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Introduction There is a balance between oxidative stress, antioxidant capacity and immune response. Their roles in physiological and behavioural mechanisms are important for the maintenance of the organism’s internal equilibrium. This study aimed to evaluate the antioxidant effects of the exogenous alga Spirulina platensis (Arthrospira platensis) in a stress-induced rat model, and to describe its possible mechanism of action. Material and Methods Thirty-six adult male Sprague Dawley rats were separated into four groups: control (C), stress (S), S. platensis (Sp), and S. platensis + stress (SpS). The rats in groups Sp and SpS were fed with 1,500 mg/kg b.w./day Spirulina platensis for 28 days. All rats were exposed to prolonged light phase conditions (18 h light : 6 h dark) for 14 days. The SpS and S groups were exposed to stress by being kept isolated and in a crowded environment. Blood samples were obtained by puncturing the heart on the 28th day. The effect of stress on serum corticosterone, oxidative stress markers (TOS, TAC, PON1, OSI) and immunological parameters (IL-2, IL-4, IFN-ɣ) were tested. Also, the brain, heart, intestines (duodenum, ileum, and colon), kidney, liver, spleen, and stomach of the rats were weighed. Results Serum corticosterone levels were higher in the S group than in the C group, and significantly lower in the SpS group than in the S group. Mean total antioxidant capacity were lower in the S group than in the C group, and Spirulina reversed this change. Although not significantly different, IL-2 was lower in the S group than in the C group. However, in the SpS group, IL-2 increased due to Spirulina platensis mitigating effects of stress. Conclusion Male rats fed a diet with Spirulina platensis could experience significantly milder physiological changes during stress, although stress patterns may be different. Exogenous antioxidant supplements merit further investigation in animals and humans where the endogenous defence mechanism against stress may not be sufficient.

BACKGROUND: Although radioiodine theraphy (RAIT) is thought to affect blood cells and oxidative stress, hemorheological alterations following dose-dependent RAIT remains unknown. OBJECTIVE: The aim of this study was to determine the effects of RAIT on hemorheological and oxidative stress parameters in patients with differentiated thyroid cancers (DTC). METHODS: Totally 31 DTC patients (mean age 46.32±11.15 years) and 26 healthy controls (mean age 50.50±6.22 years) were included. Venous blood samples were collected from each patient before and after treatment (7th day, 1th month and 6th month). Erythrocyte aggregation-deformability and oxidative stress parameters were determined. p < 0.05 was considered as statistically significant. RESULTS: Erythrocyte deformability of the patients determined at 16.87 and 30 Pascal were significantly lower than healthy individuals. Erythrocyte aggregation index (AI) of the patients was higher, whereas erythrocyte aggregation half-time (t½) was lower compared to control. Erythrocyte deformability values and AI were not significantly different from the pre-and post-radioiodine treatment groups. There was no statistically significant difference between the oxidative stress parameters before and after the treatment. CONCLUSIONS: Patients were in a worse hemorheological condition compared to healthy individuals. After RAIT, RBC deformability and aggregation were not affected and no significant change in oxidative stress parameters was detected. © 2020-IOS Press and the authors. All rights reserved.

Background The enzymatic method of caeruloplasmin measurement is based on copper-dependent oxidase activity. The advantage of the oxidase determination is that it has a much lower detection limit compared with immunoassay-based methods. It has found its application in both the diagnosis of Wilson’s disease and also in the monitoring of patients’ response to treatment. Methods The method previously described in literature was adapted for use on a 96-well plate. Caeruloplasmin oxidase activity results were derived from the equation: caeruloplasmin oxidase activity = (A15−A5) × 185 U/L. Results Repeatability (intra-batch) imprecision ranged from 6 to 15% and intermediate (inter-batch) imprecision varied from 7 to 16% for caeruloplasmin oxidative activities of 14, 29, 45 and 99 U/L. Between 3 and 92 U/L, the assay appeared linear with a regression coefficient R2 = 0.9958. The lower limit of quantification was 4 U/L. Samples were stable over a five-week period at 4℃ and for at least four freeze–thaw cycles. There was a statistically significant difference between the areas under ROC curve for copper-to-caeruloplasmin ratios between caeruloplasmin oxidative activity and immunoassay-based methods ( P Conclusions Using the oxidative assay provides a cost-effective means of estimating caeruloplasmin concentrations. The method is easily adaptable to a 96-well plate format that facilitates high throughput of samples in a busy laboratory. The enzymatic method is more sensitive and specific for differentiating between Wilson’s and non-Wilson’s when compared with immunoassay-based methods.

