Influence of ischemic preconditioning on blood coagulation, fibrinolytic activity and pancreatic repair in the course of caerulein-induced acute pancreatitis in rats.

UNLABELLED: Previous studies have shown that ischemic preconditioning protects several organs, including the pancreas, from ischemia/reperfusion-induced injury. The aim of the investigation was to determine whether ischemic preconditioning affects the course edematous pancreatitis. METHODS: In rats, ischemic preconditioning was performed by short-term clamping the celiac artery. Acute pancreatitis was induced by caerulein. The severity of acute pancreatitis was evaluated between the first and tenth day of inflammation. RESULTS: Ischemic preconditioning applied alone caused a mild pancreatic damage. Combination of ischemic preconditioning with caerulein attenuated the severity of pancreatitis in histological examination and reduced the pancreatitis-evoked increase in plasma lipase and pro-inflammatory interleukin-1beta. This effect was associated with an increase in plasma level of anti-inflammatory interleukin-10 and partial reversion of the pancreatitis-evoked drop in pancreatic DNA synthesis and pancreatic blood flow. In secretory studies, ischemic preconditioning in combination with induction of acute pancreatitis attenuated the pancreatitis-evoked decrease in secretory reactivity of isolated pancreatic acini to stimulation by caerulein. In the initial period of acute pancreatitis, ischemic preconditioning alone and in combination with caerulein-induced acute pancreatitis prolonged the activated partial thromboplastin time (APTT), increased plasma level of D-dimer and shortened the euglobulin clot lysis time. The protective effect of ischemic preconditioning was observed during entire time of experiment and led to acceleration of pancreatic regeneration. CONCLUSIONS: Ischemic preconditioning reduces the severity of caerulein-induced pancreatitis and accelerates pancreatic repair; and this effect is related to the activation of fibrinolysis and reduction of inflammatory process.

Markers of Antioxidant Defense in Patients with Type 2 Diabetes.

AIMS: Diabetes is considered a state of increased oxidative stress. This study evaluates blood concentrations of selected markers of antioxidant defense in patients with type 2 diabetes. METHODS: The study included 80 type 2 diabetes patients and 79 apparently healthy controls. Measured markers included ferric reducing ability of plasma (FRAP), reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyltransferase (GGT) and uric acid serum, and plasma and/or hemolysate levels. RESULTS: FRAP, uric acid, CRP, and GGT levels were significantly higher in patients with diabetes. Plasma and hemolysate GR was significantly higher whereas GPx activity was significantly lower in patients with diabetes. There were no significant differences in antioxidant defense markers between patients with and without chronic diabetes complications. Fasting serum glucose correlated with plasma GPx, plasma and hemolysate GR, FRAP, and serum GGT, and HbA1c correlated with serum GGT. Only FRAP and serum uric acid were significantly higher in obese (BMI > 30 kg/m(2)) patients with diabetes than in nonobese patients. CONCLUSIONS: Some components of antioxidant defense such as GR, uric acid, and GGT are increased in patients with type 2 diabetes. However, the whole system cannot compensate for an enhanced production of ROS as reflected by the trend toward decreased erythrocytes GSH.

Myeloperoxidase inactivation in the course of catalysis of chlorination of taurine.

Myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was isolated from leukocytes of patients with chronic granulocyte leukemia. In the presence of H2O2 and Cl- at pH 4.0-6.6 the myeloperoxidase catalyses chlorination of taurine to monochloramine taurine and simultaneously undergoes inactivation. The myeloperoxidase inactivation rate depends on the concentration of H2O2 and Cl-: both the initial rate of chlorination and myeloperoxidase inactivation rate increase with increasing concentration of H2O2. However, an increase in concentration of Cl- results in a decrease in enzyme inactivation. At a given H2O2 concentration, myeloperoxidase inactivation is a first order reaction, which implied that the enzyme may react with a substrate a limited number of times.

Oxidation of amino acids and peptides in reaction with myeloperoxidase, chloride and hydrogen peroxide.

