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Biomedical subjects

E Reiner

Publications and source records attributed to E Reiner.

At least 19 recordsLinked to original sources
PubMed2001

Activated clotting time as a screening test prior to catheter-based cardiovascular procedures.

The activated clotting time (ACT) was investigated as a rapid, inexpensive, point-of-service screening test for coagulation abnormalities prior to catheter-based procedures. A total of 963 patients were screened by obtaining a history, standard coagulation profile, and activated coagulation time. The prevalence of normal patients (normal ACT and coagulation profile) was 94% (sensitivity = 91%; specificity = 27%). A normal ACT had a positive predictive value of 95%. The ACT was an acceptable screening test due to its ability to predict positively a low rate of bleeding complication and normal coagulation studies. Patients with ACT > 150 sec should be further evaluated with a screening coagulation panel. Additionally, given its low specificity, coagulation studies should be obtained in high-risk patients since an abnormal ACT does not effectively correlate with abnormal coagulation studies.

Blood Coagulation Disorders↗
PubMed2001

Serum paraoxonase activities in hemodialyzed uremic patients: cohort study.

AIM: To determine whether paraoxonase activity, paraoxonase phenotypes, and lipid status are altered in uremic patients on long-term hemodialysis treatment as compared to healthy population. METHODS: Patients (n = 69) and control subjects (n = 145) were from the area of Slavonski Brod, Croatia. Paraoxon was used as a substrate for measuring basal or sodium chloride-stimulated (NaCl-stimulated) paraoxonase activity, and phenylacetate for measuring arylesterase activity. The double substrate method was used to assign phenotypes. Cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-cholesterol) were determined by methods routinely used in medical-biochemical laboratories. Enzyme activities are expressed as international units per liter of serum or per mmol of HDL-cholesterol (HDL-standardized activities). RESULTS: Basal and NaCl-stimulated paraoxonase activity, as well as arylesterase activity expressed per serum volume, were significantly lower in the hemodialyzed uremic patients compared to the controls; 69% (p < 0.001), 73% (p < 0.001) and 49%, (p < 0.001), respectively. However, basal and NaCl-stimulated paraoxonase activity standardized for HDL-cholesterol concentrations were not significantly reduced in the hemodialyzed uremic patients as compared to controls (86%, p = 0.614 and 87%, p = 0.720, respectively), contrary to arylesterase activity, which remained significantly lower (72%, p < 0.001). The distribution of paraoxonase phenotypes in hemodialyzed uremic patients and controls was as follows: AA 45% and 39%, AB 37% and 48%, BB 18%, and 13%, respectively. CONCLUSION: Patients on long-term hemodialysis have decreased paraoxonase/arylesterase activity, which might indicate a greater risk of premature atherogenesis.

Adolescent↗
PubMed2001

Organochlorine compounds in human milk and food of animal origin in samples from Croatia.

Human milk and food of animal origin (milk, meat, and fish) have been analysed over several decades for the DDT-complex, PCBs, HCB, and HCH-isomers. Human milk has also been analysed for PCDDs and PCDFs. Human milk samples were collected in six inland sites and one island, and food samples in marketplaces and individual households throughout Croatia. All human milk samples contained DDE and PCBs, and almost all food samples contained the DDT-complex. All analysed pools of human milk contained PCDDs and PCDFs. Levels of most analysed compounds show a decreasing trend over the past decade. The obtained data were the basis for the calculation of the daily intake of organochlorine compounds in breast-fed infants and adults.

Croatia↗
PubMed2000

Cholinesterase: substrate inhibition and substrate activation.

The relationship between activities and substrate concentrations (pS-curves) was analysed for reactions of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8). Catalytic constants Km, Kss, Vm, n and b were calculated from the Michaelis, Haldane, Hill and Webb equations in order to assess whether a given substrate also acts as an inhibitor or activator. It is suggested that the term substrate inhibition should only be attributed to substrates revealing bell-shaped pS-curves, while the terms apparent substrate inhibition or apparent substrate activation should relate to calculated values of the catalytic constants.