Studies on the oxidation products of organic pollutants and their toxicity in textile dyeing sludge after the sludge was treated by the advance oxidation processes were limited, since textile dyeing sludge was a complicated mixture. For the first time, simulated sludge was used to study the degradation mechanism of 3,3′-dimethoxybenzidine (DMB) during the combined ultrasound-Mn(VII) treatment. The toxicity of DMB and its products was also evaluated. The results indicated that the compositions and microstructures of polyaluminium chloride (PAC)- and polyferric sulphate (PFS)-based simulated sludge were similar to those of real textile dyeing sludge. The optimum conditions of ultrasound-Mn(VII) treatment were: a KMnO4 dosage of 40 μM, an ultrasound power density of 0.36 W cm−3, and a reaction time of 20 min. 98.24% of DMB and 63.04% of total organic carbon (TOC) in the simulated sludge were removed. Six products, that is, 2-nitroanisole, 3-methoxy-4-nitrophenol, vanillylmandelic acid, vanillyl alcohol, m-anisic acid, and benzoic acid, were identified by GC-MS and LC-MS-MS. It was noted that all of these identified products were also detected in the real textile dyeing sludge after the ultrasound-Mn(VII) treatment. All of them were less toxic than DMB. Moreover, 53.30% and 54.80% of toxicity toward the alga Desmodesmus subspicatus and the bacterium Vibrio fischeri were removed in simulated sludge, respectively. Therefore, simulated sludge was helpful for studying a pollutant's degradation mechanism in the complex sludge mixtures. The results would also provide some useful suggestions for the sludge disposal after the sludge was treated by the advance oxidation processes.

Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine > pyrogallol > guaiacol and was found to be a homotetramer of 155 kDa with each subunit having a size of 38 kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase–heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol.

Herein we report a label-free microRNA (miRNA) biosensor in which the formation of a thin insulating film is used to amplify the analytical signal. Briefly, the biosensor is made of an oligonucleotide-coated gold electrode. After hybridizing with a target miRNA, free capture probe (CP) strands on the biosensor are removed by a nuclease digestion. A second hybridization with an oligonucleotide-tailed DNAzyme is performed to introduce the DNAzyme to the biosensor. The DNAzyme triggers the polymerization of 3,3′-dimethoxybenzidine (DB) in the presence of H2O2 and the hybridized miRNA-CP duplexes serve as templates to guide the deposition of poly (3,3′-dimethoxybenzidine) (PDB). The formation of the insulating PDB film alters the impedance of the biosensor, rendering it readily distinguishable by electrochemical impedance measurements. The accumulative nature of the PDB deposition drastically improves the detectability of the biosensor. A proof-of-concept study is conducted on the detection of miRNAs in total RNA extracted from cultured cells.

In this study, laccase was immobilized on nylon 6,6/Fe

Spectrophotometric method with three systems were developed here for the determination of gold(III) using o-dianisidine, aniline sulphate and catechol. Gold(III),in the system 1 it oxidizes o-dianisidine, in the system 2 it oxidizes catechol followed by its coupling with o-dianisidine, in the system 3 it oxidizes catechol followed by its coupling with aniline sulphate forming dye products with respective λ max 446 nm, 540 nm, and 505 nm. All the three systems were optimized and analytical parameters were calculated. The molar absorptivity values were 9.27 × 10 4 , 1.97 × 10 4 and 1.62 × 10 4 respectively for the systems 1, 2 and 3 with the corresponding Sandell sensitivity values (μg cm − 2 ), 0.0021, 0.0096 and 0.011. The optimized systems were used for the determination of gold present in some forensic jewellery and pharmaceutical samples and the results obtained were compared with the results of all samples determined by Inductively Coupled Plasma – Atomic Emission Spectrometric method and a few of them were also complemented by Energy Dispersive X–Ray Fluorescent spectral analysis.

Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The property of amino-functionalized mesoporous silica was characterized by N(2) adsorption-desorption and thermogravimetric (TG) analysis. The feature of the silica-based matrix before and after myoglobin adsorption was identified by fourier transform infrared (FTIR) and UV/VIS measurement. With o-dianisidine and H(2)O(2) as the substrate, the peroxidase activity of adsorbed myoglobin was determined. With Micrococus lysodeilicus as the substrate, the antibacterial activity of covalently tethered lysozyme was measured. Results demonstrated that the final product not only presented peroxidase activity of the myoglobin but yielded antibacterial activity of the lysozyme.

The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H(2)O(2) required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r=4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase.

By reactions of indium chloride tetrahydrate with three bis-Schiff bases ligands (N, N’-bis (3-methoxy-salicylidene)benzidine = L1; N, N’-bis (3-methoxysalicylidene)-2, 2’-dimethyl- benzidine =L2; N, N’-bis (3-methoxysalicylidene)-o- dianisidine = L3), respectively, three novel corresponding complexes 1~3 were obtained. The complexes were characterized by infrared spectrum, molar conductance, elemental analysis and thermal gravimetric analysis. The chemical formulae of the complexes 1~3 were determined as (InCl3)2Ln (1, n = 1; 2, n = 2; 3, n = 3). All the complexes are constructed with binuclear indium ions. Every complex consists of two central In (III) ions, one corresponding bis-Schiff base ligand and six Cl-.

Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K(M) (weaker substrate binding) and k(cat) (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally 'strained'.

Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs.

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it ‘guards’ heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.

