Pharmacotherapeutic monitoring of dipyrone in northeastern Brazilian donkeys (Equus asinus).

This study aimed to monitor the effects of dipyrone following multiple administrations in northeastern donkeys. Ten castrated male donkeys, aged 6.4 ± 3 years and weighing 130.6 ± 9.8 kg, were administered dipyrone (25 mg/kg IV) every 12 h, resulting in six administrations (D1 to D6) per animal. Blood samples were collected over a 72 h monitoring period. A validated UHPLC-MS/MS method was employed to determine the plasma concentrations of the 4- methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The calculated pharmacokinetic variables of 4-MAA after D1 and D6 were, respectively: Cmax (µg/mL) = 163.60 ± 179.72 and 178.79 ± 196.94; T1/2beta (h) = 2.65 ± 0.65 and 3.37 ± 1.03; and AUC0-t (µg/mL × h) = 240.38 ± 130.87 and 373.52 ± 78.85. The same variables for 4-AA were: Cmax, (µg/mL) = 0.44 ± 0.27 and 0.90 ± 0.31, T1/2beta (h) = 14.77 ± 13.13 and 35.97 and AUC0-t (µg/mL × h) = 3.20 ± 0.43 and 27.73 ± 11.99. Concentrations of 4-MAA exceeded the minimum concentration required for 50% inhibition of cyclooxygenases 1 and 2. However, an accumulation of 4-AA, was observed. Further clinical studies are necessary to ascertain the implications of these findings on the pharmacodynamic response to dipyrone in northeastern donkeys.

Deep eutectic solvent-based adhesive tape extraction combined with enzyme inhibition assay for the determination and distinction of dithiocarbamate pesticides in food samples.

A simple, green extraction method of dithiocarbamate (DTC) pesticides in food samples was developed using adhesive tapes and a green deep eutectic solvent (DES). A rapid and convenient determination and distinction method of DTC pesticides was established using tyrosinase inhibition assay. First, DTC pesticides were extracted by pasting and peeling off the adhesive tape, then eluted by the DES synthesized from xylitol and ethylene glycol. Second, determination of DTC pesticides was conducted by inhibiting the activity of tyrosinase which can catalyze the oxidation of catechol. Less colored products were generated in the reaction system (tyrosinase, catechol, and 4-aminoantipyrine), leading to weak absorbance. In addition, different DTC pesticides (ziram, propineb, zineb, mancozeb, thiram, metiram, and ferbam) were successfully distinguished by sensor arrays (tyrosinase, phenolic compounds, and 4-aminoantipyrine) through principal component analysis. The limit of detection was found to be 0.2 µg kg-1, and the limit of quantification was 0.6 µg kg-1. The recoveries ranging from 89.4% to 103.8% were obtained in vegetable, fruit, and cereal, with a relative standard deviation of less than 4.2%. The method is simple, rapid, and convenient and shows good application prospects in the determination of pesticides in a variety of food samples.

Pharmacokinetic profiles of the two major active metabolites of metamizole, 4-methylaminoantipyrine (MAA) and 4-aminoantipyrine (AA), after intravenous injection in cats.

This study aimed to determine the pharmacokinetic profile of two active metabolites of metamizole (dipyrone), N-methyl-4-aminoanthypyrine (MAA) and 4-aminoanthypyrine (AA), after intravenous administration in cats. Eight healthy mixed-breed cats were intravenously administered metamizole (25 mg/kg). Blood samples were collected at predetermined time points for up to 48 h after administration. Information on behavioral changes in the animals and adverse effects was collected. Plasma aliquots were processed and analyzed using the ultra-performance liquid chromatography tandem mass spectrometry technique. A validated UPLC-MS/MS method was used to characterize the pharmacokinetics of MAA and AA. Salivation was identified as an adverse clinical sign. The mean maximal plasma concentrations of MAA and AA were 29.31 ± 24.57 µg/mL and 1.69 ± 0.36 µg/mL, with half-lives of around 4.98 and 14 h, respectively. The area under the plasma concentration curve values were 28.54 ± 11.33 and 49.54 ± 11.38 h*µg/mL for MAA and AA, respectively. The plasma concentration of MAA was detectable for up to 24 h and was smaller than AA. AA was detectable for >48 h. Results suggest that metamizole is converted into active metabolites in cats. Further PK/PD and safety studies should be performed before defining the dose or administration intervals for clinical use.

