Determination of thyroxine in pharmaceuticals using flow injection with luminol chemiluminescence inhibition detection.

A simple flow injection method is reported for the determination of thyroxine, based on its inhibition effect on luminol-iron(II) chemiluminescence in alkaline medium in the presence of molecular oxygen. The detection limits (2s) for d- and l-thyroxine are 0.08 and 0.1 mg/L, respectively, with a sample throughput of 100/h. The calibration data for d- and l-thyroxine over the range 0.2-1.0 mg/L gives correlation coefficients (r(2)) of 0.9915 and 0.984 with relative standard deviations (RSD; n = 4) in the range 1.2-2.8%. The effects of some organic compounds was studied on luminol-iron(II) CL system for thyroxine determination. The method was applied to pharmaceutical thyroxine tablets and the results obtained (in the range 50.5 +/- 2.0-51.6 +/- 1.2 microg l-thyroxine/tablet) were in reasonable agreement with the value quoted.     

Model components of luminol chemiluminescence generated by PMNL

The production of activated oxygen species (AOS) by neutrophils (PMNL) is thought to play a key role in the host defence against invading microorganisms. However, the oxygen metabolites are toxic not only to the invading bacteria but also to the surrounding tissue. The oxidative metabolites production can be evaluated by means of chemiluminescent methods. In this study, the possibility of a new analytical approach for quantitative assessment of chemiluminescent kinetics (AOS generation) of isolated PMNL was estimated.

Based on the assumption that the kinetics of luminol-amplified chemiluminescence (LCL) of stimulated PMNL possesses a time-probabilistic nature, this kinetics was described with three components. These components, obtained from different investigated systems, were analyzed and a conclusion was made that the first and the second component represent the processes resulting in extra-and intracellular myeloperoxidase (MPO)-dependent light emission (AOS generation), respectively. The second component was found to be completely dependent on the stimulus ingestion. The third component was not completely MPO-dependent and complicated for interpretation. This component was weakly dependent on the stimulus ingestion, and presents at least some intracellular processes different from those presented by the second component.

A conclusion is made that the examined approach for analysis of LCL kinetics allows an assessment of extra-and intracellularly generated quantities of AOS by stimulated PMNL. The assessment could be done for emitting systems in which no additional modificators are used.     

Enhancement and inhibition of luminol chemiluminescence by phenolic acids

We explored the behaviour of a series of phenolic acids used as enhancers or inhibitors of luminol chemiluminescence by three different methods to determine if behaviour was associated with phenolic acid structure and redox character. All the phenolic acids inhibited chemiluminescence when hexacyanoferrate(III) was reacted with the phenolic acids before adding luminol. The redox character of these compounds was clearly related to structure. When hexacyanoferrate(III)-luminol-O₂ chemiluminescence was initiated by phenolic acid-luminol mixtures some phenolic acids behaved as enhancers of chemiluminescence, and others as inhibitors. We propose a mechanism to explain these findings. We found direct relationships between the redox character of the phenolic acids and the enhancement or inhibition of the chemiluminescence of the luminol–H₂O₂–peroxidase system and we propose mechanism to explain these phenomena.     

Kinetic characteristics of luminol chemiluminescence caused by chloramine compounds

The decaying part of the kinetic curves of luminol chemiluminescence (0.02 mM) induced by N-chlorphenylalanine is approximated by an exponential dependence, which varies insignificantly as chloramine concentration is changed from 0.2 to 0.7 mM. On the whole, the chemiluminescence of luminol is a result of its oxidation, which occurs in three stages with the formation of two intermediate products. N-Chlorphenylalanine is involved in the process at the initial stage. The reciprocal of the time the luminescence reaches a maximum increases linearly with the growth of N-chlorphenylalanine concentration. According to the calculations using the equations that reflect three stages of luminol conversion in the presence of excess chloramine, the rate constant for the initial stage is about 10(3) l/(mol.min). The rate constant for one stage of the conversion of luminol oxidation product is approximately 0.2 min-1, and the rate constant of the other is severalfold greater. Luminol chemiluminescence induced by low concentrations of N,N-dichlortaurine is more durable. Probably, it is composed of two types of emission one of which slowly decays.     