This paper describes the applicability of in vitro digestion models as a tool for consumer products in (ad hoc) risk assessment. In current risk assessment, oral bioavailability from a specific product is considered to be equal to bioavailability found in toxicity studies in which contaminants are usually ingested via liquids or food matrices. To become bioavailable, contaminants must first be released from the product during the digestion process (i.e. become bioaccessible). Contaminants in consumer products may be less bioaccessible than contaminants in liquid or food. Therefore, the actual risk after oral exposure could be overestimated. This paper describes the applicability of a simple, reliable, fast and relatively inexpensive in vitro method for determining the bioaccessibility of a contaminant from a consumer product. Different models, representing sucking and/or swallowing were developed. The experimental design of each model can be adjusted to the appropriate exposure scenarios as determined by the risk assessor. Several contaminated consumer products were tested in the various models. Although relevant in vivo data are scare, we succeeded to preliminary validate the model for one case. This case showed good correlation and never underestimated the bioavailability. However, validation check needs to be continued.

A peroxidase (POX)-containing fraction was purified from buckwheat seed. The POX consisted of two isozymes, POX I and POX II, that were purified 6.6- and 67.4-fold, respectively. Their molecular weights were estimated to be 46.1 kDa (POX I) and 58.1 kDa (POX II) by gel filtration. While POX I and II each oxidized quercetin, o-dianisidine, ascorbic acid and guaiacol, only POX II oxidized ABTS. Kinetic studies revealed that POX I and II had lower K(m) values for quercetin (0.071 and 0.028 mM), ABTS (0.016 mM for POX II) and ascorbic acid (0.043 and 0.029 mM) than for o-dianisidine (0.229 and 0.137 mM) and guaiacol (0.288 and 0 ). The optimum pHs of POX I and II for various substrates were almost the same, except for quercetin; pH 8.0 for POX I and pH 4.5 for II. Their optimal temperatures were 30 degrees C (POX I) and 10 degrees C (POX II), and POX I was more stable than POX II above 30 degrees C.

Peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1-100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.

A novel "ready-to-use" glucose test strip based on a polyurethane hollow nanofiber membrane was fabricated through facile co-axial electrospinning. By utilizing glucose oxidase and horseradish peroxidase in the core-phase solution, and a chromogenic agent either in the core solution (in which case 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) was used) or in the shell-phase solution (in which case o-dianisidine was used) for co-axial electrospinning, in situ co-encapsulation of the two enzymes within the hollow nano-chamber and incorporation of chromogenic agents either inside the nano-chamber or in the shell of the hollow nanofibers was realized. Such unique "all-in-one" feature enabled the prepared hollow nanofiber membrane-based test strips to be applied either as colorimetric sensors in solution or as an optical biosensor operated in the "dip-and-read" mode. When used as a colorimetric biosensor in solution, the test strip with o-dianisidine as chromogenic agent shows an excellent linear response range between 0.01 mM to 20 mM and a high apparent lumped activity recovery of 62.1% as compared to the reaction rate of the free bi-enzyme system. While the activity recovery of the test strip with ABTS as chromogenic agent is only 18.0%, and the test strip is found to be unstable due to spontaneous-oxidation of the ABTS. The o-dianisidine test strip was also applied as an optical biosensor, visible rufous color was quickly developed on the surface of the membrane upon dropping 10 mu L of glucose sample, and an excellent correlation between differential diffusive reflectance of the test strip at 440 nm and glucose concentration was obtained in the range of 0.5-50 mM. The test strips also exhibited excellent long-term storage stability with a half-life at 25 degrees C as long as four months.

Objectives: To develop a new, colorimetric and automated method for measuring total oxidation status (TOS). Design and methods: The assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. The oxidation reaction of the assay was enhanced and precipitation of proteins was prevented. In addition, autoxidation of ferrous ion present in the reagent was prevented during storage. The method was applied to an automated analyzer, which was calibrated with hydrogen peroxide and the analytical performance characteristics of the assay were determined. Results: There were important correlations with hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide solutions (r = 0.99, P b 0.001 for all). In addition, the new assay presented a typical sigmoidal reaction pattern in copper-induced lipoprotein autoxidation. The novel assay is linear up to 200 μmol H2O2 Equiv./L and its precision value is lower than 3%. The lower detection limit is 1.13 μmol H2O2 Equiv./L. The reagents are stable for at least 6 months on the automated analyzer. Serum TOS level was significantly higher in patients with osteoarthritis (21.23 ± 3.11 μmol H2O2 Equiv./L) than in healthy subjects (14.19 ± 3.16 μmol H2O2 Equiv./L, P b 0.001) and the results showed a significant negative correlation with total antioxidant capacity (TAC) (r = �0.66 P b 0.01). Conclusions: This easy, stable, reliable, sensitive, inexpensive and fully automated method that is described can be used to measure total oxidant status.

