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At least 271 records · Page 15Linked to original sources

PubMed2000

Sensitive and nonenzymatic measurement of hydrogen peroxide in biological systems.

The increasing demand in detecting H(2)O(2) under various experimental conditions is only partly fulfilled by most conventional peroxidase-based assays. This article describes a sensitive and nonenzymatic H(2)O(2) assay that is based on the chemiluminescence reaction of luminol with hypochlorite. It allows the determination of H(2)O(2) down to nanomolar concentrations. Actual H(2)O(2) concentrations rather than a turnover of H(2)O(2) can be determined in monolayer cultures, perfusates, suspensions of intact cells, organelles, and crude homogenates. One of the strengths of this assay is that it may be used to assess fast enzyme kinetics (catalase, glutathione peroxidase, oxidases) at very low H(2)O(2) concentrations. Its use together with a glucose oxidase/catalase system appears to be a powerful tool in studying signal functions of H(2)O(2) in various biological systems on a quantitative basis. Several applications are discussed in detail to demonstrate the technical requirements and analytical potentials.

Animals↗

PubMed2005

Reduction of chemical reaction networks using quasi-integrals.

We present a numerical method to identify possible candidates for quasi-stationary manifolds in complex reaction networks governed by systems of ordinary differential equations. Inspired by singular perturbation theory, we examine the ratios of certain components of the reaction rate vector. Those ratios that rapidly approach a nearly constant value define a slow manifold for the original flow in terms of quasi-integrals, that is, functions that are nearly constant along the trajectories. The dimensionality of the original system is thus effectively reduced without reliance on a priori knowledge of the different time scales in the system. We also demonstrate the relation of our approach to singular perturbation theory which, in its simplest form, is just the well-known quasi-steady-state approximation. In two case studies, we apply our method to oscillatory chemical systems: the 6-dimensional hemin-hydrogen peroxide-sulfite pH oscillator and a 10-dimensional mechanistic model for the peroxidase-oxidase (PO) reaction system. We conjecture that the presented method is especially suited for a straightforward reduction of higher dimensional dynamical systems where analytical methods fail to identify the different time scales associated with the slow invariant manifolds present in the system.

Hemin↗

PubMed2006

Limitation of glucose oxidase method of glucose estimation in jaundiced neonates.

The most widely used method for estimation of plasma glucose is that adopted by Trinder's using glucose oxidase-peroxidase (GOD-POD) system. This method gives much lower blood glucose values with blood samples of neonatal jaundice (plasma bilirubin level > 10 mg/dL) of age 10 +/- 5 daysthan with samples of neonates of the same age group without jaundice or older children suffering from other diseases like acute respiratory distress, septicemia.

Glucose↗

PubMedDate not supplied

[The enzymatic activity of IgG preparations in viral hepatitis].

The activity of the enzymatic activity of the preparations of IgG1, IgG2 and IgG4, isolated from the blood of patients with acute virus hepatitis B and chronic viral hepatitis C resulting in cirrhosis, was studied. The blood samples were found to have DNAase activity significantly exceeding that of immunoglobulins isolated from the blood sera of healthy donors, as well as peroxidase, oxidase and esterase activities, whose level did not significantly differ from those of the donor blood sera. The interaction of IgG preparations with the cations of different metals was studied. The study revealed that the addition of CuSO4 solution at the final concentration of 4.7 x 10(-5) M to the blood samples led to a significant increase in activity in comparison with the initial one (on the average, 7.8 +/- 2.97 times) in all 14 samples. The activity thus observed was partially inhibited by the addition of the solution of staphylococcal protein A. As noted in the course of this study, high DNAase and peroxidase activities of Ig were most frequently observed in patients with cirrhosis of the liver. The difference in the levels of IgG activity between patients with cirrhosis of the liver and patients with virus hepatitis, but no signs of cirrhosis, is not significant.

Acute Disease↗

PubMed1967

Prevention of induced atherosclerosis by peroxidase.

Hepatocatalase peroxidase, an active peroxidase-oxidase subunit isolated from beef-liver catalase, prevents cholesterol deposition and aortic atherosclerosis in cholesterol-fed rabbits and has no apparent toxicity or undesirable de effects. No allergic or immunological reactions have been observed. The participation of this enzymatic subunit in homeostatic control mechanisms and its potential pharmacological value in the control of human atherosclerosis are suggested.

