Antiradical Activity of Hydrolysates and Extracts from Mollusk A. broughtonii and Practical Application to the Stabilization of Lipids.

The antiradical properties of hydrolysates and hydrothermal extracts of bivalve mollusks (Anadara broughtonii) from the Far Eastern Region of Russia and their influence on lipid oxidation in mayonnaise were investigated. The radical binding activity of hydrolysates and extracts of A. broughtonii varies from 55% to 89%. The maximum radical-binding activity was observed for acid hydrolysates. The antiradical efficiency of acid hydrolysates is 35%-41% of the BHT (butylhydroxytoluene) index. The antiradical activity depends on the (method of) technological and biotechnological processing of raw materials. Acid and enzymatic hydrolysates and hydrothermal extracts of A. broughtonii in mayonnaise slow down the process of oxidation of lipids and hydrolysis of triglycerides. Acid hydrolysates reduce the speed of oxidation and hydrolysis of lipids in mayonnaise more efficiently than the enzymatic hydrolysates.

The proteome analysis of rat platelet with nano-liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique.

Recently, the proteomic analysis has become an ideal tool to study the structure and function of platelets. We proposed a nano-liquid chromatography (nano-LC) technique coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) for rat platelet proteome analysis. In this study, we attempted to analyze the rat platelet proteome in two different subcellular fractions: cytosol and membrane. Platelet-rich plasma was collected from healthy rats. The platelet samples were extracted with Subcellular Proteome Extraction Kit to collect subcellular compartments. For further investigations, platelet lysate, cytosol and membrane fractions were used. Enzymatic digestion of proteins was performed using Filter Aided Sample Preparation method with trypsin as a proteolytic enzyme. Tryptic peptides were analyzed using nano-LC-MALDI-TOF/TOF-MS. Platelet proteins identification was performed using the Mascot engine. We identified 238 proteins in the platelet lysate, 210 in the cytosol, and 148 in the membrane fraction. Among them, 45 were unique for platelet lysate, 55 for cytosol, and 34 for the membrane fraction. The gene ontology analysis showed that there were differences in the proteome of cytosol and membrane fractions related to the molecular functions, i.e. coagulative activity. Our results may suggest that the membrane or cytosol location of the proteins with coagulative activity may be responsible for the acute or delayed platelet response to an agonist. The nano-LC-MALDI-TOF/TOF-MS method can be used for identifying proteins of subcellular fraction in rat platelets.

Toxicological aspects of soluble titanium - a review of in vitro and in vivo studies.

Owing to the widespread use of titanium dioxide (TiO2) in various food, household and cosmetic products and in industry, the number of publications concerned with the potential toxic effects and health risks related to this substance in both bulk and nano form is steadily increasing. On the other hand, there is also a third form of titanium, ionic, which is produced by the biodegradation of Ti alloy implants. Thus, the growing population of implant recipients should raise concerns about the impact of soluble Ti on human health. Today, the mechanism of this metal's action on the human body is still not fully understood. This review aims to explore the health risks associated especially with long-term use of titanium implants. The paper provides an analysis of data available from the last 10 years concerning investigations of the adverse effects of Ti ions, including in vitro models of various tissues, in vivo studies of implant corrosion in animals and humans, and potential pathomechanisms of the adverse health impact induced by soluble titanium.

Diazepam and its metabolites in the mothers' and newborns' hair as a biomarker of prenatal exposure.

UNLABELLED: Pregnant women are exposed to benzodiazepines for therapeutic purposes during gestation. The goal of this study was to evaluate prenatal exposure to benzodiazepines. Time of exposure during course of pregnancy is a significant aspect of fetal exposure to drugs. Benzodiazepine concentration assay in hair of mothers and newborns exposed prenatally to these drugs was performed in the studies. Development, validation and evaluation of benzodiazepine determination method in mothers and their newborns enables assessment of health risks for the child and implementation of adequate therapeutic procedures. We used A LC-ESI-MS/MS method that allowed determination of diazepam (the main benzodiazepine used by pregnant women was diazepam) and its metabolites (nordazepam, oxazepam) in hair of mothers and newborns. LOQ 10 pg/mg of hair was used in the study. RESULTS: concentration of nordazepam was higher than parent drug (diazepam) and higher in newborns' hair when compared to mothers'. The mean concentrations of diazepam in mothers' hair were 31.6±36.0 and 34.1±42.4 pg/mg in the second and third trimester of pregnancy respectively. The mean concentration of diazepam in newborns' hair was higher and reached levels of 53.3±36.5 pg/mg. The mean concentration of nordazepam in the mothers' hair corresponding to the second and third trimester was 52.9±48.1 and 89.9±122.8 pg/mg, respectively. Nordazepam in the newborns' hair was detected at the mean level of 108.1±144.2 pg/mg. It was concluded that diazepam and nordazepam are permanently incorporated into the hair structure. Presence of diazepam and its metabolites in newborn's hair confirms that these benzodiazepines permeate placental barrier. Segmental analysis of mothers' hair enabled the assessment of drug administration time. Diazepam and its metabolites determined in hair of newborns may serve as biomarkers of prenatal exposure to these drugs. The performed LC-MS/MS analysis was accurate enough to determine even low concentrations of benzodiazepines, at the level of few pg/mg of hair. Levels of diazepam detected in hair of newborns were higher than levels determined in mothers. This may confirm the fact, that fetus's ability to metabolize diazepam is scarce. Nordazepam was found in higher concentrations in hair of newborns than in hair of mothers, which may suggest that it is cumulated in child's organism. Other metabolites of diazepam--oxazepam and temazepam--were detected in very few cases, in low concentrations.