Hollow nanoshells of gold entrapping an enzyme, horseradish peroxidase (HRP), in the cavity of the nanoshell have been prepared in the reverse micelles by leaching out silver chloride (AgCl) from Au(shell)AgCl(core) nanoparticles with dilute ammonia solution. The particles have been characterised by dynamic laser light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electron diffraction. The particle size is below 100 nm diameter, depending upon the size of the aqueous core of reverse micelles in which these particles have been prepared. This soft-chemical method for the preparation of such particles allows the entrapped enzyme to remain active inside the hollow gold nanoparticles. Small substrate molecules such as o-dianisidine can easily enter through the pores of the nanoshell and can undergo enzymatic oxidation by H2O2. The enzyme kinetics follows Michaelis-Menten mechanism. When the substrate is chemically conjugated with dextran molecule (10 kDa), the enzymatic reaction is practically completely prevented perhaps by the inability of dextran-o-dianisidine conjugate to penetrate the pores of the nanoshells. However, HRP did not show any activity when trapped inside solid gold nanoparticles.

The activity and kinetics of carrot peroxidase were determined by using pyrogallol, guaiacol and o -dianisidine as hydrogen donors and peroxidase inactivation was studied by thermal and microwave heating. The kinetics of peroxidase showed characteristics which were dependent upon the identity and concentration of the hydrogen donor used. With pyrogallol and guaiacol, the true K m was found to be 0.34 and 1.4 mM for hydrogen peroxide, respectively, whereas the apparent K m with o -dianisidine was 7.7 × 10 −3 mM. The lowest K m and highest V max / K m with o -dianisidine exhibited the greater tendency of the enzyme toward hydrogen peroxide and the specificity of the competing substrate, o -dianisidine. Thermal treatment of carrot peroxidase was done in the range of 35–75 °C for 0.5–180 min. Inactivation kinetics of peroxidase showed a biphasic first-order model, while at 75 °C, peroxidase showed monophasic first-order behaviour. Kinetic parameters, k and E a , were determined for heat labile and heat resistant fractions of peroxidase. Biphasic behaviour of enzyme inactivation was observed for the microwave treatment at 70 and 210 W, whereas at 350 and 700 W microwave powers enzyme inactivation was monophasic. Microwave heating was found to be more effective for inactivating the enzyme than thermal treatment and additionally vitamin C retention was higher in microwave treated samples compared to heat treatment.

Due to limitations of the existing preparative methods of hollow nanoparticles by either heating at high temperature (>600 °C) or by using strong acid, alkali, or an organic solvent, it was not possible up till now to encapsulate any sensitive organic molecule like enzyme or others inside the cavity of hollow nanoparticles. We have demonstrated a much softer method of preparing hollow silica nanoparticles with horseradish peroxidase (HRP) inside the cavity by synthesizing HRP-doped core-shell silica-coated silver chloride nanoparticles and finally leaching out silver chloride with dilute ammonia at low temperatures. TEM pictures showed the hollow cavity inside the nanoparticles. The enzyme entrapped in these particles was active. The turnover number of HRP entrapped into these hollow particles and dispersed in aqueous buffer (pH 7.2) ( k cat = 2.56 × 10 6 s −1 ) was found to be less than that of free enzyme in aqueous buffer ( k cat = 6.133 × 10 7 s −1 ) but higher than that of HRP entrapped in solid-core silica nanoparticles and dispersed in aqueous buffer ( k cat = 1.05 × 10 5 s −1 ). The result showed that hollow nanoparticles could be prepared using soft chemical methods and sensitive chemicals like active enzyme could be entrapped in the cavities and it retains its activity.

o-Dianisidine (3,3'-dimethoxybenzidine) is applied in the production of some dyes and also used in analytical tests. However, this compound is anticipated to be a human carcinogen. An analytical strategy utilizing square wave voltammetry for the determination of o-dianisidine is presented. An electrochemical system was consisted of three electrodes: carbon paste working electrode, platinum wire counter electrode and silver-silver chloride (Ag/AgCl) reference electrode. However, square wave voltammograms of direct measurements of o-dianisidine were found to be hardly reproducible, exhibiting few peaks due to some labile short-lived intermediates with the only exception of a quite stable peak at +0.7 V vs. Ag/AgCl. Quantitative determination of o-dianisidine gave satisfactory results only when the carbon paste working electrode was replaced by deoxyribonucleic acids (DNA) electrode obtained by immobilization of double-stranded (ds) DNA on carbon electrode. Square wave voltammogram of DNA showed two peaks attributed to adenine and guanine and the latter was used as analytical signal. After interaction with o-dianisidine, guanine oxidation peak was reduced to the extent related to the concentration of the analyte. Initial reduction of guanine peak took place already at the concentration of o-dianisidine equal to 0.4 microM; high concentrations (above 100 microM) of the analyte quenched completely a guanine response. The presented electrochemical system enables a specific detection of o-dianisidine by the presence of an oxidation peak at +0.7 V and its quantitative determination by measuring a reduction of guanine peak by means of a DNA sensor.

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.