The Peroxidase-Coupled Assay to Measure MAO Enzymatic Activity.

MAO activity measurement is generally performed following different spectroscopy methods, in most cases using peroxidase as a coupled reaction catalyst. In the presence of horseradish peroxidase (HRP), the assay follows the oxidation of the typical MAO substrate (aromatic amines) which generates hydrogen peroxide as a secondary product. There are several chromogens and fluorogens that, in the presence of hydrogen peroxide, are converted by HRP to detectable products. In the present chapter we describe the spectrophotometric 4-aminoantipyrine assay as well as the fluorogenic assay with the Amplex® Red chemical probe. These methods are applied on MAO activity and Michaelis-Menten curve determinations as well as inhibitory activity experiments.

ZIM, a Norbornene Derived from 4-Aminoantipyrine, Induces DNA Damage and Cell Death but in Association Reduces the Effect of Commercial Chemotherapeutics.

Cancer incidence is increasing, and the drugs are not very selective. These drugs cause adverse effects, and the cells become resistant. Therefore, new drugs are needed. Here, we evaluated the effects of ZIM, a candidate for chemotherapy, and 4-AA alone and in association with commercial chemotherapeutic agents. Subsequently, the results of ZIM and 4-AA were compared. Male Swiss mice were treated with doses of 12, 24, or 48 mg/kg ZIM or 4-AA alone or in association with cisplatin (6 mg/kg), doxorubicin (16 mg/kg), and cyclophosphamide (100 mg/kg). Biometric parameters, DNA damage (comet and micronuclei), cell death, and splenic phagocytosis were evaluated. DNA docking was also performed to confirm the possible interactions of ZIM and 4-AA with DNA. 4-AA has been shown to have low genotoxic potential, increase the frequency of cell death, and activate phagocytosis. ZIM causes genomic and chromosomal damage in addition to causing cell death and activating phagocytosis. In association with chemotherapeutical agents, both 4-AA and ZIM have a chemopreventive effect and, therefore, reduce the frequency of DNA damage, cell death, and splenic phagocytosis. The association of 4-AA and ZIM with commercial chemotherapeutic agents increased the frequency of lymphocytes compared to chemotherapeutic agents alone. Molecular docking demonstrated that ZIM has more affinity for DNA than 4-AA and its precursors (1 and 2). This was confirmed by the lower interaction energy of the complex (-119.83 kcal/mol). ZIM can break the DNA molecule and, therefore, its chemotherapeutic effect can be related to DNA damage. It is considered that ZIM has chemotherapeutic potential. However, it should not be used in combination with cisplatin, doxorubicin, and cyclophosphamide as it reduces the effects of these drugs.

Novel Schiff base scaffolds derived from 4-aminoantipyrine and 2-hydroxy-3-methoxy-5-(phenyldiazenyl)benzaldehyde: Synthesis, antibacterial, antioxidant and anti-inflammatory.

The synthesis of four new azo-Schiff base ligands from 2-hydroxy-3-methoxy-5-(phenyldiazenyl)benzaldehyde and 4-aminoantipyrine is described in this study. The molecular structures of all the scaffolds were confirmed using NMR spectroscopies such as 1 H and 13 C, as well as FT-IR and Mass spectroscopy. After successful synthesis and characterization of all the ligands, their in vitro antibacterial, antioxidant and anti-inflammatory activities were carried out by using standard protocols. Results revealed that all the four ligands (L1-L4) possessed excellent biological potency.