Acetylcholinesterase↗
PubMed2000

Comparison of protocols for measuring activities of human blood cholinesterases by the Ellman method.

This paper presents a protocol for routine assays of human blood cholinesterase activities which separates erythrocytes from plasma by centrifugation and measures acetylcholinesterase activity in unwashed erythrocytes and butyrylcholinesterase activity in the plasma. The recommended substrate for both enzymes is 1.0 mM acetylthiocholine. The protocol is compared with other two recommended protocols for the activity measurements of the two enzymes using the Ellman method. The paper discusses the advantages and disadvantages of each and concludes with a proposal for an international agreement between laboratories for the evaluation of a standardized protocol.

Acetylcholinesterase↗
PubMed1999

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.

Acetylcholinesterase↗
PubMed1999

Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4'-bipyridine and by a coumarin derivative.

Inhibition of recombinant mouse wild type AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8), and AChE peripheral site-directed mutants and human serum BChE variants by 4,4'-bipyridine (4,4'-BP) and the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) was studied. The enzyme activity was measured with acetylthiocholine as substrate. Enzyme-inhibitor dissociation constants for the catalytic and peripheral sites were evaluated from the apparent dissociation constants as a function of the substrate concentration. Inhibition by 4,4'-BP of AChE, BChE and the AChE mutant Y72N/Y124Q/W286A, was consistent with inhibitor binding to both catalytic and peripheral sites. The dissociation constants for the peripheral site were about 3.5-times higher than for the catalytic site. The competition between CHMC and substrate displayed two binding sites on the AChE mutants Y72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site were on average two-times higher than for the catalytic site. CHMC displayed binding only to the catalytic site of Y72N/Y124Q/W286A mutant and only to the peripheral site of w.t. AChE and the human usual BChE. Modelling of the 4,4'-BP and CHMC binding to wild type mouse AChE substantiated the difference between the inhibitors in their mode of binding which was revealed in the kinetic studies.

Acetylcholinesterase↗
PubMed1999

Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants.

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degrees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges: the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (nH) calculated from Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1) and 0.89 and 4.9 mM (K2) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.

Binding Sites↗
PubMed1999

3-Hydroxyquinuclidinium derivatives: synthesis of compounds and inhibition of acetylcholinesterase.

Four compounds were prepared: 3-hydroxy-1-methylquinuclidinium iodide (I), 3-(N,N-dimethylcarbamoyloxy)-1-methylquinuclidinum iodide (II), and two conjugates of I and II with 2-hydroxyiminomethyl-3-methylimidazole in which two parts of the molecule were linked by -CH2-O-CH2- (III and IV). III and IV are new compounds and their synthesis and physical data were given. All compounds were tested as inhibitors of human erythrocyte acetylcholinesterase (EC 3.1.1.7, AChE). The enzyme activity was measured in 0.1 M phosphate buffer (pH 7.4) at 10 and 37 degrees C with acetylthiocholine (ATCh) as the substrate. The obtained enzyme/inhibitor dissociation constants were between 0.05 and 0.5 mM at 10 degrees C and between 0.2 and 0.6 mM at 37 degrees C. At both temperatures compounds III and IV had higher affinities for the enzyme than compounds I and II and this difference was more pronounced at 10 than at 37 degrees C. The carbamates II and IV were also progressive AChE inhibitors. For compound II the rate constants of inhibition were 6300 and 2020 M(-1) min(-1) at 37 and 10 degrees C, respectively. Compound IV was a very weak carbamoylating agent with rate constants of inhibition of 100 and 63 M(-1) min(-1) at 37 and 10 degrees C, respectively. The oxime group in compounds III and IV hydrolyzed ATCh at rates of 23 and 3.2 M(-1) min(-1) at 37 and 10 degrees C, respectively.

Acetylcholinesterase↗
PubMed1999

Paraoxonase and arylesterase activities in the serum of two hyperlipoproteinaemic patients after repeated extracorporal lipid precipitation.

