**3. Postmortem diagnostics of fatal insulin poisoning**

#### **3.1. Medicolegal autopsy**

**2.2. Homicide-suicide**

110 Ultimate Guide to Insulin

According to the information of the Prosecutor's Office, a 63-year-old man was supposed to kill his wife and dog and then commit suicide. Such situation in forensic medicine is called homicide-suicide or dyadic death. In the apartment a farewell letter and empty packages after insulin, NovoMix 30 Penfill (a mixture of fast and long-acting insulin analogue), were revealed (**Figure 2**). External medicolegal examination and forensic autopsies carried out at the Department of Forensic Medicine and Forensic Toxicology of the Medical University of Silesia in Katowice did not explain the cause of death. However, potential injection sites on the thighs and the shoulder of woman were revealed (**Figure 3**). Different biological materials for additional tests—biochemical, chemical-toxicological, and histopathological and for forensic genetics—were taken. Due to the inability to quickly determine insulin level in body fluids and the site of injection using the reference chromatographic methods [17–20], the determination of this hormone was ordered to two clinical diagnostic laboratories (by chemiluminometric and immunoradiometric methods). In addition, C-peptide (short 31-amino-acid

**Figure 3.** Numerous supravital point wounds and surrounding bruises on the thighs—potential insulin injection sites.

A classic postmortem macroscopic examination of the corpses (forensic autopsy) usually does not explain the cause and mechanism of death [23, 24]. Typically a feature of acute cardiorespiratory failure and nonspecific lesions related to the age of victim (e.g., atherosclerotic changes in vessels) can be found. For this reason, additional laboratory tests are necessary in each case. In addition to routinely collected sections from internal organs and body fluids (blood and urine), it is worth to take at least the sample of vitreous humor (VH) and the samples from potential injection sites for both histopathological and directed toxicological analyses.

#### **3.2. Forensic histopathology and immunohistochemistry**

A detailed histological examination of all internal organs, especially of the pancreas and liver, aiming at detection of insulinoma (tumor of the pancreas that is derived from β cells and secretes insulin) and morphological symptoms of hypoglycemia, respectively, should be always performed by experienced pathologist. The content of glycogen (multibranched polysaccharide of glucose that serves as a form of main energy storage) in the liver may be evaluated by the Periodic Acid-Schiff (PAS) or Best's Carmine staining. Its low amounts can indirectly confirm insulin overdose, as it was presented in the first case [15]. Another useful option is to perform IHC staining for the presence of insulin at the injection site. It is not necessary to buy special antibodies. These routinely used in clinical histopathology can be successfully used for this purpose, as we demonstrated in the second case [16].

instrumental method widely used in analytical chemistry. This was a significant advance in postmortem biochemistry. Our own experience shows that in cases where suicide by insulin poisoning is suspected, determination of its concentration in the vitreous humor and nonbiological material using the immunoradiometric assay (IRMA) gives the opportunity, similarly as the LC-MS/MS analysis, of objective confirmation of the poisoning, so both methods can be

Insulin in Forensic Medicine and Toxicology http://dx.doi.org/10.5772/intechopen.76691 113

In our department, we have analyzed material consisted of 93 samples of vitreous humor taken during forensic autopsies. Analysis revealed that in 86 vitreous humor samples (92.5%), the concentration of insulin, determined with IRMA, was below the limit of detection of this method (below 0.5 μIU/ml). The concentration of insulin in vitreous humor was determined only in seven cases (range of results, 1.42–24.42 μIU/ml). We have described above one of

The IRMA method is known as sensitive, specific, and relatively cheap in comparison to modern methods, but it requires adequate apparatus for the measurement of radioactivity and some experience in its interpretation. It is worth knowing that the studies on insulin determination using antibody-radiolabeled antigen reaction in the late 1950s were the beginning of a new medical discipline—radioimmunology [36]. Until the introduction of radioimmunoassay (RIA), death caused by insulin overdose was extremely difficult to prove [4]. In turn, the huge advantage of modern chromatographic methods (LC-MS/MS) is the possibility of differentiation between different types of insulins (human or animal insulin and their synthetic derivatives/analogues). In 2015, Palmiere et al. have presented preliminary results of postmortem determination of insulin using chemiluminescence enzyme immunoassay (CLEIA). Their conclusion was that the analysis of vitreous humor with CLEIA may provide suitable data, similar to analysis with LC-MS/MS and immunoradiometric assay, to support the hypothesis

Regardless of the method used in toxicological investigation, an analytically confirmed higher level of insulin in the vitreous humor plays an important and even a decisive role in structuring the final medicolegal opinion about the cause of death. This is the reason why the vitreous humor should be routinely collected and analyzed during forensic autopsy in every case with