Design, synthesis and in vivo evaluation of 1,4-dioxo-2-butenyl aryl amine derivatives as a promising anti-inflammatory drug prototype.

Inflammation is a natural response of the organism to an infection, trauma, or cellular stress. Pain is the first symptom of acute and chronic inflammation. The standard class of medication to treat inflammatory pain is the nonsteroidal anti-inflammatory drug (NSAID). These drugs are associated with severe side effects such as gastric ulcers, gastritis, or internal bleeding. One of NSAIDs, Dipyrone® (metamizole) is largely used in many European and South American countries despite its dubious effectivity and its withdrawal from the market of several countries. Here, aiming to identify a new anti-inflammatory drug prototype based on Dipyrone® structure, a set of 27 molecules were virtually screened, and 4 compounds containing the active metabolite 4-aminoantipyrine and 1,4-dioxo-2-butenyl fragment were selected for docking, synthesis, and biological evaluation. The selection was based on the number of H-bonds and π- π stacking interactions between the inhibitor and the amino acids within the binding site of the enzyme. Carrageenan-induced paw edema, acetic acid-induced writhing, and formalin assays were used to evaluate inflammation and pain response. The selected compounds 1-4 inhibited the involvement of biogenic amines in the formation of paw edema. Compounds 1-4 also reduced pain in the inflammatory response phase. It has to be noted that 4-AA may cause agranulocytosis, which should be borne in mind when developing drug candidates of similar structure. Our new drug prototypes based on 4-aminoantipyrine and 1,4-dioxo-2-butenyl moieties open a gate for developing a prototype of nonsteroidal anti-inflammatory drugs.

Double-signal quantification of amoxicillin based on interaction with 4-aminoantipyrine at copper and nitrogen co-doped carbon quantum dots as an artificial nanozyme.

An one-pot hydrothermal method was developed for synthesis of carbon quantum dots co-doped with copper and nitrogen (Cu, N@CQDs). The synthesized Cu, N@CQDs has unique advantages such as high fluorescence quantum yield (39.1%) and high catalytic activity. Oxidative coupling of amoxicillin (AMX) with 4-aminoantipyrine (4-NH2-APE) in the presence of H2O2 as an oxidant to produce pink quinoneimine chromogen was carried out with the aid of Cu, N@CQDs as a peroxidase-like catalyst. This system was used for the colorimetric and fluorometric assays of AMX with reliable results. Colorimetric method is based on the measurement of a pink-colored product at λmax = 505 nm while the fluorometric assay is based on the quenching of the fluorescence emission of Cu, N@CQDs at 440 nm after excitation at 370 nm. For the colorimetric method, the absorption intensity linearly increased over the concentration range 4.3-110.0 µM with LOD (S/N = 3) of 1.3 µM. For the fluorometric method, the emission intensity of Cu, N@CQDs linearly decreased upon addition of AMX in the concentration range 0.2-120.0 µM with a limit of detection (LOD, S/N = 3) of 0.06 µM. The proposed system was applied to the determination of AMX in different real samples such as pharmaceutical capsules, human serum, milk, and conduit water samples with recoveries in the range 95.8-104.1% and relative standard deviation (RSD %) less than 4.1%.

Pharmacokinetics of metamizole (dipyrone) as an add-on in calves undergoing umbilical surgery.

