Natasa Trutic

Determination of veterinary antibiotics in bovine urine by liquid chromatography-tandem mass spectrometry.

A follow-up of antibiotics (tetracyclines, fluoroquinolones, cephalosporins, penicillins and amphenicols) in the bovine urine is important for two reasons: to understand if they are still present in organism, and whether their occurrence in urine might be considered as an environmental risk. A validated HPLC-MS/MS method (Decision 2002/657/EC) for antibiotics determination in bovine urine was developed. CCα and CCβ were in the range of 0.58-0.83 and 0.55-1.1 ng mL(-1), respectively. Recoveries were 92-108%, with inter-day repeatability below 12%. Analysis of bovine urine revealed frequent presence of tetracyclines, which was related with animals age. The cause, most presumably, might be found in different therapeutic protocols applied for veal calves and young bulls enrolled in this study. Most abundant was oxytetracycline with highest level in veal calves (1718 ng mL(-1)) vs. young bulls (2.8 ng mL(-1)). Our results indicate the necessity of antibiotics monitoring in bovine urine before animals undergo further processing in the food industry.

Monte Carlo QSAR models for predicting organophosphate inhibition of acetycholinesterase

A series of 278 organophosphate compounds acting as acetylcholinesterase inhibitors has been studied. The Monte Carlo method was used as a tool for building up one-variable quantitative structure–activity relationship (QSAR) models for acetylcholinesterase inhibition activity based on the principle that the target endpoint is treated as a random event. As an activity, bimolecular rate constants were used. The QSAR models were based on optimal descriptors obtained from Simplified Molecular Input-Line Entry System (SMILES) used for the representation of molecular structure. Two modelling approaches were examined: (1) ‘classic’ training-test system where the QSAR model was built with one random split into a training, test and validation set; and (2) the correlation balance based QSAR models were built with two random splits into a sub-training, calibration, test and validation set. The DModX method was used for defining the applicability domain. The obtained results suggest that studied activity can be determined with the application of QSAR models calculated with the Monte Carlo method since the statistical quality of all build models was very good. Finally, structural indicators for the increase and the decrease of the bimolecular rate constant are defined. The possibility of using these results for the computer-aided design of new organophosphate compounds is presented.

Tetrahydro-metabolites of cortisol and cortisone in bovine urine evaluated by HPLC–ESI-mass spectrometry

Interconversion of hormonally active cortisol (F) into the corresponding inactive 11-keto form, cortisone (E), is catalyzed by 11beta-hydroxysteroid dehydrogenases (11β-HSDs). With a view to estimating in vivo activities of some 11β-HSD isoforms, the measurement of urinary F and E and their tetrahydro metabolites (tetrahydrocortisol, THF, allotetrahydrocortisol, ATHF, tetrahydrocortisone, THE) has been suggested. The basic knowledge of THF, ATHF and THE levels in farm cattle is limited. Therefore the aim of this study was first to optimize a simple and quick method to determine F and E tetrahydro-metabolites in bovine urine by HPLC-mass spectrometry with electrospray ionization (HPLC-ESI-MS) and then to apply the method to real urine of bovines treated with prednisolone. The samples underwent filtration, deconjugation, solid-phase extraction (SPE) and the relevant analytes were measured by HPLC-ESI-MS. The method described in this paper is simple and efficient, featuring good linearity (up to 0.996) and reproducibility (6.8-12.5%, CV). Especially, good LODs were obtained, from 1.63 to 2.67 ppb, depending on the analyte. The chromatographic conditions were optimized in order to obtain a resolution which would allow to simultaneously measure two diastereoisomers, i.e. THF and ATHF. In our study, ATHF turns out to be below the detection limit, while for 18 samples tested the contents of examinated metabolites were as followed: THF (12.5±4.8 ppb), THE (10.9±5.5 ppb), F (11.6±3.3 ppb) and E (5.0±2.2 ppb). When the method was applied to the subject treated with prednisolone a major increase in the concentration of tetrahydro metabolites was observed before the slaughter, mainly due to stress conditions; prednisolone treatment, most presumably, influenced the 11β-HSD activity, as indicated by the decrease in the F/E ratio. This work may provide a useful methodological contribution to the future definition of F, E, THF, ATHF and THE urinary baseline values in order to obtain indirect evaluations of HSDs activity in farm cattle and possible applications in screenings for suspected abuse of synthetic corticosteroids in bovines.

Acute-phase protein gene expression in rat liver following whole body X-irradiation or partial hepatectomy.