An interesting observation, so far unused in the forensic practice, is an increase in the expression of certain genes stimulated by insulin, especially in hyperinsulinemic conditions. This is a potentially promising area for further research. An example might be the changes of neuropeptide Y (NPY) gene expression and its release during hypoglycemic stress. Han et al. found that subcutaneous insulin injection produced an immediate increase in plasma NPY immunoreactivity and delayed increases in adrenal and neuronal NPY mRNA and adrenal NPY immunoreactivity in rats [38]. They have concluded that these results suggest that NPY may play a role in insulin-induced hypertension. Another example can be increased vascular resistance in the equine digit and overexpression of endothelin-1 (ET-1) in the laminar tissue

used in forensic practice [32].

of insulin overdose [37].

an "insulin" background [29, 37].

**3.4. Forensic molecular biology**

due to the short-term hyperinsulinemia [39].

these cases, where insulin was used to commit suicide [15].

#### **3.3. Forensic toxicology and thanatobiochemistry (postmortem biochemistry)**

In routine forensic practice, usually antemortem blood samples of the victim, who sometimes is hospitalized before the death, are unavailable for forensic toxicologists, so they can analyze only postmortem biological material taken during autopsy and nonbiological specimens revealed at the crime scene, like syringes, ampoules, vials, or remnants of the infusion solution and tubings [24–26].

What is important from the medicolegal point of view is that the interpretation of insulin levels in the postmortem biological material is difficult and still in doubt [27–31]. The number of published papers dealing with this problem is relatively low. The time of survival after insulin injection depends on many different factors: type of insulin (differentiated onset of action and insulin half-life), method of administration (injection or insulin infusion pump), anatomical localization of injection sites on the body (different rate of absorption), etc. [32]. It certainly influences the insulin levels detected in the postmortem biological material. Unfortunately, in the forensic practice, investigators usually do not know that time, because the cadavers not infrequently are found after a long time since death at an advanced stage of late postmortem changes, for example, when the victim lived alone or the killer committed suicide [21].

Additionally, insulin determination in postmortem blood has a low diagnostic and testimonial value, mainly because of ongoing thanatochemical processes of autolysis and putrefaction [8, 24]. The main barrier that prevents receiving correct and trustworthy results of insulin determinations in postmortem blood with radioimmunological methods is the blood hemolysis (rupturing of red blood cells and the release of their contents into surrounding plasma). This fact was confirmed in the literature and by our own studies performed in the Department of Forensic Medicine and Forensic Toxicology in Katowice [32, 33].

Fortunately, insulin crosses the blood-retinal barrier and may be identified in the VH, which is generally very valuable alternative material for many different chemical-toxicological analyses [34, 35]. The advantage of this material is that it is easy to obtain during typical forensic autopsy. Another advantage is anatomical isolation, useful especially in the case of advanced autolytic and putrefactive changes *in corpore*. It has also a very low cell count, so there is a small postmortem metabolism of glucose and other substances by surviving cells.

In 2011 Thevis et al. have published the first successful mass spectrometry-based analysis of postmortem material (VH) related to an insulin poisoning case [17]. The natural levels of insulin in vitreous humor determined by the authors were below the liquid chromatography– tandem mass spectrometry (LC-MS/MS) limit of detection. LC-MS/MS is modern advanced instrumental method widely used in analytical chemistry. This was a significant advance in postmortem biochemistry. Our own experience shows that in cases where suicide by insulin poisoning is suspected, determination of its concentration in the vitreous humor and nonbiological material using the immunoradiometric assay (IRMA) gives the opportunity, similarly as the LC-MS/MS analysis, of objective confirmation of the poisoning, so both methods can be used in forensic practice [32].

In our department, we have analyzed material consisted of 93 samples of vitreous humor taken during forensic autopsies. Analysis revealed that in 86 vitreous humor samples (92.5%), the concentration of insulin, determined with IRMA, was below the limit of detection of this method (below 0.5 μIU/ml). The concentration of insulin in vitreous humor was determined only in seven cases (range of results, 1.42–24.42 μIU/ml). We have described above one of these cases, where insulin was used to commit suicide [15].

