Grzegorz Buszewicz

The influence of ethanol on the level of ketone bodies in hypothermia

Presently available possibilities of macro- and microscopic diagnosis of death from hypothermia are very limited as the changes observed are either weakly specific (ecchymoses in the mucous membrane of the stomach, histological features of haemorrhagic pancreatic necrosis, cardiomyocyte necrosis or decreased content of glycogen in hepatocytes) or represent only local action of low temperatures (frostbites, violet patches in the region of knees and elbows, red livores) and they may not be present in cases of death from cooling at environmental temperature close to zero or higher. The study evaluated the usefulness of acetoacetic acid (Ac-Ac), beta-hydroxybutyric acid (beta-HBA) and acetone determinations in blood, urine and vitreous humour for diagnosis of death from hypothermia. These three substances called ketone bodies, are easily assimilated energetic substrates that get oxidized preferentially before glucose and fatty acids. In hypoglycaemia (also hypothermia-induced one), the tissues dependent on glucose (e.g. the brain) cover most of their energetic needs from oxidation of these compounds. The analysis of 16 cases of death in circumstances suggesting hypothermia (mainly of the alcohol abusers) showed that the degree of ketosis was inversely proportional to the blood ethanol concentration. This relation may result from stimulation of insulin release and a decrease in the release of its antagonists by ethanol, as well as from inhibition of free fatty acid (FFA) beta-oxidation due to increase in the NADH/NAD ratio. So, the antiketonaemic effects of ethanol (together with its influence on the dilatation of the peripheral vessels and inhibition of shivering thermogenesis by muscle relaxation), explain increased sensitivity of intoxicated persons to low temperatures.

Anticonvulsant activity of phenobarbital and valproate against maximal electroshock in mice during chronic treatment with caffeine and caffeine discontinuation.

We evaluated the protective activity (expressed as ED50, values in mg/kg) of phenobarbital (PB, 120 min before testing) and valproate (30 min) alone or combined with caffeine in male mice with seizures induced by maximal electroshock (MES). Both antiepilep‐tic drugs (AEDs) were administered by intraperitoneal (i.p.) injection in a single dose to mice receiving intraperitoneal caffeine either in a single dose 30 min before the test or as pretreatment every 12 h for 3 and 14 days. In addition, we determined the ED50, values of the AEDs 24 and 72 h after 14–day treatment with caffeine. Finally, we studied the influence of a challenge dose of caffeine, injected in mice 24 and 72 h after 14 days of treatment with caffeine, on the protective activity of PB or VPA. Caffeine in a single dose of 23.1 mg/kg reduced the anticonvulsant effect of PB. Its protective activity was further impaired after 3 and 14 days of caffeine treatment. The ED50, for VPA was significantly increased both by the single dose of caffeine and by chronic treatment. The anticonvulsant activity of PB and VPA measured 24 and 72 h after 14–day treatment with caffeine did not differ from control values, but a challenge dose of caffeine injected 24 or 72 h after daily injections for 14 days resulted in a significant reduction in the protective activity of both AEDs. Measurement of the total plasma levels of caffeine, VPA, and PB did not suggest pharmacokinetic interactions as an explanation for our results. Our results indicate that chronic caffeine exposure may progressively reduce the antiepileptic potency of VPA and PB.

Pharmacodynamic and pharmacokinetic interactions between common antiepileptic drugs and acetone, the chief anticonvulsant ketone body elevated in the ketogenic diet in mice

Purpose:  Acetone is the principal ketone body elevated in the ketogenic diet (KD), with demonstrated robust anticonvulsant properties across a variety of seizure tests and models of epilepsy. Because the majority of patients continue to receive antiepileptic drugs (AEDs) during KD treatment, interactions between acetone and AEDs may have important clinical implications. Therefore, we investigated whether acetone could affect the anticonvulsant activity and pharmacokinetic properties of several AEDs against maximal electroshock (MES)–induced seizures in mice.

