Elsa Reiner's research while affiliated with Institute for Medical Research and Occupational Health and other places

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002-250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine 'slides down' the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π-π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme-substrate, enzyme-inhibitor and enzyme-substrate-inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.

A gas-chromatographic method on capillary columns is described for measuring concentrations of total PCBs and of six PCB conge-ners, PCB-28, PCB-52, PCB-101, PCB-138, PCB-153 and PCB-180, in human blood serum. Recovery of compounds was evaluated, and the repeatability and reproducibility of the results tested on sam-ples analysed on the same day and over a period of two years. The method was verified in an international AQA study in three rounds of measurements. The method was applied for the analysis of 45 serum samples collected in Zagreb, Croatia. All samples contained PCB-138 and PCB-153; the incidence of the other congeners was between 80 and 98%.

The distribution and time trend of organochlorine pesticide (OCP), polychlorinated biphenyl (PCB), and polychlorinated dibenzo-p-dioxin/polychlorinated dibenzofuran (PCDD/PCDF) concentrations in human milk samples from Croatia collected in 1981-2003 are presented. Between 1981/1982 and 1987/1989, the concentrations of HCB, beta-HCH, DDE, and total PCBs decreased about 50%, while for the last decade, the concentrations have been decreasing very slowly. In 2002/2003 the range of PCB congeners and OCPs was from below the limit of determination to 332 ng g(-1) milk fat. PCDD/PCDF concentrations in human milk samples collected in 1981-2000 ranged between 5.2 and 26.7 pg I-TEQ g(-1) milk fat and showed a decreasing trend.

The aim of this study was to differentiate the EDTA-sensitive from the EDTA-insensitive human serum esterases by evaluating their catalytic constants, K(M) and V(m), for the hydrolysis of phenylacetate (PA). Measurements were done at 37 degrees C in 0.1 M Tris/HCl buffer pH 7.4 and 8.4. The K(M,sen) and K(M,ins) constants were significantly different, 0.97 and 2.7 mM respectively, confirming that two esterases hydrolyse PA. The pH of the medium had no effect on K(M) values, and also no effect on V(m,sen) while V(m,ins) was two fold higher at pH 8.4 than at 7.4 further confirming the existence of two different enzymes. The stability of the esterases in aqueous media was also studied. EDTA-sensitive activity in buffer without CaCl(2) was extremely unstable; the time-course of inactivation followed a two-phase reaction kinetics, indicating that two EDTA-sensitive esterases hydrolyse PA. The EDTA-insensitive activity remained constant in aqueous media under the same experimental conditions.

The first association of the Croatian biochemists was established in 1957 as the Section of Biochemistry within the Croatian Chemical Society. In 1976, the Section became the Croatian Biochemical Society. The Society acted under that name until 2003 when the name changed to Croatian Society of Biochemistry and Molecular Biology. The Section, and later the Society, joined the Federation of European Biochemical Societies (FEBS) in 1965 and the International Union of Biochemistry (IUB), later International Union of Biochemistry and Molecular Biology (IUBMB), in 1977. Until 1992 the membership in these international associations was indirect, i.e. through the Union of the Chemical (and later Biochemical) Societies of Yugoslavia. Since 1992, our Society is a direct member of both international associations. The Society is led by a president and eight members elected every second year at annual assemblies. When founded 1976, the Society had 85 members. At its 30th anniversary in 2006, the Society had 433 members. The seat of the Society is in Zagreb. Branches are in Rijeka, Split and Osijek. The main activity of the Society is to organise lectures by speakers from Croatia and abroad, and to organise national and international professional and scientific meetings. Since 1974, the Society publishes a bulletin Informacije (in Croatian) to disseminate information obtained from Croatian sister societies, and from FEBS and IUBMB.

IntroductionLevels in Human Serum and UrineLevels in Human Milk and Fat TissueLevels in FoodPCB Pattern in Human and Animal SamplesCalculation of the Intake of Organochlorine Compounds by Adults and Breast-Fed InfantsConclusion

Application of Recombinant DNA Methods for Production of Cholinesterases as Organophosphate Antidotes and Detectors To develop new avenues for synthesizing novel antidotes for organophosphate poisoning and for detection of the organophosphates, we have turned to recombinant DNA methods to synthesize cholinesterases with unusual properties. For antidotal therapy we describe mutations of the native mouse and human enzymes that allow for enhanced rates of oxime reactivation. Such enzymes, when localized in the circulation, would enable the circulating cholinesterase to become a catalytic rather than simply a stoichiometric scavenger. Hence, "oxime-assisted catalysis" provides a means for scavenging the organophosphates in the circulation thereby minimizing their tissue penetration and toxicity. Accordingly, the oxime antidote or prophylactic agent has a dual action within the circulation and at the tissue level. Second, through a novel chemistry, termed freeze-frame, click chemistry, we have used organophosphate conjugates of acetylcholinesterase as templates for the synthesis of novel nucleophilic reactivating agents. Finally, acetylcholinesterase can be modified through cysteine substitution mutagenesis and attachment of fluorophores at the substitution positions. When linked at certain locations in the molecule, the attached fluorophore is sensitive to organophosphate conjugation with acetylcholinesterase, and thus the very target of insecticide or nerve agent action becomes a detection molecule for organophosphate exposure.

Mechanisms of Organophosphate Toxicity and Detoxication with Emphasis on Studies in Croatia This review comprises studies on the mechanisms of toxicity and detoxication of organophosphorus (OP) compounds done in Croatia in different research areas. One area is the synthesis of antidotes against OP poisoning and their in vivo testing in experimental animals. In vitro studies included in this review focus on the mechanisms of reversible inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), protection of cholinesterases from inhibition by OPs, and reactivation of phosphylated cholinesterases. The third area comprises distribution profiles of BChE and paraoxonase (PON) phenotypes in selected population groups and the detection of OPs and metabolites in humans. Finally, methods are described for the detection of OP compounds in human blood and other media by means of cholinesterase inhibition.