Influence of tobacco smoke on the pharmacokinetics of citalopram and its enantiomers.

The purpose of this study was to evaluate the influence of tobacco smoke on the pharmacokinetics of citalopram (CIT) and desmethylcitalopram (DCIT) and its enantiomers on an animal model. High performance liquid chromatography (HPLC) with a diode array detector (DAD) was used for the identification and quantification of the studied compounds. The HPLC quantification of racemic mixtures of CIT was performed on a C18 column. The limits of detection (LOD) and quantification (LOQ) were: 7 and 10 ng/ml respectively. HPLC separation of citalopram enantiomers (S- and R-CIT) was performed on a Chirobiotic V column. The limits of detection (LOD) and quantification (LOQ) were: 6 and 15 ng/ml for R- and S-CIT respectively. The experiment was carried out on male Wistar rats. The rats were exposed to tobacco smoke for five days (6 hours per day). After the exposure, citalopram was administered in a dose of 10 mg/kg intragastrically. In the control group (non-exposed animals), citalopram was administered in the same way and at an equal dose. The blood of the animals was collected at nine time points. It was found that tobacco smoke exposure inhibits the biotransformation of citalopram. The half-life of the racemic mixture of citalopram after intragastric administration was increased by about 287%. Changes in the pharmacokinetic parameters of S-citalopram (active isomer) show a similar tendency to those of the racemic mixture. The pharmacokinetics of R-citalopram showed no statistically important differences after tobacco smoke exposure. Alterations in the pharmacological parameters of desmethylcitalopram presented an opposite trend to the parent drug. After exposure to tobacco smoke, the induction of metabolism of this compound was observed.

Effect of maternal tobacco smoking or exposure to second-hand smoke on the levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine of mother and the first urine of newborn.

Tobacco smoking during pregnancy is associated with a variety of negative consequences not only for the mother, but also for the developing fetus. Many studies have shown that carcinogens contained in tobacco smoke permeate across the placenta, and are found in fetus. The aim of the study was to determine the prenatal exposure to tobacco-specific carcinogenic N-nitrosamines on the basis of measurements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine of smoking and second-hand smoke (SHS) exposed women and in the first urine of their newborns. A questionnaire documenting demographics and socio-economical data, smoking habits and exposure to SHS was completed by 121 delivering women near or at term. Maternal concentrations of cotinine and NNAL were measured in urine of the mother and the first urine of her newborn infant by liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean concentration of cotinine was 439.2 ng/mg creatinine and NNAL concentration in urine of smoking women was 74.0 pg/mg creatinine, and for her newborn 78.6 pg/mg creatinine. Among mothers exposed to SHS, cotinine and NNAL mean concentration were 23.1 ng/mg creatinine, and 26.4 pg/mg creatinine. In newborns of SHS exposed mothers during pregnancy the mean concentration of NNAL was 34.1 pg/mg creatinine, respectively. Active tobacco smoking as well as passive exposure to smoking during pregnancy is an important source of tobacco specific N-nitrosamines to the fetuses as evidenced by increased concentrations of this carcinogen. Determination of NNAL in maternal urine samples can be a useful biomarker of prenatal exposure of newborn to carcinogenic nitrosamines.

The influence of simultaneous whole body exposure to cigarette smoke and low-protein diet feeding on fertility, reproduction, and progeny of rats.