Alkaline Phosphatase↗

PubMed1976

Sources of error in glucose determinations in neonatal blood by glucose oxidase methods, including Dextrostix.

An investigation was carried out to examine why a glucose oxidase-peroxidase-orthodianisidine method for plasma glucose, without protein precipitation, gave low results for neonatal blood. The magnitude of the difference between the results with and without protein precipitation was examined in a clinical neonatal series, and in sera to which bilirubin, hemolysate, pure hemoglobin, and uric acid had been added. Systematic linear inhibition was demonstrated with bilirubin, and the results suggested that high concentrations of hemolysate and uric acid could also interfere. Use of alkaline protein precipitants eliminated the inhibition. Dextrostix test results for neonatal blood are compared with results of conventional glucose analyses and possible sources of discrepancy examined.

Bilirubin↗

PubMed1990

A comparison of chromogen test strip (Chemstrip bG) and serum glucose values in newborns.

Although glucose oxidase-peroxidase chromogen test strips are frequently used to estimate serum glucose values in newborns, previous studies have not evaluated multiobserver variability of test strip readings and have included few infants with hypoglycemia. We compared values of 272 samples of serum glucose with values simultaneously obtained by chromogen test strips (Chemstrip bG) in newborns. The diagnostic sensitivity of a chromogen test strip less than 2.2 mmol/L for predicting a serum glucose level less than 1.9 mmol/L was 86% (95% confidence interval [CI], 75% to 94%), with 78% specificity (95% CI, 73% to 84%). The positive predictive value in our specimens, with a 21% prevalence of serum glucose levels less than 1.9 mmol/L, was 52% (95% CI, 41% to 62%), with a negative predictive value of 95% (95% CI, 91% to 100%). Fifty-eight of our serum glucose values were less than 1.9 mmol/L and the levels obtained by chromogen test strip were greater than or equal to 2.2 mmol/L in 8 of these cases. Review of these 8 cases showed that a delay in performing the laboratory glucose oxidase serum glucose could account for the discrepancy in 2 cases. Chromogen test strips are readily available and easy to use, but more sensitive, specific, accurate, and precise methods of serum glucose screening in newborns are needed.

Blood Glucose↗

PubMed1996

Chaos control by electric current in an enzymatic reaction.

We apply the continuous delayed feedback method of Pyragas to control chaos in the enzymatic Peroxidase-Oxidase (PO) reaction, using the electric current as the control parameter. At each data point in the time series, a time delayed feedback function applies a small amplitude perturbation to inert platinum electrodes, which causes redox processes on the surface of the electrodes. These perturbations are calculated as the difference between the previous (time delayed) signal and the actual signal. Unstable periodic P1, 1(1), and 1(2) orbits (UPOs) were stabilized in the CSTR (continuous stirred tank reactor) experiments. The stabilization is demonstrated by at least three conditions: A minimum in the experimental dispersion function, the equality of the delay time with the period of the stabilized attractor and the embedment of the stabilized periodic attractor in the chaotic attractor.

Algorithms↗

PubMed1975

New, simple maltogenic assay for mechanized determination of alpha-amylase activity in serum and urine.

We report a new method for the mechanized determination of serum and urinary alpha-amylase by use of a continuous-flow system, based on the measurement of maltose formed by incubating the sample with amylodextrin at pH 7 and 40 degrees C. After dialysis, maltose is converted enzymatically to glucose, which is measured by Trinder's glucose oxidase-peroxidase method [J. Clin. Pathol. 22, 246 (1969)]. The reaction is linear for amylase activities up to 1400 Somogyi units/dl (2560 U/liter) and for maltose concentrations through 1500 mg/dl. No blank assay is required; consequently precision is improved and the automated system is simplified. Calibration with primary maltose standards increases accuracy and reliability. Common reducing substances in serum and urine do not interfere at their normal concentrations. There is a linear correlation between the results of this method and those of chromogenic and iodometric methods for normal and pathologic sera and urines. The chromogenic method yields significantly higher results and the iodometric method significantly lower results than this maltogenic method for elevated amylase activities. The normal range is 40-140 Somogyi units/dl (73-256 U/liter).