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H₂O₂ formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C₁₀-C₁₈) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H₂O₂ standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.     

Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.     

Autofluorescence of viable cultured mammalian cells.

The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.     

A luminol chemiluminescence method for sensing histidine and lysine using enzyme reactions

The analysis of free amino acids in urine and plasma is useful for estimating disease status in clinical diagnoses. Changes in the concentration of free amino acids in foods are also useful markers of freshness, nutrition, and taste. In this study, the specific interaction between aminoacyl–tRNA synthetase (aaRS) and its corresponding amino acid was used to measure amino acid concentrations. Pyrophosphate released by the amino acid–aaRS binding reaction was detected by luminol chemiluminescence; the method provided selective quantitation of 1.0–30 μM histidine and 1.0–60 μM lysine.     

Mathematical model for the luminol chemiluminescence reaction catalyzed by peroxidase

A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship. (c) 1993 Wiley & Sons, Inc.     

Characteristics of luminol chemiluminescence induced by the catalytic action of myeloperoxidase

The effects of pH, luminol myeloperoxidase and hydrogen peroxide concentrations on the intensity of luminol chemiluminescence induced by myeloperoxidase catalysis were investigated. It was found that the intensity of luminescence is proportional to the enzyme concentration (up to 8.10(-8) M) and reaches the saturation level at higher enzyme concentrations. The dependence of chemiluminescence intensity on [H2O2] is bell-shaped: at H2O2 concentrations above 1.10(-4) M the luminescence is inhibited with a maximum at neutral values of pH. Luminol at concentrations above 5.10(-5) M inhibits this process. It was demonstrated that the effects of singlet oxygen, superoxide and hydroxyl radicals on the chemiluminescence reaction are insignificant. Luminol oxidation in the course of the myeloperoxidase reaction is induced by hypochlorite.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

On-line electrocatalyzed luminol chemiluminescence determination of d-glucose and dissolved oxygen in simulated mammalian cell bioreactor perfusion fluid using solid phase reagent modules

On-line instrumentation and methods for the chemiluminescence based real-time monitoring of d-glucose and O₂ levels in mammalian cell bioreactor perfusion fluid are described. The unit processes required for the analysis include: pH adjustment using solid phase flow-through modules, immobilized enzyme catalyzed oxidation of glucose by molecular oxygen to produce hydrogen peroxide, controlled release of luminol using a solid phase flow-through module, electrocatalyzed luminescence using gold electrodes, and photodetection of chemiluminescent emissions. Calibration curves for d-glucose and dissolved O₂ in simulated bioreactor perfusion fluid have been generated using fully integrated reagentless test systems from 0–800 mg l^–1 and 0–10 mg l^–1, respectively.     

Mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite

Two different mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite were found. Most substances investigated in these experiments acted by scavenging NaOCI. This mechanism was independent of the concentration of hydrogen peroxide and the incubation time between luminol and inhibitors. The most potent inhibitors were substances containing SH groups. Compounds with amino groups as a target for HOCI/OCI^? to yield chloramines were much less effective inhibitors. Another mechanism of inhibition was found for catalase. It depended on the presence of hydrogen peroxide in the incubation medium and the incubation time between luminol and catalase. The enzyme inhibited the luminescence by removing H₂O₂ at molar concentrations much smaller than those found for all other inhibitors. Our results confirm the present models of the mechanism of generation of luminescence in luminol oxidation.     

Scavenger effect of flavonols on HOCl-induced luminol chemiluminescence.

