vitreous humour, blood and urine) should be obtained if there is any suspicion of disturbances of glucose metabolism. Parameters of major relevance are sum value and haemoglobin A1c, found to be elevated in most cases of fatal coma (above 415 mg/dL and 12.1%, respectively). The level of free acetone usually exceeds 21 mg/L and urine glucose concentration exceeds 500 mg/dL. Correct diagnostic procedure always requires the combination of a minimum of three positive values (i.e. increased sum value, haemoglobin A1c positive, and ele­vated acetone concentration or increased sum value) and several indicative findings within macromorphology and histology.

In medical forensics, the diagnosis of fatal diabetic coma can only be made as a diagnosis of exclusion. Consequently, other mechanisms of death (e.g. intoxications) have to be ruled out. However, overlap with other causes of death appears to be rather typical and common. Because the whole diagnostic pro­cedure can only be carried out as a diagnosis of exclusion, the only area of overlap causing problems is that with ‘natural causes of death’ because myocardial infarction or pulmonary embolism may both represent real complications of diabetic coma and can also cause metabolic decompensation to pre­existing diabetes mellitus. Differentiation may be problematic especially in cases with acute myocardial infarction, but con­trary to such acute changes, the situation is different with chronic alterations, for example in narrowing coronary arterio­sclerosis or myocardial scars. With such preconditions, the higher the relevance of positive biochemical findings, the more intensive they appear to be (very high sum value and acetone level, etc.).

Postmortem biochemical examinations can also help in cases without morphological causes of death outside the field of diabetes mellitus so that specimens of bodily fluids should also be obtained. Often analyses of certain parameters sensibly complement postmortem morphological diagnostics as in cases of liver disease, chronic renal failure and electrolyte imbalance. Preliminary studies have also been carried out on the usefulness of other body compartments (e.g. synovial fluid) for a range of examinations as well as for further biochemical parameters (e.g. troponin T).

Importantly, urea levels in blood and CSF are likely to be elevated in the case of chronic kidney disease and furthermore slightly following death, but this increase has been found to be considerably lower in liquor compared to blood. Postmortem diagnosis of renal insufficiency can be made with urea levels above 200 mg/dL (urea–nitrogen in excess of 93 mg/dL). Cre­atinine concentrations seem to remain widely unaltered in all

studied the expression of a series of molecules for diagnosing hypertrophic cardiomyopathy. The most promising results for postmortem diagnosis are provided by proteomics as they allow the establishment of effect markers and markers of response to a series of stimuli that may provide the key to the solution of many problems encountered in forensic pathology. Proteomics is the study of the structure, quantity and function of proteins and it provides information on the interaction networks of cells and also of intracellular and extracellular proteins. It is esti­mated that a proteome contains around 100 000 proteins and their corresponding post­transcriptional derivatives. The refer­ences provide examples ranging from the study of protein deg­radation as an indicator for death data, to the differential diagnosis of vital and postmortem wounds and their data or the diagnosis of some causes of death. As an example the authors’ group are going to analyse advances made in diagnos­ing submersion.

The first advances in this field were provided by genomics when a series of studies were made on a series of supposed deaths due to submersion by inhibition, to search for the pres­ence of possible cardiac rhythm pathologies to explain the process. In a study of autopsy samples from 165 corpses found in water, Lunetta et al. (2003) detected a slight prevalence of long QT syndrome (0.61%, confidence interval 95: 0.02–3.33). Similarly, Tester et al. (2005) carried out an interesting study investigating KCNQ1 and RyR2 mutations that could provide an explanation for some submersion cases which are difficult to explain. One very significant field is the applica­tion of the study to aquaporins in the alveolar cells of the lungs for differential diagnosis between freshwater and saltwater submersion. To date, aquaporin 1 and aquaporin 5 have been studied. Whereas the study by Hu et al. (2004) was carried out on mice, the Hayashi et al. (2009) investigations included mate­rial taken from human autopsies (28 cases) in addition to rats used for experimentation. These authors found that the expres­sion of aquaporin 5 (AQP5) in type I alveolar cells was sup­pressed in freshwater submersion, as part of the processes for preventing haemodilution. This phenomenon does not occur in either saltwater submersion or the postmortem immersion of a body.

Zhao et al. (2006) used polymerase chain reaction to study the expression of HIF-1a in the kidneys and vascular endothe­lial growth factor (VEGF) mRNA for different causes of death. Twenty­seven cases of submersion are included, and they show the usefulness of VEGF mRNA as an indicator of acute circula­tory collapse. Using immunohistochemical techniques this research team studied the expression of the same substances in cardiac tissue: the mRNA of the hypoxia inducible myocardial factor (HIF) 1a, erythropoietin (Epo) and VEGF for different causes of death, but with a special interest in deaths with a cardiac origin. They demonstrated their usefulness for differ­ential diagnosis between cardiac deaths and deaths by submer­sion or other violent asphyxiation. The authors’ research team has carried out a study of different biochemical markers and their application for diagnosing vital submersion.

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Kärkela, J., Bock, E. & Kaukinen, S. (1993) CSF and serum brain­

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bodily fluids postmortem. The most reliable examinations are possible in CSF with a level below 1.6 mg/dL, expected to be typical in individuals without kidney disease. However, post­mortem biochemistry can only represent one pillar of the pro­cedure in establishing the cause of death, as such results are unsuitable to be used as the only diagnostic criterion.

A combined spectrum of postmortem biochemical values is of most relevant meaningfulness regarding the diagnosis of fatal metabolic disturbances. These may be strongly indicative of chronic or acute mechanisms and diseases, although no clinical diagnoses on postmortem findings can be made. Exclu­sion of any competitive mechanism is of special importance. A final diagnosis regarding the cause of death can only be made by the inclusion of medical history, macromorphology, histo­logical findings, postmortem biochemical results and toxicol­ogy, and as a diagnosis of exclusion only.

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