Adult↗

PubMed1996

Stochastic Resonance and time advance coding in chemical reactions.

Stochastic Resonance (SR) is a phenomenon which may be found in nonlinear systems close to an excitation threshold. SR is a means for enhancing a weak periodic subthreshold signal from its noisy background by adding stochastic fluctuations, i.e. in biological and physical systems. It has been proposed that SR is important for the ability of neural systems to detect weak periodic signals. In the present work we show experimentally that SR occurs in two nonlinear chemical reactions, namely in the enzymatic Peroxidase-Oxidase (PO) reaction and in the Belousov-Zhabotinskii (BZ) reaction. A small sinusoidal signal with increasing noise is imposed on the focal steady state near a subcritical Hopf bifurcation. When the threshold is crossed beyond a certain noise amplitude, the system responds with spikes. The resulting interspike histogram and the plot of the signal to noise ratio, which is evaluated from the respective Fourier spectra, pass through a maximum at an optimal external noise level. An alternative way to cross the excitation threshold without noise is the variation of the bias value of the sinusoidal signal. The variation of the bias value causes the spikes to appear earlier if the sinusoidal function is moved closer towards the threshold. This so-called time advance coding is shown experimentally for the first time in the BZ reaction by imposing sinusoidal flow rate variations using different bias values. The phenomenon has been proposed by Hopfield to be a means for analog pattern recognition.

Artifacts↗

PubMedDate not supplied

Enhanced chemiluminescent assays for acetylcholine.

Acetylcholine and choline chemiluminescent assays have limitations when these compounds are detected in small areas of mammalian nervous tissue. Use of 7-dimethyl-aminonaphthalene-1,2-dicarbonic acid hydrazide (7-DMAN), instead of luminol, gives a threefold increase in emitted light in the chemiluminescent assay for acetylcholine based on the coupled choline oxidase-peroxidase reaction. Addition of light enhancers, such as para-iodophenol or D-luciferin, to luminol or 7-DMAN further increased the light emission. Under these conditions the detection limit for acetylcholine was 650 femtomoles. This enhanced chemiluminescent assay should be convenient for the detection of in vivo and in vitro acetylcholine release from mammalian neurons.

Acetylcholine↗

PubMed1994

A spectrophotometric assay for glucosidase I.

A spectrophotometric assay for glucosidase I using the synthetic trisaccharide alpha-D-Glc 1-->2 alpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)8COOCH3 is reported. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The trisaccharide is specific for glucosidase I and provides all the necessary structural features for correct interaction in the enzyme active site. The utility of the assay for monitoring enzyme activity during isolation and for use in kinetic and inhibition studies (i.e., with 1-deoxynorjirimycin) is demonstrated.

1-Deoxynojirimycin↗

PubMed1992

Peroxidase-promoted oxidation and peroxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine. A new pathway for its metabolic degradation.

Spectral data provide the first evidence that lactoperoxidase, a model enzyme for most mammalian peroxidases, catalyzed the one-electron oxidation and/or peroxidation of 5,7-dihydroxytryptamine. This process correlates with the production of superoxide radicals as is evident from the observed inhibitory effect of superoxide dismutase on product formation. 5,7-Dihydroxytryptamine is a classical peroxidase-oxidase substrate acting as a one-electron donor for enzyme compounds I, II and III. The one-electron peroxidatic oxidation of this serotonergic neurotoxin, responsible for the selective degeneration of central (5-hydroxytryptamine) neurons, is a fast process requiring measurement on the ms time scale. Attention is drawn to the biochemical and toxicological implications, because this fast reaction results in formation of known cell damaging species: free radicals, superoxide radicals and quinoidal products probably involved in the toxic action of 5,7-dihydroxytryptamine.

5,7-Dihydroxytryptamine↗

PubMed2004

Identification and characterization of a novel cassava (Manihot esculenta Crantz) clone with high free sugar content and novel starch.