Oxidation was studied of N-acetyl derivatives of cystine, cysteine, methionine and glycyltryptophan employing the myeloperoxidase-Cl--H2O2 system at pH 4.5, 6.0 and 7.0. Moreover, oxidation of pentapeptide composed of Leu-Trp-Met-Arg-Phe-COOH with myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and hypochlorite was also studied. It was found that amino-acid derivatives having an amino group bound to an acetyl residue react with functional groups of the side-chain. The -SH groups of N-acetylcysteine and the -SS- group of cystine oxidize to cysteic acid. Methionine residues oxidize to methionine sulphoxide, and tryptophan residues to a derivative of 2-oxoindolone. The same reaction products were obtained when respective amounts of hypochlorous acid were used instead of myeloperoxidase, Cl- and H2O2. Differences in the stoichiometry of reactions of myeloperoxidase-mediated oxidation and hypochlorite oxidation suggest differences in the reaction mechanisms of both studied systems. Interaction of the studied pentapeptide with myeloperoxidase-Cl(-)-H2O2 system as well as with hypochlorite showed that in the peptide molecule individual amino acids oxidize consecutively according to their susceptibility to oxidation. No splitting of peptide bonds was observed. Therefore, a modified peptide with methionine sulphoxide and and oxidized tryptophan incorporated into the molecule was obtained.

Apolipoprotein E is highly susceptible to oxidation by myeloperoxidase, an enzyme present in the brain.

Apolipoprotein E, the most common apolipoprotein found in the brain, is linked to several pathologies like Alzheimer's disease. Apolipoprotein E directly binds to beta-amyloid with a strong affinity. Myeloperoxidase, a protein secreted by neutrophils and involved in the inflammatory process, is also present in the brain. In vitro myeloperoxidase oxidation of recombinant human apolipoprotein E leads to fragmentation of the protein with low concentrations of hydrogen peroxide and polymerization with higher concentrations. Comparison with bovine serum albumin shows a higher susceptibility of apolipoprotein E to myeloperoxidase oxidation, which may have importance in the Alzheimer's disease process.

Chlorination of N-acetyltyrosine with HOCl, chloramines, and myeloperoxidase-hydrogen peroxide-chloride system.

N-acetyl-L-tyrosine (N-acTyr), with the alpha amine residue blocked by acetylation, can mimic the reactivity of exposed tyrosyl residues incorporated into polypeptides. In this study chlorination of N-acTyr residue at positions 3 and 5 in reactions with NaOCl, chloramines and the myeloperoxidase (MPO)-H2O2-Cl- chlorinating system were invesigated. The reaction of N-acTyr with HOCl/OCl- depends on the reactant concentration ratio employed. At the OCl-/N-acTyr (molar) ratio 1:4 and pH 5.0 the chlorination reaction yield is about 96% and 3-chlorotyrosine is the predominant reaction product. At the OCl-/N-acTyr molar ratio 1:1.1 both 3-chlorotyrosine and 3,5-dichlorotyrosine are formed. The yield of tyrosine chlorination depends also on pH, amounting to 100% at pH 5.5, 91% at pH 4.5 and 66% at pH 3.0. Replacing HOCl/OCl- by leucine/chloramine or alanine/chloramine in the reaction system, at pH 4.5 and 7.4, produces trace amount of 3-chlorotyrosine with the reaction yield of about 2% only. Employing the MPO-H2O2-Cl- chlorinating system at pH 5.4, production of a small amount of N-acTyr 3-chloroderivative was observed, but the reaction yield was low due to the rapid inactivation of MPO in the reaction system. The study results indicate that direct chlorination of tyrosyl residues which are not incorporated into the polypeptide structure occurs with excess HOCl/OCl- in acidic media. Due to the inability of the myeloperoxidase-H2O2-Cl- system to produce high enough HOCl concentrations, the MPO-mediated tyrosyl residue chlorination is not effective. Semistable amino-acid chloramines also appeared not effective as chlorine donors in direct tyrosyl chlorination.

Action of myeloperoxidase-hydrogen peroxide-chloride system on the egg white lysozyme.