Free fatty acids as markers of death from hypothermia

The possibilities of using morphological markers of fatal hypothermia are limited; therefore, other diagnostic criteria of deaths from hypothermia are being researched. The initiation of protective mechanisms against adverse effects of low temperatures results in activation of hormonal systems and development of characteristic biochemical changes that can be impaired by alcohol intoxication. The aim of the study was to assess the usefulness of determinations of the profile of free fatty acid concentrations as potential markers of hypothermia-related deaths, particularly in intoxicated victims. The study group consisted of blood samples collected during autopsies of 23 victims of hypothermia. The control group included blood samples collected from 34 victims of sudden, violent deaths at the scene of an incident (hangings and traffic accidents) and 10 victims who died because of post-traumatic subdural hematomas with prolonged agony. The study and control groups were divided into three subgroups according to blood alcohol concentrations: 0.0-0.99; 1.0-2.99 and ≥3.0‰. Statistical analysis in the individual subgroups demonstrated significant increases in concentrations of palmitic, stearic and oleic acids (P<0.05), independent of blood ethanol concentration. Palmitic, stearic and oleic acids can be considered the potential markers of fatal hypothermia, including the cases of intoxicated individuals.

Glucocorticosteroids as markers of death from hypothermia

In the course of hypothermia, biochemical changes occur that are associated with stimulation of protective thermogenic mechanisms as well as mobilization of internal energy resources mediated by the hormone system. The objective of the investigation was the assessment of validity of determinations of cortisol, cortisone and corticosterone as hypothermia markers in cases of fatal hypothermia combined with concomitant insobriety of the victims. The experimental group consisted of blood samples collected in the course of medico-legal autopsies of 23 hypothermia victims. The controls included blood samples originating from 34 victims of violent sudden deaths (deaths by hanging and traffic road accidents at the scene) and from ten individuals deceased after prolonged agony in consequence of post-traumatic subdural hematomas. In both groups, three subgroups were distinguished that included cases with ethanol levels within the following ranges: 0.0-0.99, 1.0-2.99 and ≥3.0‰. The comparison of determination results showed that irrespectively of blood ethanol concentration, cortisol, cortisone and corticosterone levels seen in hypothermia victims were significantly higher as compared to the controls (P<0.001).

Low dose of bupropion significantly enhances the anticonvulsant activity of felbamate, lamotrigine and topiramate in mice

Experimental evidence indicates that bupropion hydrochloride, an antidepressant and a first-line smoking cessation aid, exerts dose-dependently anticonvulsant and convulsant effects. In this study, chronic bupropion pretreatment intraperitoneally (i.p.) for 14 days in a dose of 5 mg/kg reduced the ED(50) (i.e. the dose protecting 50% of mice against electroconvulsions) of lamotrigine, topiramate, and felbamate from 4.58, 60.95, and 48.79 (antiepileptic+vehicle) to 3.01, 41.68, and 37.28 mg/kg (antiepileptic+bupropion), respectively, against maximal electroshock-induced seizures in mice. Bupropion significantly increased the plasma and brain concentrations of lamotrigine. Plasma concentration of topiramate was elevated, however, the brain concentration of the drug was not affected. Neither plasma nor brain concentrations of felbamate were elevated by bupropion administration. Bupropion did not exacerbate motor coordination impairment caused by the antiepileptic drugs in the rotarod test. Chronic administration of bupropion significantly potentiates the protective activity of lamotrigine, topiramate, and felbamate against maximal electroshock-induced seizures. A pharmacokinetic interaction is responsible for the effect of bupropion co-administered with lamotrigine.

The Genetic Predisposition and Its Impact on the Diabetes Mellitus Development in Patients with Alcoholic Chronic Pancreatitis