This preliminary clinical investigation of the pharmacokinetic behavior of the main metamizole (dipyrone) metabolites 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA) in calves undergoing umbilical surgery is part of an already published main study. A single intravenous dose of metamizole was added to ketamine/xylazine/isoflurane anesthesia. Eight Simmental calves weighing 90 ± 10.8 kg and aged 47.6 ± 10.4 days received 40 mg/kg metamizole intravenously 10 minutes prior to general anesthesia. Blood samples were collected over 24 hours and analyzed for 4-MAA and 4-AA. Meloxicam was additionally given twice: 2.5 hours pre- and 20.5 hours postsurgically. The pharmacokinetic profile of 4-MAA was best fitted to a two-compartment model and was characterized by a fast distribution half-life and slow elimination half-life (t½alpha = 5.29 minutes, t½beta = 9.49 hours). The maximum concentration (Cmax 101.63 µg/mL) was detected at the first measurement time point 15 minutes after administration. In contrast, 4-AA showed fast, high and biphasic plasma peak concentration behavior in five calves (2.54-2.66 µg/mL after 15-30 minutes, and 2.10-2.14 µg/mL after 2-3.5 hours) with a t½beta of 8.87 hours, indicating a rapid distribution and subsequent redistribution from well-perfused organs. Alternatively, three calves exhibited a slower and lower monophasic plasma peak concentration (1.66 µg/mL after 6.5 hours) with a t½beta of 6.23 hours, indicating slow accumulation in the intravascular compartment. The maximum concentration and area under the plasma concentration curve (AUC) of 4-AA were lower than those of 4-MAA. This metabolic behavior supports our already published data on clinical monitoring and plasma cortisol concentrations (PCCs). Compared to those of saline controls, lower PCCs correspond to the t½alpha of 4-MAA. Data on Tmax and t½beta also match these clinical observations. However, further studies are required to assess the exact analgesic mechanism and potency of the metamizole metabolites in calves.

Construction of a colorimetric sensor array based on the coupling reaction to identify phenols.

Phenols are harmful to the human body and the environment. Since there are a variety of phenols in actual samples, this requires a sensor which possesses the ability to simultaneously distinguish them. Herein, we report a colorimetric sensor array, which uses two nanozymes (Fe-N-C nanozymes and Cu-N-C nanozymes) as electronic tongues for fingerprint identification of six phenols (2,4,6-trichlorophenol (2,4,6-Tri), 4-nitrophenol (P-np), phenol (Phe), 3-chlorophenol (3-CP), 4-chlorophenol (4-CP), and o-nitrophenol (O-np)) in the environment. Nanozymes catalyzed the reaction of hydrogen peroxide, different phenols and 4-aminoantipyrine (4-AAP) to produce different color variations. These signal changes as fingerprints encouraged us to develop a pattern recognition method for the identification of phenols by linear discriminant analysis (LDA). The six phenols at 50 nM have their own response patterns, respectively. Surprisingly, this sensor array had distinguished the six phenols in actual samples successfully.

Microwave assisted Biology-Oriented Drug Synthesis (BIODS) of new N,N'-disubstituted benzylamine analogous of 4-aminoantipyrine against leishmaniasis - In vitro assay and in silico-predicted molecular interactions with key metabolic targets.

Biology-Oriented Drug Synthesis (BIODS) deals with the simple chemical transformations on the commercially available drugs in order to enhance their new and diversified pharmacological profile. It opens new avenues for the rapid development of drug candidates for neglected tropical diseases (NTDs). Leishmaniasis is one of the NTDs which spread by the bite of sandflies (plebotomine). It ranges from cutaneous self-healing leishmaniasis to life threatening visceral leishmaniasis, known as kala-azar. The current treatment options include the use of pentamidine, miltefosine, and amphotericin B drugs. Unfortunately, all currently available drugs are associated with adverse effects, such as severe nephron- and cardiotoxicity, pancreatitis, and hepatotoxicity. This warrants the development of new drugs against leishmaniasis. Moreover, emergence of resistance against the current medications further worsens the conditions. With this objective, new N, N'-disubstituted benzylamine derivatives of ampyrone (4-aminoantipyrine) were synthesized by using ultrasonication, and microwave assistance. All derivatives were found to be new, except 1, 4, and 11. All the compounds were evaluated for their anti-leishmanial activity, and cellular cytotoxicity. Among them, compounds 4, 5, 8, and 9 showed a significant anti-leishmanial activity in vitro, in comparison to standard drug, miltefosine (IC50 = 25.78 ± 0.2 µM). These compounds were also docked against various metabolic enzymes to predict their interactions and mechanism of action, and were found to act via targeting important enzymes of various metabolic pathways.