We studied the effect of total body X-irradiation and partial hepatectomy on the acute phase protein gene expression in rat liver. Male rats of AO strain were irradiated with high X-ray doses, without any visible tissue damage. In contrast, partial hepatectomy consisted of surgical removal of 40% liver tissue. The changes in liver mRNA concentrations for positive acute-phase reactants including cysteine protease inhibitor, alpha(1)-acid glycoprotein, fibrinogen and haptoglobin, and albumin as a negative reactant were monitored by Northern blot and slot-blot hybridizations using corresponding [32P]dCTP labeled cDNA probes. While in the first 24 h after the partial hepatectomy, liver mRNA levels for the positive acute-phase reactants increased, briefly followed by an immediate decrease, the duration and timing of the acute-phase responses to the whole body X-irradiation were slightly different and lasted for as long as 72 h. Although both treatments induced the mRNA expression of acute-phase reactants in rat liver, the observed variations in the duration and intensity of the changes in mRNA levels for the acute-phase proteins in these two types of tissue damage suggest the involvement of specific mechanisms in a fine tuning of the non-specific acute-phase responses to meet the unique requirements of the particular injury.

Monte Carlo method based QSAR modeling of maleimide derivatives as glycogen synthase kinase-3β inhibitors

The Monte Carlo method was used for QSAR modeling of maleimide derivatives as glycogen synthase kinase-3β inhibitors. The first QSAR model was developed for a series of 74 3-anilino-4-arylmaleimide derivatives. The second QSAR model was developed for a series of 177 maleimide derivatives. QSAR models were calculated with the representation of the molecular structure by the simplified molecular input-line entry system. Two splits have been examined: one split into the training and test set for the first QSAR model, and one split into the training, test and validation set for the second. The statistical quality of the developed model is very good. The calculated model for 3-anilino-4-arylmaleimide derivatives had following statistical parameters: r(2)=0.8617 for the training set; r(2)=0.8659, and r(m)(2)=0.7361 for the test set. The calculated model for maleimide derivatives had following statistical parameters: r(2)=0.9435, for the training, r(2)=0.9262 and r(m)(2)=0.8199 for the test and r(2)=0.8418, r(av)(m)(2)=0.7469 and ∆r(m)(2)=0.1476 for the validation set. Structural indicators considered as molecular fragments responsible for the increase and decrease in the inhibition activity have been defined. The computer-aided design of new potential glycogen synthase kinase-3β inhibitors has been presented by using defined structural alerts.

Erosive Effect of Different Soft Drinks on Enamel Surface in vitro: Application of Stylus Profilometry

Objective: To assess the erosive potential of various soft drinks by measuring initial pH and titratable acidity (TA) and to evaluate enamel surface roughness using different exposure times. Materials and Methods: The initial pH of the soft drinks (group 1: Coca-Cola; group 2: orange juice; group 3: Cedevita; group 4: Guarana, and group 5: strawberry yoghurt) was measured using a pH meter, and TA was measured by titration with NaOH. Enamel samples (n = 96), cut from unerupted human third molars, were randomly assigned to 6 groups: experimental (groups 1-5) and control (filtered saliva). The samples were exposed to 50 ml of soft drinks for 15, 30 and 60 min, 3 times daily, during 10 days. Between immersions, the samples were kept in filtered saliva. Enamel surface roughness was measured by diamond stylus profilometer using the following roughness parameters: Ra, Rq, Rz, and Ry. Data were analyzed by one-way ANOVA, Tukeys post hoc and Student-Newman-Keuls post hoc tests. Results: The pH values of the soft drinks ranged from 2.52 (Guarana) to 4.21 (strawberry yoghurt). Orange juice had the highest TA, requiring 5.70 ml of NaOH to reach pH 7.0, whereas Coca-Cola required only 1.87 ml. Roughness parameters indicated that Coca-Cola had the strongest erosion potential during the 15 min of exposure, while Coca-Cola and orange juice were similar during 30- and 60-min exposures. There were no significant differences related to all exposure times between Guarana and Cedevita. Strawberry yoghurt did not erode the enamel surface regardless of the exposure time. Conclusion: All of the tested soft drinks except yoghurt were erosive. Erosion of the enamel surfaces exposed to Coca-Cola, orange juice, Cedevita, and Guarana was directly proportional to the exposure time.

Assessment of thiamine content in some dairy products and rice milk

The levels of thiamine were compared between different types of milk, fermented milk products and rice milk. The analysed products were mainly from the markets, so vitamin analysis may be a good parameter for the quality of commercial food. Thiamine concentrations were determined by high performance liquid chromatography (HPLC) using a reversed-phase C-18 column with fluorescence detector. The average thiamine contents were 0.46 µg/mL for ultra high temperature (UHT), 0.39 µg/mL for pasteurized and 0.38 µg/mL for chocolate milk. Unprocessed cow milk had average thiamine concentration of 0.47 µg/mL. The highest thiamine content (11.95 µg/mL) was found in rice milk. Sour cream samples had higher concentration of thiamine (0.54 µg/mL) than yogurt (0.45 µg/mL) and sour milk (0.47 µg/mL). In the opened UHT milk packages, which were stored in refrigerator for 10 days, significant thiamine loss was not observed. Investigated products contained appropriate amounts of thiamine and had satisfactory nutritional value with respect to vitamin B1.