The enzyme system composed of human neutrophilic myeloperoxidase (H2O2-oxidoreductase, EC 1.11.1.7), H2O2 and Cl-, at pH 4.5 interacts with egg white lysozyme (EC 3.2.1.17) in several stages. In the first stage, occurring at lysozyme to H2O2 molar ratio of 1:1.4-1.8, the lysozyme loses its enzyme activity but does not yield any derivative distinguishable from the native protein on polyacrylamide gel electrophoresis (PAGE). The second stage of oxidation begins at lysozyme to H2O2 molar ratio above 1:5, producing a change in the lysozyme spectrum at 260-290 nm, and yielding protein derivatives with molecular masses equal to multiples of 14.3 kDa, i.e. the lysozyme molecular mass. This implies that an excessive oxidation of lysozyme by the myeloperoxidase-H2O2-Cl- system produces cross-linking of lysozyme molecules to di-, tri-, tetra-, and pentameric structures. At lysozyme to H2O2 molar ratio exceeding 1:12 a water insoluble white product, which consists of a set of lysozyme cross-linked derivatives, is obtained.

Teaching clinical chemistry in central European countries--past and present.

Central Europe is traditionally referred to as the area occupied by the former Eastern Germany, Poland, Slovakia, Czech Republic and Hungary. In all of these countries great emphasis is placed on teaching clinical chemistry and biochemical pathology, both at undergraduate and postgraduate levels. In Czech Republic and in Poland analysis of blood, urine, body fluids, exudates and secretions as well as the fundamentals of interpretation of morbid states in biochemical terms are taught as an independent subject taking from 60 to 90 h of lectures, seminars and practical training. In Hungary, the fundamentals of clinical chemistry and biochemical pathology are included in courses of biochemistry, pathology and in clinical subjects, such as internal medicine and pediatrics. The postgraduate study of clinical biochemistry, which yields in all mid-European countries a certificate of specialisation in laboratory diagnostics (Poland), or clinical pathology (Czech Republic, Hungary), is based on at least 5 years experience in laboratory medicine and then extended studies including clinical biochemistry, haematology, cytology, microbiology, as well as the fundamentals of toxicology and immunology. A basic background in clinical practice is also required. In all countries in the area there also exists a well developed postgraduate education for laboratory workers without a medical background. These people can apply for a certificate in medical analytics (Poland), but they cannot work as clinical pathologists or laboratory diagnostic consultants.

Preanalytical factors in human plasma ascorbate assay.

Estimation of vitamin C (ascorbic acid) in various samples of fresh and frozen human plasma has shown that freezing in a regular freezer at -25 degrees C causes an approximately 14% loss of ascorbate in the sample. Freezing the same samples in a deep freezer at -75 degrees C causes less of an ascorbate loss amounting to about 9%. On the other hand, using the dry ice alcohol bath freezing, which shortens the freezing process to a fraction of seconds produced loss of ascorbate by 3.5% only. The storage time of previously frozen samples at -25 degrees C or -75 degrees C, from 2 to 14 days does not produce noticeable differences in the sample ascorbate concentration. Loss of ascorbate in samples frozen in the dry ice alcohol bath may be acceptable assuming analytical variability of ascorbate assay amounting to about 4% and broad biological variability of ascorbate concentration in various clinical conditions. Use of frozen plasma samples for ascorbate assays may essentially facilitate running this analysis in clinical laboratories.

Immunoglobulin cleavage by hypochlorous acid treatment.

IgA, IgG and IgM were cleaved by hypochlorous acid treatment. The apparent calculated molecular masses of three polypeptides obtained from IgA were 81.1. 25.8 and 13.9 kDa. The amounts of released IgA fragments were proportional to the amount of HOCl employed. At a HOCl:IgA molar ratio above 320:1, a profound degradation of IgA polypeptide chains occurred, resulting in a yellow-coloured product. The HOCl treatment of IgG resulted in similar effects, the liberation of three fragments, one of them being of a size slightly larger than that of the light chain (30.4 kDa). The treatment of IgM with HOCl also produced three fragments: one corresponding to the monomeric IgM molecule, the second to the light chain (26.4 kDa) and the third of a size smaller than the heavy chain. The optimal protein/HOCl ratios for the degradation of IgG and IgM were 375:1 and 808:1, respectively.

