Čedomila Milin

Dose- and time-dependent effects of luteolin on carbon tetrachloride-induced hepatotoxicity in mice

Carbon tetrachloride (CCl(4)) is a well-known model compound for producing chemical hepatic injury. This study investigated the protective effects of the flavonoid luteolin on the CCl(4)-induced hepatotoxicity in mice. Luteolin dissolved in dimethyl sulfoxide (DMSO) was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose, and once daily for 2 consecutive days. Two hours after the final treatment, the mice were treated with CCl(4) (20 mg/kg, i.p.). CCl(4)-induced hepatotoxicity was reduced in a dose- and time-dependent manner, as determined by decreased serum aminotransferase activities and liver histopathology. CCl(4) intoxication resulted in an overexpression of heat shock protein gp96 in the mice liver, which was strongly attenuated by luteolin pretreatment. Luteolin has also decreased oxidative stress produced by CCl(4), as suggested by improvement in the Cu/Zn superoxide dismutase activity. The effect of luteolin on myeloperoxidase, an indicator of inflammatory cell infiltration, was also investigated. Treatment of the mice with luteolin resulted in a significant decrease in the myeloperoxidase activity. The hepatoprotective effect of luteolin against CCl(4) hepatotoxicity was higher in animals pretreated with luteolin for 2 consecutive days. This suggests that the protection might be due to induction of some adaptive mechanisms. The data indicate that luteolin could be effective in protecting mice from the hepatotoxicity produced by CCl(4).

Determination of standard zinc values in the intact tissues of mice by ICP spectrometry.

The most commonly encountered difficulties for the quantitative measurement of zinc in biological samples are the limited sample amount, total and effective digestion of connective and fatty residues, and low zinc concentrations. These problems often lead to the determination of lower zinc values than actually present, so that the sample preparation, digestion, and analytical procedure deserve careful attention.In this short communication, a new method for microwave tissue disintegration is described. The authors have obtained consistent and reproducible results with tissue samples of 0.5 g or less.

Kinetic characterisation of enzymatic esterification in a solvent system: adsorptive control of water with molecular sieves

Abstract The kinetics of enzymatic esterification of glycerol with oleic acid, in equimolar ratio, catalyzed by immobilized Mucor miehei lipase in a solvent system in the presence of the molecular sieves was carried out at 37°C at different Lipozym and solvent ( n -hexane) concentrations and the molecular sieve contents were studied in a batch stirred-tank reactor (BSTR). The reactions were followed by the determination of reaction conversions during 45 h. The experimental data of enzymatic esterification of glycerol with oleic acid in a solvent system in the presence of molecular sieves showed minimal deviation from the calculated value in the irreversible second order kinetic model. On the basis of the experimental data, we found an empirical correlation between concentrations of Lipozym , concentrations of solvent ( n -hexane), contents of the molecular sieve and the reaction rate constant, k 1 .

Physico-chemical characteristics of olives and olive oil from two mono-cultivars during various ripening phases

The aim of this study was to find a correlation between the chemical and physical parameters in the pulp of the olive, in order to identify the optimum period during the 1998 harvesting period. For this reason various parameters have been monitored: the amount of oil in the pulp during the ripening phase obtained by solvent extraction with n-hexane, the acidity, the peroxide value, the composition of fatty acids and the saturated/unsaturated fatty acid ratio, the composition of the unsaponifiable fraction, the antioxidant capacity, the percentage of oil and the puncture test in two local Croatian olive cultivars. The chemical parameters change during the ripening, in order to control the oxidative changes of the cellular homeostasis of the whole lipophilic fraction that needs a stronger safeguard of the double bonds against the oxidative stress. The modification of the physical characteristics, observed with the puncture test, is related to the chemical changes which occur in the olive during the ripening period and the monitoring of these physical and chemical parameters can help to increase nutritional quality (as antioxidant capacity) and allow for an acceptable amount of oil.