The effect of heparin-induced extracorporal lipid precipitation (HELP) on the activities of paraoxonase (EC 3.1.8.1) and arylesterase (EC 3.1.1.2) was studied in serum of a patient with hyperlipoproteinaemia (A) and of a patient with non-insulin dependent diabetes mellitus and hyperlipoproteinaemia (B). The enzyme activities were measured spectrophotometrically (Tris-HCl buffer, pH 7.4, 37 degrees C) with paraoxon and phenylacetate as substrates of paraoxonase and arylesterase, respectively. Both patients underwent HELP applications once a week over a period of 7 weeks. Over that period no overall change was observed either in enzyme activities or in the lipid and protein content of the sera. However, each HELP session caused an immediate decrease of EDTA-insensitive arylesterase activity (on average 56% in A and 42% in B), while EDTA-sensitive arylesterase remained almost unaltered. Paraoxonase remained unchanged in A, but decreased in B (approximately 60%). Of the atherogenic lipoprotein parameters, the most pronounced decrease was found in VLDL-cholesterol and in triglycerides (on average 45% in A and 32% in B), while the anti-atherogenic HDL-cholesterol decreased < 10%. Possible implications of the effect of HELP on the enzyme activities studied remain to be explained.

Aryldialkylphosphatase↗
PubMed1998

Quinuclidinium-imidazolium compounds: synthesis, mode of interaction with acetylcholinesterase and effect upon Soman intoxicated mice.

Four compounds were prepared: 3-oxo-1-methylquinuclidinium iodide (I), 2-hydroxyiminomethyl-1,3-dimethylimidazolium iodide (II) and two conjugates of I and II linked by -(CH2)3- (III) and -CH2-O-CH2- (IV). The aim was to evaluate separately the properties of I and II as opposed to III and IV, which contain both moieties in the same molecule. All four compounds were reversible inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7). The enzyme/inhibitor dissociation constants for the catalytic site ranged from 0.073 mM (II) to 1.6 mM (I). The dissociation constant of I for the allosteric (substrate inhibition) site was 4.8 mM. Possible binding of the other compounds to the allosteric site could not be measured because II, III and IV reacted with the substrate acetylthiocholine (ATCh) and at high ATCh concentrations the non-enzymic reaction interfered with the enzymic hydrolysis of ATCh. The rate constants for the non-enzymic ATCh hydrolysis were between 23 and 37 l/mol per min. All four compounds protected AChE against phosphorylation by Soman and VX. The protective index (PI) of I (calculated from binding of I to both, catalytic and allosteric sites in AChE) agreed with the measured PI; this confirms that allosteric binding contributes to the decrease of phosphorylation rates. The PI values obtained with III and IV were higher than those predicted by the assumption of their binding to the AChE catalytic site only. The toxicity (i.p. LD50) of compounds I, II, III and IV for mice was 0.21, 0.68, 0.49 and 0.77 mmol/kg body wt. respectively. All four compounds protected mice against Soman when given (i.p.) together with atropine 1 min after Soman (s.c.). One-quarter of the LD50 dose fully protected mice (survival of all animals) against 2.52 (IV), 2.00 (I and III) and 1.58 (II) LD50 doses of Soman.

Acetylcholinesterase↗
PubMed1998

Organochlorine pesticides and PCBs in human milk collected from mothers nursing hospitalized children.

Organochlorine compounds in milk were analyzed in samples collected over a nine-year period (1987-1995) from nursing mothers (N = 139) whose children were hospitalized for various disorders. All samples contained p,p'-DDE and PCBs; the median concentrations were 318 micrograms/kg milk fat and 220 micrograms/kg milk fat resp. Higher levels were found in mothers (N = 12) nursing neonates with impaired neurodevelopmental competencies or an inappropriate arousal reaction. No difference was observed between mothers nursing children with respiratory or gastrointestinal diseases, urinary tract infections or other infectious diseases, anemias, prolonged neonatal hyperbilirubinaemias or when children were with dermatological findings, congenital malformations or healthy.