Synthesis and Biological Evaluation of 4-Aminoantipyrine Analogues.

OBJECTIVES: The aim of the present study is to carry out a simple synthesis of aminoantipyrine analogues and exploration of their antibacterial, cytotoxic, and anticonvulsant potential. METHODS: The compounds were characterized employing multi-spectroscopic methods. The in vitro pharmacological response of a series of bacteria was screened employing serial dilution method. The derivatives were screened against maximal electro-shock for their anticonvulsant activity. Molecular docking was carried out to optimize the interaction of the compounds with HPV16-E7 receptors. Further, the in vitro cytotoxicity was tested against human cervical cancer (SiHa) cell lines. RESULTS: The compounds show protection against maximal electroshock, esp. 3-nirto- and 4- methyl-3-nitrobenzamido derivatives. In addition, they reveal appreciable DNA cleavage activities and interactions with HPV16-E7 protein receptors, esp. 3,5-dinitro- and 4-methyl-3-nitrobenzamido derivatives. Furthermore, they show potent activity against cervical cancer cells (LD50 value up to 1200 in the case of 4-methyl-3-nitrobenzamido derivative and an inhibition of a maximum of ~97% of cells). CONCLUSION: The simply synthesized aminoantipyrine derivatives show a variety of biological activities like antibacterial and anticancer effects. In addition, this is the first study demonstrating that 4-aminoantipyrine derivatives show an anticonvulsant activity.

Pharmacokinetic properties of metamizole active metabolites in Northeastern Brazilian donkeys (Equus asinus).

Metamizole (MT), also known as dipyrone, is an analgesic and antipyretic drug labeled for use in humans and domestic animals in some countries. As with other drugs, the administration of MT in donkeys is based on studies carried out with horses. In the present report, we aimed to determine the pharmacokinetics of the two main metamizole active metabolites (N-methyl-4-aminoantipyrine [MAA] and 4-aminoantipyrine [AA]) following 10 (M10 ) and 25 mg/kg (M25 ) IV metamizole doses in Northeast Brazilian donkeys (n = 10). Blood was collected at predetermined times within over 48 h; MAA and AA plasma concentrations were determined by a validated LC-MS/MS method. The metabolites were quantifiable in the M10 until 12 h and M25 until 24 h after drug administration. As expected, AUC0→t , AUC0→∞, and Cmax demonstrated significant differences increases in metamizole metabolites profiles when groups were compared. No adverse effects were observed. This study indicates the need for an extremely sensitive analytical method to adequately characterize the pharmacokinetics of active metabolites of MT, MAA, and AA. In conclusion, the method developed in this research was able to measure the active metabolites of metamizole and with that it was possible to establish their pharmacokinetic profile. Furthermore, after projection of the minimum MAA concentrations, it is possible to infer that the dose of 10 mg/kg will be used on donkeys at 6 h intervals, while the M25 group at 12 h intervals. However, clinical studies are needed to assess this hypothesis.

Extracellular Oxidase from the Neonothopanus nambi Fungus as a Promising Enzyme for Analytical Applications.

The extracellular enzyme with oxidase function was extracted from the Neonothopanus nambi luminescent fungus by using mild processing of mycelium with ß-glucosidase and then isolated by gel-filtration chromatography. The extracted enzyme is found to be a FAD-containing protein, catalyzing phenol co-oxidation with 4-aminoantipyrine without addition of H2O2, which distinguishes it from peroxidases. This fact allowed us to assume that this enzyme may be a mixed-function oxidase. According to gel-filtration chromatography and SDS-PAGE, the oxidase has molecular weight of 60 kDa. The enzyme exhibits maximum activity at 55-70 °C and pH 5.0. Kinetic parameters Km and Vmax of the oxidase for phenol were 0.21 mM and 0.40 µM min-1. We suggest that the extracted enzyme can be useful to develop a simplified biosensor for colorimetric detection of phenol in aqueous media, which does not require using hydrogen peroxide.