Effect of indomethacin on motor activity and spinal cord free fatty acid content after experimental spinal cord injury in rabbits.

Study design:Determination of functional and biochemical parameters as well as the effect of specific therapies on these parameters, in the experimental model of neurotrauma in rabbits.Objective:To assess the effect of indomethacin (0.1–3.0 mg/kg for 9 days), a potent inhibitor of endogenous prostaglandin synthesis, on the motor activity and on the spinal cord tissue concentration of free palmitic, stearic, oleic, arachidonic and docosahexaenoic acids in an experimental model of a spinal cord injury in rabbits.Setting:Faculty of Medicine, University of Rijeka, Croatia.Methods:The animals were randomly divided into nine experimental groups, four sham and/or vehicle-treated and five indomethacin-treated (including one sham-operated and four injured groups). Laminectomy was followed by contusion of the spinal cord, using a modification of the technique of Albin. Motor activity was controlled daily during the course of the next nine postoperation days and scored using Tarlovs system. Spinal cord samples from the impact injury site were taken and frozen in liquid nitrogen. Total lipids were isolated and purified by a modification of the method of Folch. Free fatty acids (FFAs) were separated from the total lipid extract by preparative thin-layer chromatography, converted to the corresponding methyl esters and identified using gas chromatography, using nonadecanoic acid as the internal standard.Results:The concentrations of all analysed free fatty acids were increased in the spinal cord after neurotrauma, in comparison to control tissues. Treatment of injured rabbits with indomethacin resulted in a significant decrease in spinal cord FFAs and exerted a positive effect on neurotrauma-induced motor impairment.Conclusion:These results indicate a mechanism whereby indomethacin protects rabbits from the sequellae of neuronal damage caused by trauma, and suggests that it may be beneficial in the therapy of neurotrauma.Sponsorship:This work was supported by the Croatian Ministry of Science and Technology (project 062019).

Effects of temperature on sorption-desorption processes of imidacloprid in soils of Croatian coastal regions

Sorption-desorption behavior of imidacloprid in six soils collected from five coastal regions in Croatia at 20, 30 and 40°C was investigated using batch equilibrium technique. Isothermal data were applied to Freundlich, Langmuir and Temkin equations, and the thermodynamic parameters ΔH°, ΔG°, ΔS° were calculated. The sorption isotherm curves were non-linear and may be classified as L-type, suggesting a relatively high sorption capacity for imidacloprid. Our results showed that the K sor F values decreased for all the tested soils as the temperature increased, indicating that the temperature strongly influences the sorption. Values of ΔG° were negative (−4.65 to −2.00 kJ/mol) indicating that at all experimental temperatures the interactions of imidacloprid with soils were spontaneous processes. The negative and small ΔH° values (−19.79 to −8.89 kJ/mol) were in the range of weak forces, such as H-bonds, consistent with interactions and partitioning of the imidacloprid molecules into soil organic matter. The ΔS° values followed the range of −57.12 to −14.51 J/molK, suggesting that imidacloprid molecules lose entropy during transition from the solution phase to soil surface. It was found that imidacloprid desorption from soil was concentration and temperature-dependent, i.e. at lower imidacloprid concentrations and temperature, lower desorption percentage occurred. Desorption studies revealed that hysteretic behavior under different temperature treatments existed, and it was more pronounced at 20°C in the soils with higher OC content. The study results emphasize the importance of thermodynamic parameters in controlling soil pesticide mobility in different geographical locations, seasons and greenhouse conditions.