Abnormalities, Drug-Induced↗
PubMed1997

Identification of the contents and the shelf-life of indicator tubes from field kits for detection of organophosphorus compounds in the air.

Indicator tubes from field kits for detection of organophosphorus compounds in the air were found on the territory of Croatia, in barracks liberated during the war years of 1991/1992, and were analysed during 1993/95. The detection kit consisted of glass indicator tubes with two sealed vials within each tube, a device for opening the tubes and breaking the vials, and an air sampling pump. The tubes were marked with serial numbers and expiry dates (1974-1992), but the description as to their contents was unavailable. The aim of this study was to identify the contents of the indicator tubes and to establish whether they were still suitable for detection of organophosphorus compounds. One vial was found to contain a cholinesterase (EC 3.1.1.7) preparation, while the other vial contained a non-thiocholine substrate and a pH-sensitive indicator (most probably cresol red). Applying DDVP as an organophosphorus cholinesterase inhibitor, it was found that all sets of indicator tubes were suitable for use regardless of the indicated expiry dates. Furthermore, the same tubes were found suitable for detection of organophosphorus compounds in water.

Air↗
PubMed1995

Catalytic properties of rabbit serum esterases hydrolyzing esterified monosaccharides.

Rabbit serum and one enzyme fraction isolated from rabbit serum by column chromatography (Fraction II) were used as catalysts in regioselective hydrolysis of radiolabelled pivaloylated monosaccharides (Piv = Me3CCO). The hydrolysis of 14C-labelled methyl 2-O-pivaloyl-(2-MP)-, 6-O-pivaloyl (6-MP)-, 2,6-di-O-pivaloyl-(2,6-DP) alpha-D- glucopyranosides and methyl 2-acetamido-2-deoxy-3,6- di-O-pivaloyl-(3,6-DPNAc) alpha-D-glucopyranosides, was studied, as well as that of the non-sugar substrates butyrylthiocholine, thiophenylbutyrate, phenylacetate and paraoxon. The specific activities of 2,6-DP, 3,6-DPNAc, butyrylthiocholine and thiophenylbutyrate were higher in Fraction II than in native sera, while those of phenylacetate and paraoxon were lower. Inhibition studies were done using the substrates mentioned and five different inhibitors, namely bis(p-nitrophenyl phosphate) (BNPP), eserine, paraoxon, HgCl2 and EDTA. The hydrolysis of 2,6-DP and 3,6-DPNAc was not inhibited by HgCl2 and only slightly by EDTA. Paraoxon, eserine and BNPP were progressive inhibitors of the hydrolysis of the two sugar substrates, and the pattern of inhibition resembled closely the inhibition of butyrylthiocholine and thiophenylbutyrate hydrolysis. This result applied to both, native serum and Fraction II. It was concluded that esterases in rabbit serum which hydrolyze pivaloylated sugar substrates belong to the category of serine esterases. Kinetic parameters (KM and Vmax), effects of temperature and pH on activity of esterases from Fraction II were also determined for the hydrolysis of sugar substrates.

Animals↗
PubMed1995

Catalytic properties and distribution profiles of paraoxonase and cholinesterase phenotypes in human sera.

Paraoxonase activities (322 healthy subjects) measured in the absence of ethylenediaminetetraacetic acid (EDTA) had a polymodal distribution profile with 60% of the subjects in the low activity mode; the activity measured in the presence of EDTA had a unimodal skewed distribution. Cholinesterase (ChE) activities (365 healthy subjects) had a unimodal, slightly skewed distribution. Patients with dementia (74) and patients with hyperlipidaemia (159) had different median paraoxonase and ChE activities than healthy subjects and all activity profiles had a higher skewness. The ChE variants usual (UU), fluoride resistant (FS) and atypical (AA) had the same affinity for the studied charged and uncharged ligands. The variants differed in rates of inhibition by the charged organophosphates and carbamates.

Aryldialkylphosphatase↗