A spectrophotometric method based on myeloperoxidase activity for the determination of leukocytes in urine is described. Red cells that may be found in urine samples were lysed by an ammonium chloride method. Leukocytes were then sedimented by centrifugation and lysed using Triton X‐100 (Sigma Chemicals Co., St. Louis, MO). Myeloperoxidase‐catalyzed oxidation of o‐dianisidine was carried out at 37°C, pH 7. The reaction was stopped with the addition of 2 M H(2)SO(4), and a stable form of oxidized o‐dianisidine in acidic solution was obtained. Solid particles that may be found in urine samples were removed by centrifugation to avoid turbidity, and absorbance values of the supernatants were recorded at 400 nm. An Average number of leukocytes were noted per number of fields by microscopic examination and were related with the absorbance values of the supernatants at 400 nm. Pearson correlation (r) between our presented spectrophotometric analysis results and visual microscopic analysis was 0.877. Roche Combur 10‐test M strips (Roche, Mannheim, Germany) and Multistix 10 SG Bayer test strips (Bayer Diagnostics, UK) were 0.645 and 0.648, respectively (P

The model compounds for horseradish peroxidase (HRP) is reported, based on the association of H 2 O 2 with iron and manganese porphyrins immobilized onto a functionalized silica gel. The models were developed in an attempt to find a biomimetical compound for systems containing heme groups. The ortho -dianisidine is a useful substrate model compound for checking the ability of degradation promoted by delignificant natural enzymes. The heterogeneous catalysts were obtained by grafting of three metalloporphyrins: (Fe(TFPP)—iron porphyrin from the 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin—and Mn(TCPP) and Fe(TCPP), iron and manganese porphyrins from the 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin), onto the modified surface of silica gel. The oxidation of ortho -dianisidine was monitored by UV-Vis spectroscopy at room temperature using different ratios of catalyst, oxidant and substrate. At high H 2 O 2 concentration, the perhalogenated iron porphyrin Fe(TFPP) produced best results, showing a turnover number of about 1100. This value was higher than those obtained for Fe(TCPP) and Mn(TCPP) systems.

The determination of leukocyte concentration in human blood depending on the detection of oxidized o‐dianisidine in acidic solution is studied. The oxidation of o‐dianisidine was carried out by peroxidase enzymes found in leukocytes. The reaction was stopped by the addition of 4N H(2)SO(4) to the solution, and a very stable, colored o‐dianisidine derivative was obtained. The calibration graph was plotted with the recorded absorbance values at 400 nm assigned to the y‐axis, and leukocyte counts in 1‐mL blood samples to the x‐axis. The equation of the calibration graph was y=0.0025x+0.0904, with a correlation coefficient of R=0.994. The coefficient of variation and P‐value of the method were 4.00% and 0.05%, respectively. J. Clin. Lab. Anal. 16:233–236, 2002. © 2002 Wiley‐Liss, Inc.

D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.

A flow injection analysis (FIA) procedure for the determination of free chlorine in industrial formulations and water samples is proposed. The manifold is provided with a gas-diffusion unit which permits the removal of interfering species and also the preconcentration of chlorine. The determination of chlorine is performed on the basis of the oxidation by o-dianisidine as a chromogenic reagent to a coloured product which can be monitored at 445 nm. The method (for a preconcentration step of 60 s) is linear over the range 0.04-1.00 mg l(-1) of chlorine, the limit of detection is 0.04 mg l(-1), the reproducibility of the procedure (as RSD of the slope) is 3.7% for a series of four independent calibrations, the precision (as RSD of a series of 30 continuous FIA peaks of 0.56 mg l(-1) of chlorine) is 1.4% and the sample throughput is 40 h(-1). A detailed comparative study of the analytical characteristics of a single mono-channel reverse FIA assembly and the same system but provided with a Fluoropore membrane filter of 0.5 microm pore size was performed to check the advantages of the new approach in terms of sensitivity, selectivity and limit of detection.

Peroxidase activities of RNAs containing 2′-amino groups, which were selected as aptamers binding to N-methylmesoporphyrin IX, were investigated. Some clones promoted the oxidation reaction of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with hydrogen peroxide (H2O2) in the presence of iron(III)-protoporphyrin (hemin), whereas others did not. Each of them had a different substrate specificity. One of the active clones promoted the oxidation of o-dianisidine and β-nicotinamide adenine dinucleotide reduced form (NADH) with H2O2 5 and 15 times faster than hemin only, respectively. On the other hand, one clone that was inactive on oxidation of ABTS exhibited the same level of activity on oxidation of o-dianisidine as that shown by the clone active on ABTS but no activity on NADH. By in vitro selection, we can produce various types of peroxidase-like non-natural RNAs. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 463–468, 2001.

Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the concentration of the protein both in its active and inactive forms. Cp activity is generally analyzed manually; the process is time-consuming, has a limited repeatability, and is not suitable for a clinical setting. To overcome these inconveniences, we have set the automation of the o-dianisidine Cp activity assay on a Cobas Mira Plus apparatus. The automation was rapid and repeatable, and the data were provided in terms of IU/L. The assay was adapted for human sera and showed a good precision [coefficient of variation (CV) 3.7 %] and low limit of detection (LoD 11.58 IU/L). The simultaneous analysis of Cp concentration and activity in the same run allowed us to calculate the Cp-specific activity that provides a better index of the overall Cp status. To test the usefulness of this automation, we tested this assay on 104 healthy volunteers and 36 patients with Wilson's disease, hepatic encephalopathy, and chronic liver disease. Cp activity and specific activity distinguished better patients between groups with respect to Cp concentration alone, and providing support for the clinical investigation of neurological diseases in which liver failure is one of the clinical hallmarks.