The most common cause of chronic pancreatitis (CP) is alcohol abuse. The aim of the present study was to identify patients with genetic predisposition to CP abusing alcohol. The question posed was whether CP manifests at a younger age and diabetes mellitus develops earlier in individuals with genetic predisposition. The study encompassed 79 patients with alcoholic chronic pancreatitis (ACP) and control group (100 persons). The following mutations were determined: R122H and N29I of PRSS1 and N34S of SPINK1 as well as E366K and E288V of SERPINA 1. No R122H and N291 mutations were observed in the group of ACP patients and in controls. Moreover, there was no E288V mutation. In 79 ACP patients, six SPINK 1 (N34S/wt) mutations were observed. In the control group, one heterozygous SPINK 1N34S gene mutation was found (P = 0.0238). Two PiZ mutations were identified in patients with ACP and one analogical mutation in controls. Amongst patients with ACP as well as SPINK1 and PiZ mutations, the onset of disease was observed earlier and developed earlier. The prevalence of SPINK1 mutation is higher in patients with ACP than in healthy populations. This mutation together with the effects of alcohol accelerates the development of ACP and of diabetes mellitus.

Comparison of SPE/d-SPE and QuEChERS-Based Extraction Procedures in Terms of Fungicide Residue Analysis in Wine Samples by HPLC-DAD and LC-QqQ-MS.

Two different extraction and clean-up protocols, based on either the SPE/dispersive SPE (d-SPE) or the quick, easy, cheap, effective, rugged, and safe approach, were optimized and compared for determination of six selected fungicides (benalaxyl, metalaxyl, triadimenol, tebuconazole, diniconazole, and epoxiconazole) in wine samples. The pilot study was performed by applying HPLC with diode-array detection, and optimized procedures were easily transferred to the LC triple-quadrupole MS system. Both extraction procedures presented good performance for all the analytes, with recoveries in the range of 70-132% and SDs ≤20%. The d-SPE clean-up step included in both procedures allows obtaining colorless extracts with the majority of coextracted matrix compounds removed. LC with electrospray ionization and tandem MS operating in the multiple reaction monitoring mode provide high sensitivity and selectivity for trace analysis. Both developed procedures were evaluated in terms of commercial wine sample analysis. In three wine samples, metalaxyl and tebuconazole residues were detected at concentrations from 0.14 to 30.7 ng/mL. Both approaches showed satisfactory feasibility for fungicide residue analysis in wine samples.

POST-MORTEM ESTIMATION OF TIME OF DEATH OF DOGS BASED ON MEASUREMENTS OF KIDNEY TEMPERATURE IN COMPARISON WITH RECTAL TEMPERATURE

Abstract The subject of the study were dogs divided into two groups according to body weight: up to 10 kg and from 10 kg to 30 kg. The aim of the study was to determine the dynamics of the post-mortem decrease in rectal and kidney temperature. The temperature was measured on both sites at the same time using a thermometer connected to a computer, under constant environmental conditions of the necropsy room. In these animals, a higher temperature in the kidneys persisted for the duration of the study. Comparative analysis between mean differences in kidney and rectal temperature in small and large dogs showed the greatest temperature amplitude in the group of small dogs, both for the kidney and the rectum. The greatest decrease in temperature, 1.2°C, was noted for the kidney in small dogs between 4 and 6 hours after death. Analysis of the dynamics of the decrease in kidney and rectal temperature for both weight groups combined, and the difference in temperature between the kidney and the rectum in the time intervals analysed showed that in the first two hours the difference between kidney and rectal temperature did not exceed 0.5°C. Two hours after death the difference in temperature between the two measurement sites was about 0.5°C after which time dropped below 0.5ºC.

Determination of ramipril in human plasma and its fragmentation by UPLC-Q-TOF-MS with positive electrospray ionization.

Abstract This report presents the application of ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with positive electrospray ionization, to determine ramipril in human plasma. First, the proteins in human plasma were precipitated using acetonitrile, then the supernatant was extracted by ethyl acetate at pH 3 and finally, the extract was analyzed using a UPLC-QTOF- MS system. The method was validated and the coefficient of determination (R2) was > 0.999, the lower limit of quantification (LLOQ) was 0.5 ng mL-1. Precision, recovery and stability were determined for three different concentrations of ramipril. RSD for this method ranged from 3.3 to 8.6 %. The intra-day mean recovery was from 65.3 to 97.3 %. In addition, the fragmentation of ramipril was studied. Due to high resolution of the spectrometer, it was possible to measure fragment masses accurately and determine their molecular and chemical formulas with high accuracy.