The aim of the present study was to evaluate the toxicological influence of different concentrations of tobacco smoke and low-protein diet on fertility, reproduction, and survival of offspring of Wistar female rats. We studied three generations, two litters in each. There were 192 animals in parent generation. Animals were exposed to three different concentrations of tobacco smoke that were reflected by the carbon monoxide (CO) concentration (500, 1000, and 1500 mg CO/m3 air) during 11 weeks (six before and two weeks during mating, and three weeks of pregnancy). Additionally, animals were divided into two subgroups that were fed normal or low-protein diet. In the present study, we concentrated on the indices describing the reproduction ability, fertility, and rearing of pups of mothers exposed to tobacco smoke. The parameters of newborns survival were estimated also. We concluded that neither tobacco smoke exposure nor the low-protein diet changed the duration of pregnancy. The tobacco smoke in all doses and low-protein diet had a negative influence on fertility, reproduction, and survival of newborns from exposed mothers. This influence was mainly attributed to the tobacco smoke exposure. The levels of carboxyhaemoglobin were increasing with increasing tobacco smoke concentration.

Determination of opiates in serum, saliva and hair addicted persons.

In the last ten years advances in analytical methods have enabled the determination of xenobiotics in alternative material such as sweat, saliva, and hair. The aim of this study was to develop an analytical method and measure the concentration of the main opiates in serum saliva and hair of subjects from a detoxification and methadone treatment programme. The analytical strategy in the presented study, based on enzymoimmunoassay screening of opiates in urine and GC/MS confirmation, meets the needs of forensic and clinical toxicology. Blood and saliva samples from thirty seven patients and hair from twenty three with a history of intravenous opiate use were collected for analysis. The ranges of morphine in serum and saliva were 0-2081 and 0-208 ng/ml respectively; corresponding concentrations of codeine were 0-580 and 0-428 ng/ml respectively. The concentration of morphine, codeine and 6-MAM in hair of addicts ranged respectively from 0-32.4, 0-12.5 and 0-2.8 ng/mg. From the clinical toxicology point of view, hair analysis is supplementary to urine, serum or saliva determination, but in drug testing at the workplace it can play a crucial role.

The bacterial flora and the changes of the N-nitrosamine concentration in the operated stomach.

The studies included 170 patients (103 men and 67 women) aged 23 to 66 years, operated on because of an ulcerous disease. Depending on the method of surgical treatment patients were divided into 5 groups (those after gastric resection with Rydygier's method, after resection with the Billroth II method, after trunk vagotomy with pyloroplasty, after highly selective vagotomy, and after gastro-enterostomy). In all patients a qualitative estimation of the nitrate-reducing bacteria was carried out, as well as a chromatographic test of the selected N-nitrosamine concentration in the gastric juice. It was shown that the changes in the quantity of the nitrate-reducing bacteria and in the N-nitrosamine concentration depended on the type of surgical intervention conducted. The largest mean content of nitrate-reducing bacteria and the highest average N-nitrosamine concentration were confirmed in the gastric juice of patients after gastroenterostomy and after gastric resection with the Billroth II method, and the lowest values--in patients after highly selective vagotomy.

Endogenous diazepam concentrations in the serum of patients with liver neoplasms.

The study included 61 patients (35 men and 26 women) ages 47 to 74 in whom a primary liver cancer was diagnosed or neoplastic metastases to the liver were confirmed in the course of a cancer of the stomach or the large bowel. In each patient the endogenous serum diazepam concentration (ESDC) was estimated chromatographically and the results obtained were compared to selected clinical traits such as the magnitude and number of neoplastic changes and their location in the liver parenchyma, the histological form of the tumor and the primary location of the cancer in the case of neoplasms of the alimentary canal. The determination of the ESDC was also carried out in a control group made up of voluntary blood donors. Neither group examined received any medication belonging to the benzodiazepine group. From the results of the tests conducted it was confirmed that the average ESDC of patients with liver neoplasms was 65 times higher than that of the control group. Simultaneously, however, in patients with a primary liver cancer the average endogenous concentration was higher than in patients with neoplastic metastases to that organ and this was statistically significant. The location in the hepatic parenchyma of the neoplastic change as well as the primary location of the cancer remained without a statistically significant influence in the changes of ESDC. It was moreover shown that significantly high ESDC were associated in the liver mainly with increased neoplastic growth (above 3 cm in diameter) and with multiple spread (5 focuses and more).

The clinical value of the determinations in the serum of zinc concentration in women with breast cancer.