Ribonuclease from cytosolic fraction of human erythrocytes.

Ribonuclease (RNase) activity is detectable in only one third of specimens of human erythrocyte haemolysates. On the other hand, treatment of erythrocytic cytosoles with sulphosalicylic acid reveals an inhibitor-bound RNase activity which is present in all erythrocyte specimens studied. The level of the erythrocyte inhibitor-bound RNase activity is comparable to that in human lymphocytes. Isolated RNase from the cytosolic fraction of human erythrocytes is poly-C avid RNase with maximum activity at pH 6.5. The enzyme is resistant to treatment with strong acids and heating up to 95 degrees C. Molecular filtration of the erythrocyte RNase shows that it is composed of two fractions differing in molecular mass, 19 000 and 15 000. No difference in enzymic properties between these fractions was found. The general properties of erythrocyte cytosolic RNase are much like those of acid RNases of human granulocytes and lymphocytes. As the erythrocytes do not metabolize RNA no function for the inhibitor-bound RNase can be suggested. Assuming that the observed erythrocyte RNase is the residual enzyme, persisting in the cell since it was functioning in the nucleated erythrocyte precursors, one may surmise that levels of free and inhibitor-bound erythrocyte RNase activity may be related to the normality or abnormality of erythrocyte maturation.

Poly-C specific ribonuclease activity correlates with increased concentrations of IL-6, IL-8 and sTNFR55/sTNFR75 in plasma of patients with acute pancreatitis.

Plasma pancreatic-type Poly-C specific ribonuclease (P-RNase)-enzyme activity increases in patients with acute pancreatitis (AP) who develop pancreatic necrosis and severe disease course. It is considered as a marker of pancreatic tissue destruction. The aim of this study was to estimate interrelations between major inflammatory cytokines such as: interleukin 6 (IL-6), interleukin 8 (IL-8) and tumor necrosis factor soluble receptors: sTNFR55 and sTNFR75 output, and plasma P-RNase activity. The study was carried out in a group of 56 patients with AP, where 20 developed pancreatic necrosis. It was found that serum P-RNase concentration and levels of all studied inflammatory cytokines significantly increase already in the first day from diagnose of the disease (2.5 folds for P-RNase, 20 for IL-8, about 200 for IL-6 and 1.5 for receptors, respectively). In the first day from admission to hospital, P-RNase activity significantly correlated with plasma concentration of studied inflammatory cytokines. The most pronounced correlation was found for P-RNase and IL-6 in days 1-4 from diagnose, manifested by Pearson correlation r coefficients amounting to 0.86, 0.79, 0.60 and 0.57 respectively (p<0.001). Dividing the studied AP patients into two groups, varying in severity of disease a significant differences in P-RNase and IL-6, IL-8 and sTNFR55/sTNFR75 were found. In patients with acute necrotizing pancreatitis P-RNase significantly correlate with levels of major inflammatory cytokines. Carried out studies suggest that activity of P-RNase reflects severity of inflammatory reaction, which is dependent on development of pancreatic injury and tissue necrosis in AP.

Extract of grapefruit-seed reduces acute pancreatitis induced by ischemia/reperfusion in rats: possible implication of tissue antioxidants.