Investigations were undertaken aimed at distinguishing and clarifying the reactivities of the Fe(SCys) 4 and diironoxo sites of rubrerythrin (Rr) during its catalysis of substrate oxidations by either dioxygen or hydrogen peroxide. Three Rr-catalyzed reactions were investigated: (1) the ferroxidase reaction: Fe aq 2+ +O 2 →Fe aq 3+ +[O] red ; (2) the NADH peroxidase reaction: NADH+H + +H 2 O 2 →NAD + +2H 2 O; and (3) the aromatic diamine peroxidase reaction exemplified with o -dianisidine as substrate: o -dianisidine+H 2 O 2 → o -dianisidine quinonediimine+2H 2 O. A non-native bacterial oxidoreductase was used as a co-catalyst for the NADH peroxidase reaction. Residues at or near both metal sites of Rr, including those furnishing iron ligands, were mutated to assist in clarifying the metal-site reactivities. In addition a Rr with Zn 2+ substituted for iron in the Fe(SCys) 4 site was examined. The results indicate that, in reactions 1 and 2, electrons from the reductant flow initially into the Fe(SCys) 4 site of Rr, then out through the diferrous site into O 2 or H 2 O 2 . In reaction 3 oxidized Rr appears to weakly activate H 2 O 2 for oxidation of the aromatic diamine substrate. The highest turnover occurs for the NADH peroxidase reaction. It is proposed that an extra carboxylate ligand not present in other diironoxo enzymes shifts the reactivity of the diferrous site of Rr towards hydrogen peroxide and away from dioxygen.

In July 1997, the US Environmental Protection Agency (EPA) began sampling and analyzing drinking water matrices from US municipalities serving populations greater than 100,000 for low-level bromate (0.20 microgram/l) in support of the Information Collection Rule (ICR) using the selective anion concentration (SAC) method. In September 1997, EPA published Method 300.1 which lowered the Method 300.0 bromate method detection limit (MDL) from 20.0 to 1.4 micrograms/l. This paper describes the research conducted at the EPA's Technical Support Center laboratory investigating a single post-column reagent, o-dianisidine (ODA), which has been successfully coupled to EPA Method 300.1 to extend the MDL for bromate. Initial studies indicate that this method offers a MDL which approaches the EPA's SAC method with the added benefit of increased specificity, shortened analysis time and reduced sample preparation. The method provides excellent ruggedness and acceptable precision and accuracy with a bromate MDL in reagent water of 0.1 microgram/l, and a method reporting limit of 0.50 microgram/l.

The inhibitory effect of mercury(II) towards the activity of horseradish peroxidase immobilized in chitosan on polyurethane foam in the o -dianisidine oxidation reaction has been used for developing a visual test procedure for the determination of Hg(II) at 1–1000 pg ml −1 . The relative standard deviation at the lower limit of its analytical concentration range is 6% ( n =3). The peroxidase immobilized on polyurethane foam keeps its catalytic activity for more than 1.5 years. The developed procedure was applied successfully for the analysis of different types of soils.

Iron-chelating low-molecular-weight compounds or catecholate siderophores have been suggested to be involved in wood biodegradation. To help in understanding the mechanism involved in the enzyme-like activity of catecholate siderophores, the oxidative properties of 2,3-dihydroxybenzoic acid (DHBA) and 3,4-dihydroxyphenylacetic acid (DHPAA) chelated with iron were studied. The pH and catechol/Fe(III) ratios were optimized for o -dianisidine oxidation, obtaining a maximum at pH 7.0, in the absence of buffer, and a catechol/Fe(III) ratio of 1:2 to DHBA and 1:1 to DHPAA was found. Under these conditions, the catechols were able to reduce Fe(III) to Fe(II) acting like siderophore models. The Fe(III) complex of DHBA and of DHPAA degraded dioxane-lignin in 60% after 2 h and 85% after 24 h, respectively. DHBA/Fe(III) oxidized the bleaching effluent (E1) in 80% in 5 min under the studied conditions.

Objectives: Serum diamine oxidase (DAO; EC 1.4.3.6) activity is often employed as a clinical indicator of the integrity of intestinal mucosa. However, interindividual variation in enzyme activity and reference values for healthy women and men have not been studied. Design and methods: DAO activity was measured by using cadaverine coupled to O-dianisidine oxidation in 50 healthy individuals. Results: The mean activity was 7.59 ± 3.67 U/L for women and 2.38 ± 0.71 U/L for men (p Conclusions: Gender is a major determinant for DAO activity in healthy subjects.