The clinical usefulness of the determinations of the zinc serum concentration at different stages of the breast cancer diagnostic process was evaluated in the present work. On the basis of the analysis of 182 women surgically treated because of this neoplasm, a statistically significant dependence was shown between the frequency of occurrence of hypozincemia and the progression of the cancer, its histological form, and the microscopic state of the axillary lymph nodes. The clinical usefulness was also shown by the determinations of the zincemia in the post-operative monitoring of patients with breast cancer. The critical value was also determined for the microelement concentration below which a statistically significantly high risk of occurrence of distant metastases appears.

[Concentration of zinc, copper and magnesium in the serum of drug addicts]. / Stezenie cynku, miedzi i magnezu w surowicy krwi osób uzaleznionych od narkotyków.

The aim of this study was to determine the concentration of zinc, copper and magnesium in the blood serum of drug addicts in order to ascertain whether drug abuse has an effect on the concentration of these metals. The study was carried out on a group of 33 addicts undergoing treatment at the Department of Clinical Toxicology, Collegium Medicum, Jagiellonian University in Kraków. The control group consisted of 22 young, healthy, drug free, male volunteers. Analyses were performed using flame atomic absorption spectrometry. The results demonstrate that zinc serum concentrations in the examined group of addicts before therapy (mean 0.82 mg/l) were reduced in relation to the control group (mean 1.12 mg/l), and was slightly enhanced after the detoxification process (mean 0.86 mg/l). The opposite effect was observed in the case copper serum concentration. The copper level in the group of addicts on admission to hospital (mean 1.35 mg/l) increased in relation to the control group (mean 1.11 mg/l) and was lower during hospitalisation (mean 1.18 mg/l). The concentration of magnesium in the serum of drug addicts did not change during therapy and was in the same range as in the control group (mean concentrations were 20.3 mg/l, 20.2 mg/l and 20.6 mg/l respectively).

[Profile of chemical analyses of psychoactive substances in the Laboratory of the Department of Clinical Toxicology in the years 1990-1999]. / Profil analiz substancji psychoaktywnych wykonanych w latach 1990-1999 w Laboratorium Toksykologicznym Kliniki Toksykologii.

The chemical-toxicological Laboratory of the Department of Clinical Toxicology carry on the analyses of xenobiotics in biological fluid collected from poisoned patient leaving in Krakow and south-eastern Poland. The evaluation of frequency of performed analyses in the years 1990-1999 shown that among psychoactive substances, ethanol is the most common reason of acute poisoning, followed by psychotropic drugs and drugs of abuse. The level of ethyl alcohol above 2 g/L was observed in more than 40% of patients. In evaluated period the number of barbiturates analyses dropped, benzodiazepines stayed on the same level. During this time evident increase of illicit drugs (amphetamines, opiates and THC) analyses was observed.

Inter- and intra-individual variability of ethanol pharmacokinetics over a long period of time.

In most research, the inter- and intra-individual variability of time profiles of ethanol concentration has been studied over a relatively short period of time. We present a study of inter- and intra-individual variability of ethanol pharmacokinetics in five healthy volunteers (social drinkers) over a period of around eight years. The volunteers received a dose of 0.7 g/kg (men) and 0.6 g/kg (women) of ethanol in the form of vodka diluted with mineral water. The blood ethanol concentrations in a series of samples were measured by gas chromatography. For pharmacokinetic calculations, a model with first-order absorption and Michaelis-Menten elimination was applied. A linear regression technique was used to calculate Widmark's coefficient. The area under the concentration-time curve (AUC) was calculated by trapezoid rule. A tendency for pharmacokinetic parameters to alter during the study time was observed, however, these changes were random and did not correlate with the duration of the study. The highest inter- and intra-subject variability of the half time of absorption was observed. The maximal elimination velocity and Michaelis-Menten constant were also very unstable; coefficients of variation were 55.8% and 79.0%, respectively. Other calculated pharmacokinetic parameters (AUC, beta60, V/F, Cmax) were characterized by a lower variability with a coefficient of variation around 36%. Back extrapolation of plasma ethanol concentration using mean value of beta60 for each subject showed that results could be underestimated up to 26% or overestimated up to 102%.

Changes in serum copper level during detoxification of acutely poisoned drug addicts.

Although it is known that drug addicts are a high-risk group for disruption of many homeostatic processes, little is know about changes in serum trace elements concentrations after taking the psychoactive substances. The aim of the study was to check the influence of the taking homemade heroin on serum level of copper. Blood samples were taken from 30 opiate addicts, and copper concentrations were measured by the means of atomic absorption spectrophotometry. The result of the study show that in the examined group, copper serum concentrations (1.35 mg/L) upon admission to the clinic were higher than in the control group (1.11 mg/L) but decreased during hospitalization (1.18 mg/L). There was no correlation between duration of stay at the hospital and changes in serum copper concentration.