Insight into multicopper oxidase laccase from Myrothecium verrucaria ITCC-8447: a case study using in silico and experimental analysis.

The oxidation activity of multicopper-oxidases overlaps with different substrates of laccases and bilirubin oxidases, thus in the present study an integrated approach of bioinformatics using homology modeling, docking, and experimental validation was used to confirm the type of multicopper-oxidase in Myrothecium verrucaria ITCC-8447. The result of peptide sequence of M. verrucaria ITCC-8447 enabled to predict the 3 D-structure of multicopper-oxidase. It was overlapped with the structure of laccase and root mean square deviation (RMSD) was 1.53 Å for 533 and, 171 residues. The low binding energy with azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (-5.64) as compared to bilirubin (-4.39) suggested that M. verrucaria ITCC-8447 have laccase-like activity. The experimental analysis confirmed high activity with laccase specific substrates, phenol (18.3 U/L), ampyrone (172.4 U/L) and, ampyrone phenol coupling (50 U/L) as compared to bilirubin oxidase substrate bilirubin (16.6 U/L). In addition, lowest binding energy with ABTS (-5.64), syringaldazine SYZ (-4.83), guaiacol GCL (-4.42), and 2,6-dimethoxyphenol DMP (-4.41) confirmed the presence of laccase. Further, complete remediation of two hazardous model pollutants i.e., phenol and resorcinol (1.5 mM) after 12 h of incubation and low binding energy of -4.32 and, -4.85 respectively confirmed its removal by laccase. The results confirmed the presence of laccase in M. verrucaria ITCC-8447 and its effective bioremediation potential.

Simultaneous Detection of Serum Glucose and Glycated Albumin on a Paper-Based Sensor for Acute Hyperglycemia and Diabetes Mellitus.

Diabetes mellitus is one of the most common chronic diseases worldwide. Generally, the levels of fasting or postprandial blood glucose and other biomarkers, such as glycated albumin, glycated hemoglobin, and 1,5-anhydroglucitol, are used to diagnose or monitor diabetes progression. In the present study, we developed a sensor to simultaneously detect the glucose levels and glycation ratios of human serum albumin using a lateral flow assay. Based on the specific enzymatic reactions and immunoassays, a spiked glucose solution, total human serum albumin, and glycated albumin were measured simultaneously. To test the performance of the developed sensor, clinical serum samples from healthy subjects and patients with diabetes were analyzed. The glucose level and glycation ratios of the clinical samples were determined with reasonable correlation. The R-squared values of glucose level and glycation ratio measurements were 0.932 and 0.930, respectively. The average detection recoveries of the sensor were 85.80% for glucose and 98.32% for the glycation ratio. The glucose level and glycation ratio in our results were crosschecked with reference diagnostic values of diabetes. Based on the outcomes of the present study, we propose that this novel platform can be utilized for the simultaneous detection of glucose and glycation ratios to diagnose and monitor diabetes mellitus.

Synthesis, antimicrobial activity, and sustainable release of novel α-aminophosphonate derivatives loaded carrageenan cryogel.

