Ewa Czech

Comments on 'Measuring insulin in human vitreous humour using LC-MS/MS' by Thevis et al.

We have read the article by Prof. Mario Thevis and his team titled ‘Measuring insulin in human vitreous humour using LC-MS/MS’ with great interest. Congratulations to the authors on the first successful mass-spectrometry-based analysis of post-mortem material related to an insulin poisoning case. It is another step forward in the post-mortem diagnostics of insulin overdose. The vitreous humour (also called a ‘vitreous body’) is a very valuable material for chemical-toxicological analyses and easy to obtain during autopsy. Its main advantage is anatomical isolation, useful especially in the case of advanced autolytic and putrefactive changes. Insulin is a very important peptide hormone in forensic toxicology for two reasons: (1) its casual or intentional overdose may be lethal and (2) it is also one of the most frequently used anabolic doping agents in sport. The case presented of a 55-year-old non-diabetic victim who died from an insulin overdose is quite interesting, especially because of the availability of ante-mortem blood samples. We have to remember that such a situation is very rare in routine forensic practice, however, where we may analyze only post-mortem biological material and non-biological specimens, like syringes, ampoules, vials, or remnants of the infusion solution and tubings, as in the commented article. The interpretation of insulin levels in the post-mortem biological material is very difficult. 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), localization of injection sites on the body (different rate of absorption), etc. The time of survival certainly influences the insulin levels detected in the post-mortem material. Unfortunately, in forensic practice, we do not usually know that time, because the cadavers are frequently found a long time after death, for example when the victim is living alone. The processes of anteand post-mortem elimination, distribution, and insulin transport across the blood-ocular barrier need to be studied more. Further to the Thevis et al.’ article, we want to shortly present our own, so far unpublished experiences in insulin determination in autopsy blood and vitreous humour. So far we have only presented this work during the 9th National Congress of the Polish Society of Toxicology in Szczyrk in 2008. In contrast to the authors of the commented article we used immunoradiometric assay (IRMA KIT IMMUNOTECH), routinely used for in vitro determination of insulin in human serum and plasma. This method is very sensitive, specific and – what is important – relatively cheap,

The influence of L-carnitine on methanol biotransformation in rats

There persists a need for potent and safe inhibitors of alcohol dehydrogenase (ADH), to effectively treat methanol poisoning by slowing its rate of biotransformation to there toxic products, formaldehyde and formic acid. Only a few former papers have reported on the significant effectiveness of L-carnitine in treating ethanol poisoning as well as alcohol abuse. As are no reports on the effectiveness of L-carnitine in treating methanol poisoning till now, the current studies were conducted to investigate the influence of L-carnitine on both oxydative metabolism and elimination of methanol in rats. Male Sprague-Dawley rats, aged 3 months with the body weight of 200-230 g were divided into 6 groups at random, with two of the groups considered to be control. Rats were given drinking water (control) or methanol in two different doses of 3220 mg/kg b.m. or 6440 mg/kg b.m. intragastrically and 0.9% NaCl (control) or 6.2 mmol/kg b.m. of L-carnitine intraperitionelly. Within 96 hours after the administration of methanol and 0.9% NaCl or L-carnitine, the urine was collected and then the animals were decapitated. To determine methanol there were taken blood samples for clot, and to determine carnitine and its derivatives blood was taken into heparinized test tubes. During the autopsy liver was also secured. In all the experimental time points stated the methanol concentrations in blood, urine and liver homogenate were determined by a head-space gas chromatography.

Developmental toxicity of hexachloronaphthalene in Wistar rats. A role of CYP1A1 expression.

Hexachloronaphthalene (HxCN) is one of the most toxic congeners of polychlorinated naphthalenes (PCNs). This study assesses the prenatal toxicity of HxCN after daily administration at doses of 0.1-1.0mg/kg b.w. to pregnant Wistar rats during organogenesis. We evaluated also the expression of CYP1A1 mRNA and protein in the livers of dams and fetuses, as well as the placenta. The results indicate that 0.3mg/kg b.w. was the lowest HxCN toxic dose for dams (LOAEL) while a dose of 0.1mg/kg b.w. was sufficient to impair the intrauterine development of embryos/fetuses without maternal toxicity. Regardless of the applied dose, HxCN generated embryotoxic effects. Dose-dependent fetotoxic effects were associated with HxCN exposure. HxCN was found to be a strong inducer of maternal and fetal CYP1A1. Expression of CYP1A1 mRNA in the placenta appears to be the most sensitive marker of HxCN exposure.

Expression of cytochrome P450 2C and 3A in female rat liver after long-term administration of gonadoliberin analogs.

OBJECTIVES Gonadoliberin (GnRH) analogs may be expected to indirectly modify growth hormone (GH) total concentration and its 24-h secretion profile. As a consequence, changes in the levels of GH may modify the mechanism of sex-dependent cytochromes P450 (CYP450) synthesis, including the expression of transcriptional factors. The aim of the study has been to evaluate the effect of long-term administration of a low dose of GnRH analogs on hepatic expression of CYP2C and CYP3A isoforms, and the transcription factors: pregnane X receptor (PXR), hepatocyte nuclear factor 4α (HNF4α), HNF6 and signal transducers and activators of transcription 5b (STAT5b). MATERIAL AND METHODS The study was carried out on adult female Sprague-Dawley rats during a 3-month treatment with dalarelin (GnRH agonist) and cetrorelix (GnRH antagonist), at a daily intraperitoneal injection (i.p.) dose of 6 μg/kg body weight/day, and 1, 2, and 4 weeks after treatment discontinuation. The concentrations of ovarian hormones and GH in the blood serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) method, respectively. Then, the expression of hepatic CYP450s (reverse transcription polymerase chain reaction - RT-PCR, Western blot and immunohistochemistry) and transcription factors (RT-PCR) was evaluated. RESULTS We have found that cetrorelix induces changes in the circadian pattern of GH secretion and enhances GH blood concentrations. These changes may cause increased expression of both, female-specific CYP450s (especially CYP3A9), and HNF4α/HNF6 transcription factors. Decrease in GH blood concentrations, resulting from the effect of dalarelin, may promote inhibition of female-specific CYP2C12 and CYP3A9 isoforms as well as STAT5b transcription factor. Slight changes in sex-independent CYP3A1 protein expression caused by GnRH analogs were also observed. CONCLUSIONS In adult female rats, HNF4α/HNF6 and STAT5b seem to be crucial for the regulation of GnRH antagonist/GH- and GnRH agonist/GH-dependent pattern of CYP450 expression, respectively.