Chronopharmacokinetics of amitriptyline in rats.

The circadian changes in absorption, tissue distribution and elimination of amitriptyline after single intravenous (i.v.) and intragastric (i.g.) administration, as well as the differences in pharmacokinetic profile after multiple i.g. administration (at 10:00 and 22:00 h) during a 12 h dosing interval, were investigated. The circadian changes of pharmacokinetic parameters of amitriptyline such as AUC (serum and tissues), clearance (i.v. and i.g.), volume of distribution, biological half-life and bioavailability were estimated. Acrophases for clearance appeared between 19:00 and 21:00 h; the bioavailability was highest during the dark phase at around 04:00 h. Higher values of AUC in serum were observed at the beginning of the light phase. A circadian rhythm of tissue distribution (AUC, K(D)) of amitriptyline with acrophase in the dark phase was observed for brain (12 h period), lung and liver (24 h), but not for heart or kidney. After single (i.v. and i.g.) amitriptyline administration, concentrations of its major metabolite, nortriptyline, were negligible; however, after ten doses, nortriptyline serum and tissue levels were similar to the concentrations of the parent drug with higher values during the day (light phase).

Effect of hematocrit and albumin concentration on hepatic clearance of tacrolimus (FK506) during rabbit liver perfusion.

Tacrolimus is an immunosuppressive agent used for organ transplantation. Studies were performed to examine the influence of different perfusate hematocrits and albumin concentrations on hepatic extraction of tacrolimus. In vitro binding, efflux and influx between red blood cells (RBCs) and buffer or plasma, and rabbit liver perfusion with use of human erythrocytes were studied. In the range of hematocrits from 0.05 to 0.4, plasma concentrations of tacrolimus were not affected by increased albumin content. Increased hematocrit caused decreases in whole blood:plasma (buffer) concentration ratios. The binding capacity of drug with RBCs was independent of hematocrit, with a value of 440 ng/ml of RBCs; the binding affinity was 0.876 ng/ml using plasma or buffer. Diffusion of tacrolimus from RBCs to buffer was rapid with a clearance of 0.940 ml/min, and equilibration was achieved within 2 min. Diffusion in the opposite direction (buffer-RBCs) was slower with a clearance of 0.576 ml/min. In such diffusion studies, plasma produced a greater difference between efflux (1.70 ml/min) and influx (0.276 ml/min) clearances. During liver perfusion, the major factor regulating elimination of tacrolimus was hematocrit. Both well-stirred and parallel-tube models reflected a low extraction ratio drug with values of 0.15 and 0.17 for the 0.05 and 0.2 hematocrits. Intrinsic clearances were 8.43 and 17.44 ml/min for the well-stirred and parallel-tube models. Albumin had a negligible influence on liver extraction of drug. A model-building process of characterizing nonlinear RBC binding, RBC diffusion rates, and liver perfusion parameters allows the complexities of tacrolimus hepatic clearance to be dissected and shows that strong RBC binding can be artificially perceived as causing a high clearance of the drug.

[Acute oral poisoning with isopropyl alcohol in alcoholics]. / Ostre doustne zatrucie alkoholem izopropylowym u osoby uzaleznionej od alkoholu etylowego.

The symptoms and clinical course of repeated poisonings with isopropyl alcohol in ethanol addicted 43-years old man is presented in the study. The blood and urine concentrations of isopropanol and acetone and also the blood ethanol concentration are given. The biological half-life of isopropanol calculated for the first 12 hours from admission to the Clinic was 6.9 hours. The biological half-life of acetone in the first 6 hours of hospitalisation was 5 hours, and then a considerable slowing down up to 29 hours was noted. The clinical symptoms, similar to ethanol poisoning, including strongly manifested catatonia and ketonuria without metabolic acidosis indicate the isopropanol intoxication. Hypokalemia should to be taken into consideration in treatment of isopropanol poisoning. An intravenous bicarbonates should be administered carefully e.g. when rhabdomyolysis occur.

Effect of ethanol dose on its chronopharmacokinetics in the rats.

Circadian changes in the elimination of ethanol after its administration in the rats in two doses: low (1.5 g/kg) and high (4.5 g/kg) were investigated. Circadian rhythm in ethanol pharmacokinetics (AUC, CLpo, t1/2) was observed after the low dose but not after the high one. The maximum rate of elimination was in the dark (active) phase at 1 am. The maximum value of clearance was related to the minimum value of AUC. Although the maximum and the minimum values of Vmax and KM differed essentially the diurnal variation were not observed.