Grapefruit seed extract (GSE) has been shown to exert antibacterial, antifungal and antioxidant activity possibly due to the presence of naringenin, the flavonoid with cytoprotective action on the gastric mucosa. No study so far has been undertaken to determine whether this GSE is also capable of preventing acute pancreatic damage induced by ischemia/reperfusion (I/R), which is known to result from reduction of anti-oxidative capability of pancreatic tissue, and whether its possible preventive effect involves an antioxidative action of this biocomponent. In this study carried out on rats with acute hemorrhagic pancreatitis induced by 30 min partial pancreatic ischemia followed by 6 h of reperfusion, the GSE or vehicle (vegetable glycerin) was applied intragastrically in gradually increasing amounts (50-500 microl) 30 min before I/R. Pretreatment with GSE decreased the extent of pancreatitis with maximal protective effect of GSE at the dose 250 microl. GSE reduced the pancreatitis-evoked increase in serum lipase and poly-C specific ribonuclease activity, and attenuated the marked fall in pancreatic blood flow and pancreatic DNA synthesis. GSE administered alone increased significantly pancreatic tissue content of lipid peroxidation products, malondialdehyde and 4-hydroxyalkens, and when administered before I/R, GSE reduced the pancreatitis-induced lipid peroxidation. We conclude that GSE exerts protective activity against I/R-induced pancreatitis probably due to the activation of antioxidative mechanisms in the pancreas and the improvement of pancreatic blood flow.

Inactivation and denaturation of some proteins by enzyme system: myeloperoxidase, chloride and hydrogen peroxide.

Changes in biological properties of serum albumin, egg white lysozyme, human serum alpha-1 antiproteinase and human leukocyte ribonuclease in effect of interaction with the enzyme system composed of myeloperoxidase from human neutrophilic polymorphonuclear leukocytes, Cl- and H2O2 were investigated. All the studied proteins lost their biological functions and were denaturated, but the amounts of hydrogen peroxide necessary to produce these effects differed remarkably for each individual protein. The alpha-1 antiproteinase ability of binding to trypsin was abolished upon employing 1.2 mols of H2O2 per mol of alpha-1 antiproteinase. The lysozyme enzymatic activity was abolished when 1.4 mols of H2O2 per mol of lysozyme were employed. Albumin decreased its binding to specific antialbumin antibodies and entirely lost the binding properties when 2 mols and about 10 mols of H2O2 per mol of albumin were employed, respectively. On the other hand 18 mols of H2O2 per mol of human leukocyte ribonuclease were necessary to inactivate this enzyme. All the mentioned proteins were protected from losing their biological functions by excess of specific amino acids with affinity to hypochlorite: Alpha-1 antiproteinase by excess of N-acetylmethionine, lysozyme by N-acetylmethionine and N-acetyl glycyltryptophane, albumin by N-acetyl derivatives of methionine, cysteine, tryptophane and lysine, whereas ribonuclease was protected from denaturation by all above mentioned amino acid derivatives. None of the studied proteins was protected from denaturation by N-acetyl tyrosine, or phenylalanine.

Oxidative modification of protein structures under the action of myeloperoxidase and the hydrogen peroxide and chloride system.

Myeloperoxidase of neutrophilic leukocytes (MPO) at pH 4.0 to 6.5 mediated oxidation of Cl- ions, yielding hypochloride (OCl-) which then reacted with amino acids and polypeptides. Thiol and thioether groups may be oxidized to disulfide or to sulphoxides and sulphonic acids respectively. Tryptophanyl residues yielded 2-oxoindole. Epsilon amino groups of lysine produced chloramine which, however, decomposed, yielding aldehyde residues. Bovine serum albumin treated with MPO-Cl-H2O2 system yielded derivatives with a decreased affinity to antialbumin antibodies and increased electrophoretic mobility. Albumin aldehyde derivatives were also obtained. At H2O2 molar ratio with albumin 20:1, a precipitation of albumin occurred, due to the formation of new polymeric albumin derivatives. The lysozyme (LZM) lost its enzyme activity when 1.4 to 1.8 mol of H2O2 per 1 mol of LZM was used. Addition of H2O2 above molar ratio 5:1 produced LZM polymerization to di-, tri-, tetra and pentameric derivatives. IgA exposed to the MPO-Cl-H2O2-Cl- system split into light chains (molecular weight: 25.8 kDa), heavy chains (molecular weight: 81.8 kDa) and a third polypeptide which size was half the light chain size (molecular weight: 13.9 kDa). The IgA exceeding the HOCl ratio 1:350 (mg/mumol) produced both precipitation and degradation of the IgA polypeptide structure. The treatment of IgG with HOCl released a fragment corresponding to half the light chain size, the light chain, and the heavy chain, whereas HOCl treatment of IgM released only a fragment which size was smaller than the heavy chain and another fragment which size was the same as the light chain. The MPO-Cl-H2O2 system produced many specific changes in protein structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer modelling of polymer decomposition by active interactions with enzyme molecules. A simulation of RNA digestion by ribonuclease.