The IRMA method is known as sensitive, specific, and relatively cheap in comparison to modern methods, but it requires adequate apparatus for the measurement of radioactivity and some experience in its interpretation. It is worth knowing that the studies on insulin determination using antibody-radiolabeled antigen reaction in the late 1950s were the beginning of a new medical discipline—radioimmunology [36]. Until the introduction of radioimmunoassay (RIA), death caused by insulin overdose was extremely difficult to prove [4]. In turn, the huge advantage of modern chromatographic methods (LC-MS/MS) is the possibility of differentiation between different types of insulins (human or animal insulin and their synthetic derivatives/analogues). In 2015, Palmiere et al. have presented preliminary results of postmortem determination of insulin using chemiluminescence enzyme immunoassay (CLEIA). Their conclusion was that the analysis of vitreous humor with CLEIA may provide suitable data, similar to analysis with LC-MS/MS and immunoradiometric assay, to support the hypothesis of insulin overdose [37].

Regardless of the method used in toxicological investigation, an analytically confirmed higher level of insulin in the vitreous humor plays an important and even a decisive role in structuring the final medicolegal opinion about the cause of death. This is the reason why the vitreous humor should be routinely collected and analyzed during forensic autopsy in every case with an "insulin" background [29, 37].

#### **3.4. Forensic molecular biology**

be always performed by experienced pathologist. The content of glycogen (multibranched polysaccharide of glucose that serves as a form of main energy storage) in the liver may be evaluated by the Periodic Acid-Schiff (PAS) or Best's Carmine staining. Its low amounts can indirectly confirm insulin overdose, as it was presented in the first case [15]. Another useful option is to perform IHC staining for the presence of insulin at the injection site. It is not necessary to buy special antibodies. These routinely used in clinical histopathology can be

In routine forensic practice, usually antemortem blood samples of the victim, who sometimes is hospitalized before the death, are unavailable for forensic toxicologists, so they can analyze only postmortem biological material taken during autopsy and nonbiological specimens revealed at the crime scene, like syringes, ampoules, vials, or remnants of the infusion solu-

What is important from the medicolegal point of view is that the interpretation of insulin levels in the postmortem biological material is difficult and still in doubt [27–31]. The number of published papers dealing with this problem is relatively low. The time of survival after insulin injection depends on many different factors: type of insulin (differentiated onset of action and insulin half-life), method of administration (injection or insulin infusion pump), anatomical localization of injection sites on the body (different rate of absorption), etc. [32]. It certainly influences the insulin levels detected in the postmortem biological material. Unfortunately, in the forensic practice, investigators usually do not know that time, because the cadavers not infrequently are found after a long time since death at an advanced stage of late postmortem changes, for example, when the victim lived alone or the killer committed suicide [21].

Additionally, insulin determination in postmortem blood has a low diagnostic and testimonial value, mainly because of ongoing thanatochemical processes of autolysis and putrefaction [8, 24]. The main barrier that prevents receiving correct and trustworthy results of insulin determinations in postmortem blood with radioimmunological methods is the blood hemolysis (rupturing of red blood cells and the release of their contents into surrounding plasma). This fact was confirmed in the literature and by our own studies performed in the Department

Fortunately, insulin crosses the blood-retinal barrier and may be identified in the VH, which is generally very valuable alternative material for many different chemical-toxicological analyses [34, 35]. The advantage of this material is that it is easy to obtain during typical forensic autopsy. Another advantage is anatomical isolation, useful especially in the case of advanced autolytic and putrefactive changes *in corpore*. It has also a very low cell count, so there is a

In 2011 Thevis et al. have published the first successful mass spectrometry-based analysis of postmortem material (VH) related to an insulin poisoning case [17]. The natural levels of insulin in vitreous humor determined by the authors were below the liquid chromatography– tandem mass spectrometry (LC-MS/MS) limit of detection. LC-MS/MS is modern advanced

small postmortem metabolism of glucose and other substances by surviving cells.

of Forensic Medicine and Forensic Toxicology in Katowice [32, 33].

successfully used for this purpose, as we demonstrated in the second case [16].

**3.3. Forensic toxicology and thanatobiochemistry (postmortem biochemistry)**

tion and tubings [24–26].

112 Ultimate Guide to Insulin

An interesting observation, so far unused in the forensic practice, is an increase in the expression of certain genes stimulated by insulin, especially in hyperinsulinemic conditions. This is a potentially promising area for further research. An example might be the changes of neuropeptide Y (NPY) gene expression and its release during hypoglycemic stress. Han et al. found that subcutaneous insulin injection produced an immediate increase in plasma NPY immunoreactivity and delayed increases in adrenal and neuronal NPY mRNA and adrenal NPY immunoreactivity in rats [38]. They have concluded that these results suggest that NPY may play a role in insulin-induced hypertension. Another example can be increased vascular resistance in the equine digit and overexpression of endothelin-1 (ET-1) in the laminar tissue due to the short-term hyperinsulinemia [39].