Novel α-aminophosphonates 4 were synthesized via one pot three-component reaction of 4-aminoantipyrine, aldehydes, triphenylphosphite and Lewis acid catalyst. The chemical structures of all the synthesized compounds were elucidated by IR, NMR and MS spectral analysis. The antimicrobial activity of 4 was tested in vitro against pathogenic microbes such as E.coli, S.aureus, A.niger and C.albicans. Three of them (4f-h) exhibited high antimicrobial activity and were loaded to carrageenan cryogel for drug delivery studies. With the aid of cellulose nanofibrils (CNF) as reinforcing material and glyoxal as a cross-linking agent, porous cryogels with improved mechanical properties were obtained. Among all, CAR-7 presents the optimum cryogel sample, which contained around 16% CNF and 0.2 mL/15 mL polymer blend. CAR-7 demonstrated highest mechanical compressive strength, porosity (80%), and swelling capacity (75%). Sustainable release behavior over 24 h was observed for the loaded cryogels. The antimicrobial activity of cryogels against S.aureus showed marginal differences between samples. CAR-9 (loaded with 4f) showed the highest reduction percentage in number of bacterial colonies (99.94%) followed by CAR-11 (loaded with 4h, 99.3%) and finally CAR-10 (loaded with 4g, 99.29%).

Extracellular Oxidases of Basidiomycete Neonothopanus nambi: Isolation and Some Properties.

Using the original technique of treating biomass with ß-glucosidase, a pool of extracellular fungal enzymes was obtained for the first time from the mycelium of basidiomycete Neonothopanus nambi. Two protein fractions containing enzymes with oxidase activity were isolated from the extract by gel-filtration chromatography and conventionally called F1 and F2. Enzyme F1 has a native molecular weight of 80-85 kDa and does not contain chromophore components; however, it catalyzes the oxidation of veratryl alcohol with Km = 0.52 mM. Probably, this enzyme is an alcohol oxidase. Enzyme F2 with a native molecular weight of approximately 60 kDa is a FAD-containing protein. It catalyzes the cooxidation of phenol with 4-aminoantipyrine without the addition of exogenous hydrogen peroxide, which distinguishes it from the known peroxidases. It was assumed that this enzyme may be a mixed-function oxidase. F2 oxidase has Km value 0.27 mM for phenol. The temperature optimums for oxidases F1 and F2 are 22-35 and 55-70°C, and pH optimums are 6 and 5, respectively.

Mechanisms of interference of p-diphenols with the Trinder reaction.

p-Diphenols, such as homogentisic acid, gentisic acid, etamsylate, and calcium dobesilate, interfere with diagnostic tests utilizing the Trinder reaction but the mechanisms of these effects are not fully understood. We observed substantial differences both in oxidation of p-diphenols by horseradish peroxidase and their influence on oxidation of 4-aminoantipyrine and various phenolic substrates. Homogentisic acid was rapidly oxidized by the enzyme and completely blocked chromophore formation. Enzymatic oxidation of the remaining p-diphenols was slow and they only moderately inhibited chromophore formation. However, in the presence of standard substrates all tested p-diphenols were rapidly converted to p-quinones. Hydrogen peroxide consumption was significantly accelerated by homogentisic acid but not much affected by the other p-diphenols. The magnitude and mechanisms of interference caused by p-diphenols therefore depend on their structure which determines their electrochemical properties - while for homogentisic acid with an electron-donating substituent and a lower reduction potential both enzymatic oxidation and reduction of the peroxidase-generated radicals occur, for p-diphenols with electron-withdrawing substituents and higher reduction potentials only the second mechanism is significant. Correlation of the effects on the Trinder reaction with reduction potentials of interfering compounds allows prediction of such properties for a wide range of other reducing compounds based on this parameter. It also explains why compounds with very different structures but strong reducing properties show such effects.

Development of a new chromogenic method for putrescine quantification using coupling reactions involving putrescine oxidase.

Quantification of polyamines, including putrescine, is generally performed using high-performance liquid chromatography (HPLC) or gas chromatography. However, these methods are time-consuming because of sample derivatization and analytical reagent preparation. In this study, we developed a simple and high-throughput putrescine quantification method on a 96-well microtiter plate using putrescine oxidase from Rhodococcus erythropolis NCIMB 11540, peroxidase, 4-aminoantipyrine, and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt. The developed method (named as PuO-POD-4AA-TOPS method) was applicable to bacterial culture supernatants. Furthermore, putrescine concentrations determined by the developed method roughly corresponded to the concentrations determined by HPLC.