Hypochlorous acid (HOCl), the main product of the myeloperoxidase system, is a strong oxidant and a potent chlorinating agent, which can damage host tissues. In the present work, the scavenger effect of three aglycone flavonols (myricetin, quercetin and kaempferol) and of the natural glycoside flavonol, rutin, was studied towards HOCl using luminol-dependent chemiluminescence (CL). At 1 micro mol/L fi nal concentration, rutin was the most powerful scavenger of HOCl with an inhibitory luminol oxidation of 91.4% +/- 3.2%. Quercetin, kaempferol and myricetin inhibited the luminol-dependent CL at the same concentration only by 75.9% +/- 3.4%, 57.7% +/- 5.3% and 43.3% +/- 3.5%, respectively. With increasing concentration of these flavonols, a dose-dependent inhibition of luminol CL was observed. In order to prove to what extent flavonols scavenge HOCl, their concentrations that gave 50% inhibition of luminescence (IC50) were compared to IC50 values of the sulphur-containing compounds N-acetyl cysteine (NAC) and taurine. The scavenging activities of compounds tested decrease in the order: rutin > NAC > quercetin > kaempferol > taurine. The present study revealed that rutin was the most effective scavenger agent.     

Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol

The chemiluminescence of luminol (3-aminophthalhydrazide) with H₂O₂ has been used to quantify endogenous amounts of H₂O₂ in plant tissues. The reaction is linear over at least three orders of magnitude between 10^?5 and 10^?2M H₂O₂. Interference by coloured compounds in the crude extract is calibrated by a purification step with Dowex AG 1-X8. The extract is calibrated with an internal H₂O₂ standard, and the specificity verified by H₂O₂ purging with catalase. The minimum delectability for H₂O₂ of this assay is at least 1 ng, corresponding to 0.1–1 g fresh material. Data are presented for the levels of H₂O₂ in potatoes after treatment with oxygen and ethylene, in tomatoes before and after ripening and in untreated germinating castor beans as well as in beans treated with aminotriazol to inhibit catalase activity. Though data using the titanium test are generally confirmed, the method presented here has the advantage of higher sensitivity and specificity.     

Specificity of oxygen radical scavengers and assessment of free radical scavenger efficiency using luminol enhanced chemiluminescence

The selective scavenging capacities of 19 important oxygen radical scavengers were determined by adding them individually to each of the four oxy radical standards, (superoxide, hydroxy, alkoxy and hydroperoxy, and singlet O2), calculating the percent chemiluminescence inhibited, and extrapolating O2 equivalents neutralized from baseline. The sensitivity (0.01 nm/ml) and selectivity of this method not only allows identification of individual oxygen free radical species but also quantitates the efficiency of free radical scavengers.     

Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.     

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.     

Study of oxygen photoreduction in chloroplasts by the method of luminol and chlorophyll chemiluminescence]

The reduction of oxygen by irradiated chloroplasts was studied for elucidation of oxygen action site in the electron transport chain of photosynthesis. Chemiluminescence system, consisted of luminol and peroxidase, was used for registration of oxygen reduction products. In the first case chemiluminescence system was added to supernatant fraction after centrifugation of suspension of irradiated chloroplasts in order to determine H2O2 which was found to be the final product of oxygen photoreduction. In the second case when chloroplasts were illuminated in the presence of chemiluminescence system and oxygen the fact delayed luminescence of luminol was observed. This photoluminescence related also with the oxygen reduction in chloroplasts caused a possible formation of radicals HO2 (or -O2). The formation of this radicals and H2O2 was inhibited by DCMU, heating of chloroplasts at 45 degrees C for 5 min and by washing with EDTA and NH2OH. The rate of HO2 dissappearance was increased by methylviologen. The kinetics of photoluminescence of luminol and afterglow of chlorophyll in chloroplasts was identical in the interval from 20 msec to several seconds. It is suggested that oxygen reaction site is located near the reaction centre of chloroplasts.     

Kinetics of a rapid, luminol dependent chemiluminescence signal induced in HL-60 cells by amphotericin B and other stimulants.

Addition of the polyene antibiotic amphotericin B or tissue culture medium to nondifferentiated HL-60 cells in the presence of luminol induces a chemiluminescence signal that reaches a peak value within a few seconds and decays exponentially in less than a minute. The kinetics of the signal and its modulation by superoxide dismutase, catalase, and horseradish peroxidase are consistent with a series of solution biochemical processes with a rate-determining step corresponding to the disproportionation of a luminol-superoxide complex. The effects of the enzymes demonstrate that superoxide is a precursor to the rate-determining intermediate and that both catalase and peroxide enhance a reaction that competes with the rate-limiting process.