This study reports the identification of a new class of cassava (Manihot esculenta Crantz) with a storage root showing unusual free sugar accumulation and novel starch. Twenty-seven clones high in free sugar were identified under cultivation in primitive rural community areas in the Amazon. Iodine test and glucose oxidase-peroxidase reagent strips were used, in the field, for identification of starch and glucose, respectively. Five out of these 27 clones of cassava were cultivated at EMBRAPA Genetic Resources and Biotechnology and used for biochemical characterization, starch synthesis enzyme activities and gene expression analysis. Carbohydrates were fractioned into free sugar, polymerized water-soluble and -insoluble alpha-polyglucan. Clones of series CAS36 accumulate over 100 times more free sugar (mainly glucose) than commercial varieties. Monosaccharide composition analysis revealed one clone with distinct water-soluble sugars not present in the commercial cultivar. Structure analysis of the water-soluble and -insoluble alpha-polyglucan revealed the presence of a glycogen-like starch in clone CAS36.1. This clone indicated disruption in the starch synthesis pathway for enzyme activities and protein blot analyses in ADPG-pyrophosphorylase and branching enzyme, and their corresponding protein. Gene expression analysis indicated the lack of transcript for the gene coding for branching enzyme, but not for the gene coding for the ADPG-pyrophosphorylase small subunit. In addition, the pattern of distribution of sugar and starch content showed to be related to tissue age in the storage root.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1987

A rapid assay method for ammonia using glutamine synthetase from glutamate-producing bacteria.

A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture.

Adenosine Diphosphate↗

PubMed1989

A colorimetric method for the enzymatic analysis of gases: the determination of ethanol and formaldehyde vapors using solid alcohol oxidase.

A novel enzymatic approach to the direct determination of ethanol vapors in the gas phase is described. The system is composed of alcohol oxidase, peroxidase, and the color indicator 2,6-dichloroindophenol dispersed on microcrystalline cellulose (avicel). Simple devices are developed for the semiquantitative determination of ethanol in the breath. The devices are optimized to produce a sharp color change at a set time of 1 min for ethanol concentrations above the legal limit for driving (kinetic method) or a stable final color after 5 min (equilibrium method). Such color changes are detectable by simple visual observation. Using TLC plastic sheets and a transmittance densitometer, the system can also be used as a quantitative method for the determination of ethanol or formaldehyde vapors. Dehydrated enzymes may be useful for the analysis of hazardous gases.

2,6-Dichloroindophenol↗

PubMed1985

Allosteric inhibition of brain hexokinase by glucose 6-phosphate in the reverse reaction.

A study of the reverse reaction of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been performed using a photometric method based on a mutarotase-glucose oxidase-peroxidase-chromogen system to trap and visualize glucose, plus a glycerol kinase-glycerol system to trap ATP. Glucose 6-phosphate or 2-deoxyglucose 6-phosphate were used as phosphoryl donors at different concentrations of ADP. Variation of glucose 6-phosphate concentrations resulted in a biphasic curve from which apparent Km and Ki values of ca. 0.2 mM were calculated. In contrast, variation of 2-deoxyglucose 6-phosphate concentrations resulted in Michaelian kinetics with an apparent Km of 2 mM. The Km value for MgADP was 16 mM irrespective of the nature and concentration of the hexose 6-phosphate substrate. These results are fully consistent with an allosteric site for glucose 6-phosphate as an explanation for the inhibition of animal hexokinases by glucose 6-P and further indicate that the maximal rate is the parameter affected. From these observations and previous knowledge, the possible occurrence in animal hexokinases of a regulatory site for ATP to account for the competition between glucose 6-phosphate and ATP in the forward reaction is postulated.

Adenosine Diphosphate↗

PubMed1982

A procedure for the kinetic colorimetric determination of serum cholinesterase activity.

A choline oxidase-peroxidase coupled enzyme procedures is proposed for the determination of cholinesterase activity in human serum. This system is not only kinetic and colorimetric but is also relatively quick and simple to perform. The initial comparisons suggest that this method correlates well with a commonly used propionylthiocholine-dinitrobis-(nitro-benzoic acid) technique. Large amounts of bilirubin in the sample appear to have only minor deleterious effects on the assay. Since there are only two reagents that may be premixed, the procedure appears to be amenable to automation. The use of a mixture of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate and 4-aminoantipyrene in the peroxidase catalyzed indicator reaction provides for a marked increase in sensitivity over previously reported 4-aminoantipyrene-phenol systems. This augmented sensitivity provides for a relatively large reagent to sample ratio. In addition, the reagents lend themselves toward lyophilization or "dry-fill".

Cholinesterases↗