A model of 200 polymer molecules each 70 units of length with randomly located susceptible and resistant links was elaborated. The production of mono-, di-, tri-unit and other fragments in repeating random interactions of polymer (P) with the polymer--cleaving enzyme (E) was calculated. Analysis of the calculations performed that the polymer molecules with an initial count of 70-units disappeared, imitating the first order kinetics with a half-life of about 0.37 x 10(3) P/E interactions. The production of fragments 30-69 units long was small and the life of those molecules was limited to a range from 0.5 x 10(3) to 5 x 10(3) P/E interactions. The mean polymer length decreases to one third at 10(3) P/E interactions. The production of single-unit fragments followed a sigmoidal relationship. The first reaction period, with increasing number of single-unit fragments per 10(3) P/E interactions, corresponded to a decrease in the mean polymer length to about 10 units. Then the production of one-unit fragments decreased and stopped at about 10(5) P/E interactions. This model could be used for analysis of RNA fragmentation processes by any number of RNA-ribonuclease interactions. Further development of this model would be of help in understanding the effect of various nucleotide sequences on the RNA digestion process.

Ribonuclease in human serum and urine expressed in arbitrary standard units of activity.

A definition of the arbitrary unit expressing the catalytic activity of ribonuclease (RNase) in serum and urine has been proposed. The unit defines the RNase activity solely in terms of a rate of RNase mediated decomposition of RNA-substrate. The proposed unit can be also used for expressing the results of RNase determining in former studies, if their analytical procedures have been enough precisely described. This makes possible the comparison of the recent experimental data with results obtained in other studies carried out with the use employment the nonstandardized RNase determining procedures. In this paper the comparison between the values expressing RNase activity in human serum and urine, published in various studies in years 1965-1992 and recalculated in the proposed arbitrary units, is presented.

[Chemical tests during a real time physician-patient interaction--the concept and practical problems resulting from its realization]. / Badania chemiczne w rzeczywistym czasie kontaktu lekarza z pacjentem--koncepcja i problemy praktyczne wynikajace z jej realizacji.

Laboratory tests performed at the patient's bed-side in a real time of patient-physician interaction, consist a part of a program focussed on maximal shortening of the treatment time and increase the efficiency of the applied medical procedures. A whole generation of a modern chemical analysers are presently available for use in a near patients testing. However, performing a "real time" bed-side testing by persons who are not prepared for work in medical analytics effects in a numerous mistakes both of preanalytical and analytical nature, decreasing the reliability of the obtained results. Thus, implementation of near patient testing should be based on the already elaborated procedures of quality assurance and quality control. The management of the bed side testing system, and supervision of its conformity with quality procedures should be a task of a hospital central laboratory, which on the other hand is also responsible for good compliance of bed-side results with the results provided from the laboratory. Nevertheless the prones of the bed-side tests on quality impairment, the properly organized near patient's test systems are of a great value for ICU wards, admission rooms, and general practitioners working in locations distant from the professional laboratories.

[Mediators of the inflammatory response in the course of acute pancreatitis]. / Mediatory procesu zapalnego w przebiegu ostrego zapalenia trzustki.

Acute pancreatitis (AP) is one of the most frequent causes of acute inflammatory states in the abdominal cave. In majority of cases the disease, if properly treated, has a selflimiting course and terminates in several days. However, in about 20% of patients with diagnosed AP haemorrhagic and necrotic lesions of the pancreas occur what effects in multiple organ injury and high mortality of these patients. Early diagnosis of haemorrhagic and necrotizing outcome of the disease is difficult, therefore new markers of necrotizing acute pancreatitis are still under research. Cytokines mediating inflammatory process may provide some specific information on the expected outcome of acute pancreatitis. Article reviews the information about the role of inflammatory cytokines in development and outcome of acute pancreatitis.