The effect of organomercury compounds (methyl-, ethyl- and phenylmercury) on the activity of immobilized peroxidase was studied. Peroxidase was immobilized in chitosan films in wells on a polystyrene plate and on chromatography paper. The oxidation reactions of o -dianisidine, o -phenylenediamine and 3,3′, 5,5′-tetramethylbenzidine by H 2 O 2 were used as indicators. The rate of all the indicator reactions was monitored visually by measuring the time of appearance of the colour of the oxidation product. The liberating effect of organomercury compounds on peroxidase immobilized on both the above mentioned supports using o -dianisidine oxidation in the presence of the inhibitor phenylthiourea and the effect of organomercury compounds on the duration of an induction period of 3,3′, 5,5′-tetramethylbenzidine oxidation in the presence of diethyldithiocarbamate catalysed by paper-immobilized peroxidase were used for the development of enzymatic test procedures for the determination of organomercury compounds at concentrations of 0.02–1000 μM. The relative standard deviation ( n = 3) at the lower limits of their analytical concentration ranges in the proposed test procedures using o -dianisidine are 18–23% ( n = 3).

Background. Although the occupational exposure to some aromatic amines is recognized to cause bladder carcinoma, the long term effect of such exposure on the risk for disease, including other malignant tumors, remains unknown. Methods. A total of 442 dyestuff workers exposed to one or more substances including benzidine (BZ), betanaphthylamine (beta-N), alpha-naphthylamine (alpha-N), and dianisidine were followed completely until December 1992 (average time since first exposure, 39.4 years). Besides the underlying cause of death, the incidence of urothelial carcinoma was determined by periodic urologic screenings. Results. Analyses of site-specific cancer mortality revealed a remarkable increased risk for bladder carcinoma for those engaged in BZ manufacture (standardized mortality ratio [SMR] = 63.6), BZ use (SMR = 27.0) and βN manufacture (SMR = 48.4), but not for those who were exposed to alph-N. The increased risk of cancer mortality for other organs was not significant for any exposure classes. The crude incidence rate per 1000 person-years of bladder carcinoma was estimated to be 8.7 for those engaged in BZ manufacture, 2.9 in BZ use, 7.7 in beta-N manufacture and 1.0 in beta-N use. Regardless of the class or type of exposure, the adjusted incidence rate of urothelial carcinoma increased with the duration of exposure. The adjusted incidence rate for BZ manufacture remained high (3.8–12.8) during the entire observation period, whereas that for BZ use increased from 0.0 to 4.4 as the time since first exposure increased from less than 10 years to 30+ years. Conclusions. Occupational exposure to either BZ or beta-N demonstrated an extremely strong and prolonged effect on workers' risk for urothelial carcinoma, particularly for bladder carcinoma, but not for malignant neoplasms of other organs.

A total of 11 manganese peroxidase isoenzymes (MnP 1 -MnP 11 ) with isoelectric points (pIs) in the range of 4.58–3.20 were isolated from liquid- and solid-state cultures of the basidiomycete Ceriporiopsis subvermispora . In the presence of hydrogen peroxide, these isoenzymes showed different requirements for Mn(II) in the oxidation of vanillylacetone, o-dianisidine , p-anisidine and ABTS, whereas oxidation of guaiacol by any isoenzyme did not take place when this metal was omitted. K m values for o-dianisidine and p-anisidine in the absence of Mn(II) are in the range of 0.5–1.0 mM and 4.5–42.0 mM, respectively. Oxalate and citrate, but not tartrate, accelerate the oxidation of o-dianisidine , both in the presence and in the absence of Mn(II). MnPs from this fungus are able to oxidize kojic acid without externally added hydrogen peroxide, indicating that they can also act as oxidases. In this reaction, however, the requirement for Mn(II) is absolute.

The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s-1. The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1/2 = approximately 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN3, isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.

Peroxidase (E.C. 1.11.1.7., hydrogen donor oxidoreductase) is widely distributed and has been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance. However the role that it plays in metabolism is not clear due to the large number of reactions it catalyzes and the considerable number of isozymic species (2). In tomato plants, Evans and Aldridge (3) separated out six isoperoxidases and in a later paper Evans reported 12 isoperoxidases from tomato shoots (4). A homogeneous tomato fruit peroxidase isozyme was obtained by Jen et al. (5) using hydrophobic chromatography. Isozymes were not detected in Euphorbia characias peroxidase (6), in Ipomoea batatas peroxidase (7) and in Hordeum vulgare peroxidase (8). The simultaneous presence of Cu (II) amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (6,8,9). In the graminea Oryza sativa, widely distributed, an FAD amine oxidase is present that oxidizes diamines (10). In this plant we also found two isoperoxidases called perox I and II. Only perox I was purified to homogeneity and its enzymatic, physical and chemical properties have been studied.