Comparison of Endothelial Nitric Oxide Synthase and Endothelin-1 Levels in Kidneys Removed From Living Pigs After Cardiac Arrest and Brain Death

OBJECTIVES The aim of this paper was to describe differences between levels of endothelial nitric oxide synthase (NOS-3) and endothelin-1 (ET-1) in swine kidneys removed from living donors (group I) and after inducing brain death by brain herniation (group II) and cardiac arrest (group III). METHODS Each group consisted of 3 animals who underwent dual renal removal procedure; kidneys were further rinsed according to standardized procedure with Biolasol perfusion liquid, stored for 24 hours (4°C), and rinsed again. Renal specimens of 4 g mass, including renal cortex and medulla, were collected before and after perfusion (times 0 and 1), after 12 hours (time 2), and after reperfusion (time 3). Enzyme-linked immunosorbent assay was used to describe levels of NOS-3 and ET-1 in collected tissues homogenates. Mann-Whitney U test was used to compare results in groups in relation to total protein content (ng/mg), and the correlation between the 2 substances was measured with the use of Spearman rho. RESULTS Group I presented low and stable levels of NOS-3 in all time intervals (averages, 0.73, 0.99, 0.52, and 0.89, respectively). Level sof ET-1 were similar (0.87, 0.63, 0.69, and 0.86, respectively), and significant correlation between levels of the 2 substances was observed. Increased levels of NOS-3 (1.89 and 1.86) and ET-1 (1.38 and 1.49) were observed directly after removal in groups II and III and further maintained during organ storage. No correlation in group I was observed, and after perfusion significantly lower level of NOS-3 was observed in kidneys removed after brain death in relation to group III (1.77 vs 2.60). CONCLUSIONS The lowest and stable levels of NOS-3 and ET1 during storage were observed in kidneys removed from living donors. Levels of analyzed substances in this group showed correlation in subsequent time intervals.

Endothelial Nitric Oxide Synthase and Endothelin-1 Expression in the Early Post–Porcine Kidney Autotransplantation Period

BACKGROUND The aim of this study was the assessment of endothelial nitric oxide synthase (eNOS) and endothelin-1 (EDN-1) expression in porcine kidneys on the 14th and 30th days after the autotransplantation procedure. METHODS The research was conducted on 12 animals that underwent a left renal transplantation procedure with further standardized rinsing and 24-hour storage in 4°C; subsequently, the kidneys were implanted in the right retroperitoneal space after right-sided nephrectomy. Removed kidneys were examined (group 0). Six randomly chosen animals (group 1) were under observation for 14 days and 6 others (group 2) for 30 days. RESULTS After these observation periods, euthanasia was performed on the animals and 4-g samples were collected from the renal cortex and medulla. The Western blot technique was used to detect the eNOS and EDN-1 expression at the protein level. The obtained results are presented as absolute values of integrated optical density. Stable graft function was observed in all animals from the 2nd day after the procedure. eNOS in group 1 reached the mean value of 1.064 and was statistically significantly lower than in group 2 (2.085) or in the control group 0 (3.318). In the case of EDN-1 expression on 14th day after transplantation, the medium level was reported (0.248), which was similar to group 0 (0.216), whereas group 2 presented values 2 times higher (0.743). CONCLUSIONS A lowered eNOS level in the organ was observed on the 14th day after autotransplantation of a pig kidney; further enzyme normalization is associated with increased EDN-1 expression.

Cytochrome P450 3 A Expression in Pigs Livers After 24-Hour Preservation in Biolasol Solution Depending on the Type of Transgenesis

BACKGROUND An insufficient number of organs for transplantation shows the need for the development of new technologies. Xenotransplantation might be the answer. OBJECTIVE To determine if the type of transgenesis influences the level of CYP3A4, which takes an active part in xenobiotics metabolism in livers after 24-hour storage, depending on the kind of solution used for preservation. MATERIALS AND METHODS The experiment was carried out on 30 livers of Polish White Landrace divided into 5 groups depending on transgene type. The following human genes were transferred: α1,2-fucosyltransferase (groups I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringers solution (group I) or Biolasol solution (II-V). Reperfusion/reflush was performed. CYP3A29 isomer concentration was analyzed in liver specimens collected twice: 30 minutes after perfusion and 30 minutes after reperfusion/reflush. Expression of mRNA CYP3A29 was marked using RT-PCR analysis and of protein CYP3A29 using Western blotting technique. RESULTS The most significant decrease in protein CYP3A29 expression after 24-hour preservation was observed in group I (55.88% decrease), while the least significant was observed in group IV (10.44% decrease). mRNA expression evaluation was similar: the most significant decrease was observed in group I (87.8% decrease) and the least significant in group III (4.6% decrease). CONCLUSION α1,2-Fcosyltransferase transgene seems to influence mRNA and protein CYP3A expression in case of liver grafting and preservation for transplantation. CYP3A expression was also influenced by the kind of preservation solution used.