Hysteretic Behavior of Imidacloprid Sorption-Desorption in Soils of Croatian Coastal Regions

Sorption and desorption are important processes that influence the transport, transformation, and bioavailability of imidacloprid in the soils. Equilibrium batch experiments were carried out using six coastal Croatian soils. The equilibrium sorption and desorption experimental data showed the best fit to the Freundlich equation. Sorption parameters predicted with the Freundlich model, KF sor and 1/n ranged from 2.92 to 5.74 (mg/kg)/(mg/L)1/n, and 0.888 to 0.919, respectively. The sorption of imidacloprid was found to be sensitive to organic carbon (OC) content. The highest sorption was observed in Krk soil (OC 4.74%) and the lowest in Zadar soil (OC 1.06%). Fitted desorption parameter values, KF des , were consistently higher than those associated with sorption. The opposite trend was observed for the exponential parameter 1/n. Results also suggested that imidacloprid sorption-desorption by soil is concentration-dependent, i.e. at lower imidacloprid concentrations a greater sorption percentage and lower desorption percentage occurred. Desorption studies revealed that there was a hysteresis effect in all the tested soils. Hysteresis coefficient values (H) varied from 0.656 to 0.859. The study results emphasize that the controlled application of imidacloprid is obligatory, especially in soils with a low organic carbon content, in order to minimize a risk of environmental and groundwater pollution.

Effect of Olive Oil- and Corn Oil- Enriched Diets on the Tissue Mineral Content in Mice

The mineral content (zinc, iron, magnesium, and calcium) in the liver, spleen, and thymus of male Balb/C mice was analyzed. Animals were fed, over 21 d, diets enriched with corn oil (FCO diet) or olive oil (FOO diet) (5% addition to standard pellet, w/w). Olive oil with predominant oleic acid (C18:1, n-9) had a quite different composition than corn oil, in which linoleic acid (C18:2, n-6) prevails. The zinc and magnesium tissue concentrations were not changed in either group. The calcium concentration in liver as well as the calcium concentration in spleen increased in mice fed both the FCO and FOO diets. Furthermore, mice fed both the FOO and FCO diets had increased spleen iron concentration. Mice fed the FCO diet had increased thymus calcium concentration compared to controls. The results show the effect of diets with unsaturated, particularly polyunsaturated fatty acids, on the calcium and iron concentration in some organs.

Monitoring the esterification of sorbitol and fatty acids by gas chromatography

Abstract Lauric acid esters of sorbitol, 1,4-sorbitan and 1,4:3,6-isosorbide were synthesized in laboratory. Esterification was carried out at 160°C with a catalyst for sorbtol cyclization and esterification, viz., p-toluenesulfonic acid. Lipid classes and polyols were separated and analysed by GC as trimethylsilyl ethers and esters. The purpose of the study was to determine the suitability of GC analysis of these products as a means of following the progress of reactions. The results confirmed the possibility of monitoring lauric acid and sorbitol esterification via the GC determination of lauric acid concentration.

Metallothionein expression and tissue metal kinetics after partial hepatectomy in mice

To better elucidate previous results showing that partial hepatectomy noticeably changes the tissue content of zinc, calcium, magnesium, and iron(II) ions in regenerating the liver, thymus, and spleen, we report on the correlation of these metal tissue kinetics in these organs with the expression of metallothionein-I+II (MT-I+II) proteins and MT-I mRNA in early postoperative period (1, 2, 6, 12, and 24 h) after one-third hepatectomy (pHx). The results showed that 2 h after pHx the regenerating liver accumulated Zn2+, Ca2+, Mg2+, and Fe2+ ions while decreasing the concentration of all these metals in the spleen and of Zn2+ in the thymus. On the 24th h, a new high accumulation of Zn2+ and Ca2+ was seen in the regenerating liver and of Zn2+, Ca2+, and Fe2+ in the spleen. Simultaneously, MT-I mRNA increased in the liver and spleen. In hepatocytes and on several spleen and thymus mononuclear lymphatic cells, the increased expression of MT proteins was found mainly in the cytoplasm and nuclei. The areas expressing MTs in regenerating liver inversely correlated with those containing apoptotic cells, suggesting that these proteins participate in tissue restoration through reduction or increase of metal ions after injury to the liver.