Made available in DSpace on 2013-08-28T14:09:25Z (GMT). No. of bitstreams: 1 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) Made available in DSpace on 2013-09-30T19:09:11Z (GMT). No. of bitstreams: 2 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) WOS000306752700025.pdf.txt: 45557 bytes, checksum: 744d1bc7b9755cb558b4b0abb89c3d2d (MD5) Previous issue date: 2012-07-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:19:33Z No. of bitstreams: 2 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) WOS000306752700025.pdf.txt: 45557 bytes, checksum: 744d1bc7b9755cb558b4b0abb89c3d2d (MD5) Made available in DSpace on 2014-05-20T14:19:33Z (GMT). No. of bitstreams: 2 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) WOS000306752700025.pdf.txt: 45557 bytes, checksum: 744d1bc7b9755cb558b4b0abb89c3d2d (MD5) Previous issue date: 2012-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis-(trifluorometanesulfonyl) imide BMIm[NTf2] was used as a novel medium for improvement of separation and quantization of 16 aromatic amines typically present as contaminants in consumer products and detected by HPLC coupled to an electrochemical detector. The aromatic amines, namely 4,4'-diaminodiphenylmethane, 4-chloroaniline, 2-methoxy-5-methyl-aniline, 3,3'-dimethylbenzidine, 2,4-diaminotoluidine, 2-chloro-4-nitroaniline, 4,4'-oxydianiline, aniline, 3,3'-dichlorobenzidine, benzidine, 4-aminobiphenyl, o-dianisidine, o-anisidine, o-toluidine, 4,4'-methylene-bis-2-chloroaniline and 2-naphthylamine are oxidized in methanol/BMIm[NTf2] at a potential around +0.68V to +0.93V vs. Ag/AgCl at a glassy carbon electrode, which is the base for their determination by HPLC/ED. Using the optimized conditions of methanol/BMIm[NTf2] 70: 30 (v/v) as mobile phase, flow-rate of 0.8 mL.min(-1), column CLC-ODS, E-ap = +1.0 V and T = 40 C analytical curves were constructed for each of the tested amines. Good linearity was obtained in the concentration range of 1.09 mg.L-1 to 217 mg.L-1, with excellent correlation coefficients. The limits of detection reached 0.021 mg.L-1 to 0.246 mg.L-1 and good relative standard deviations (RSD, n = 3) were obtained from the measurements. Satisfactory recovery for each aromatic amine was achieved, ranging from 95 to 103%. The developed method was successfully applied to determine six aromatic amines present as contaminants in commercial hair dye samples. State Univ Julio de Mesquita Filho UNESP, Inst Chem, BR-14800900 Araraquara, SP, Brazil State Univ Julio de Mesquita Filho UNESP, Inst Chem, BR-14800900 Araraquara, SP, Brazil

Reactions of peroxidase oxidation of triftazine and thioproperazine have been investigated in the presence of horseradish peroxidase using steady state kinetic methods. It has been shown that phenothiazines are slowly oxidizable substrates for horseradish peroxidase. k(cat) and K(m) values have been determined in the range of pH from 4.5 to 7.5. The study of co-oxidation of phenothiazines and o-dianisidine (ODN) revealed that in the presence of aminazine and ODN in the reaction medium both substances follow sequential oxidation. ODN oxidation was not observed until full conversion of aminazine. At pH 4.5-5.5 thioproperazine bound to the enzyme-substrate complex and caused a nticompetitive inhibition of peroxidase. At pH5.5 sequential substrate oxidation with preferential thioproperazine conversion occurred. In the range of pH from 4.5 to 7.5 triftazine did not influence ODN oxidation.

The aim of this study was to evaluate the impact of smoking on the relationship between interleukin-1 (IL-1β) and oxidation in patients with periodontitis and response to nonsurgical periodontal therapy.Data were obtained from 30 patients with generalized chronic periodontitis (15 smokers and 15 nonsmokers) and from 10 periodontally healthy controls. IL-1β level, total oxidant status (TOS) and total antioxidant status (TAS) were recorded in gingival crevicular fluid. Probing depth, clinical attachment level, gingival and plaque indices and bleeding on probing were also measured. The gingival crevicular fluid and clinical parameters were recorded at baseline and 6 wk after periodontal treatment.The study showed statistically significant improvement of clinical parameters in both smokers and nonsmokers after periodontal treatment. Moreover, the baseline IL-1β levels were significantly higher in smokers compared with nonsmokers (p0.05). After periodontal treatment, the IL-1β levels were significantly reduced in both smokers and nonsmokers (p0.05). There were no significant differences in TOS and TAS between periodontitis patients and healthy controls at baseline and 6 wk after periodontal treatment. The level of IL-1β in gingival crevicular fluid was positively correlated with TOS in both smokers and nonsmokers.Periodontal treatment improved the clinical parameters in both smokers and nonsmokers. The results confirm that periodontal therapy has an effect on IL-1β levels in gingival crevicular fluid, but not on TOS and TAS.

The sensitizing effect of riboflavin (RF) and riboflavin 5′-phosphate (FMN) on the photo-oxidation of dianisidine was studied. The rate of the photochemical reaction was monitored spectrophotometrically at 460 nm. The method allows the determination of RF and FMN in the range 1 × 10–7–5 × 10–6 mol dm–3 with a relative standard deviation of about 0.68%. The method can be successfully adapted to flow injection. Manual and flow injection methods were satisfactorily applied to the determination of RF or FMN in fortified breads and pharmaceutical preparations. A possible mechanism for the sensitized photoreaction is proposed.