Antioxidants and Antioxidant Capacity of Human Milk / Antioksidansi i antioksidativni kapacitet humanog mleka

SUMMARY Milk contains plenty of enzymatic and non-enzymatic antioxidant components that probably account for the vital antioxidant protection of the infants at early stages of life against the development of complications induced by oxygen free radicals. Indigenous milk enzymes play a key role in regulating lactogenesis, including active involution of mammary gland. Moreover, they are essential constituents of antioxidation and the innate immune system of milk. Among antioxidant enzymes, superoxide dismutase, catalase and selenium-containing glutathione peroxidase have been demonstrated in human milk. Mainly, the enzyme content of colostrum is higher than that in corresponding mature milk. Beside lipophilic antioxidant in human milk, tocopherols, carotenoids and vitamin A are of great interest. Those components demonstrate the highest levels in colostrum and decline during early lactation, despite the fact that total lipids increase. The complete list of active antioxidant components in human milk is not yet known. This review reports the main findings of enzymatic and non-enzymatic antioxidants, as well as antioxidant capacity of human milk. Synergism of action of several antioxidants helps to eliminate free radicals in newborns. Bearing in mind that milk contains a number of antioxidants, many reactions are possible and it is difficult to define the exact contribution and function of each antioxidant. Besides qualitative and quantitative analysis of human milk antioxidants, the measurement of total antioxidant capacity could be a useful tool for examination of this dynamic, complex fluid SAŽETAK Mleko sadrži mnoge enzimske i neenzimske antioksidanse koji verovatno utiču na vitalnu antioksidativnu zaštitu dece u ranoj fazi života od razvoja komplikacija izazvanih slobodnim kiseoničnim radikalima. Enzimi koji se prirodno nalaze u mleku imaju ključnu ulogu u regulaciji laktogeneze, uključujući aktivnu involuciju mlečnih žlezdi. Štaviše, oni su esencijalni činioci antioksidativnog procesa i urođenog imunog sistema mleka. Među antioksidativnim enzimima u humanom mleku određeni su superoksid dismutaza, katalaza i glutation peroksidaza koja sadrži selen. Uglavnom je sadržaj enzima u kolostrumu veći od sadržaja u odgovarajućem zrelom mleku. Od lipofilnih antioksidanasa u humanom mleku, od najvećeg značaja su tokoferoli, karotenoidi i vitamin A. Sadržaj ovih komponenti je najveći u kolostrumu, koji zatim opada tokom rane laktacije, bez obzira na činjenicu da sadržaj masti raste. Svi aktivni antioksidansi u humanom mleku još uvek nisu poznati. U ovom radu su prikazana najznačajnija saznanja o enzimskim i neenzimskim antioksidansima kao i o antioksidativnom kapacitetu humanog mleka. Sinergističko delovanje pojedinih antioksidanasa pomaže u eliminaciji slobodnih radikala kod novorođenčadi. S obzirom da mleko sadrži mnoštvo antioksidanasa, mnoge reakcije su moguće, pa je teško odrediti precizan doprinos i funkciju svakog antioksidansa. Pored kvalitativne i kvanititativne analize antioksidanasa humanog mleka, određivanje ukupnog antioksidativnog kapaciteta bi mogao da bude koristan metod ispitivanja ovog dinamičnog, složenog fluida

Physicochemical and biochemical parameters in milk of Serbian breastfeeding women

BACKGROUND/AIM This study was undertaken to determine the changes and relationships between some important milk constituents as well as physical, rheological, and biochemical parameters of milk obtained from Serbian breastfeeding mothers. MATERIALS AND METHODS Physicochemical and biochemical parameters and the concentrations of vitamins, uric acid, and minerals were determined during the three periods of lactation covering colostrum, transitional, and mature milk collected from 67 mothers who had a term-pregnancy. RESULTS Large interindividual variations regarding many parameters were found between mothers at the same period of lactation, but the average values were mostly in the expected and recommended ranges. For some parameters, our values are quite different in relation to the milk of women from other countries or data reported by other authors. CONCLUSION Differences in vitamin and mineral contents and physicochemical and rheological characteristics of milk obtained by Serbian breastfeeding mothers compared to that of mothers from other parts of the world have been found. This paper presents the measured data of some physical parameters of human milk about which there is little information in the literature.