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The ability of cyclodextrins to enhance the water solubility of lipophilic compounds is used to modify the water-based Oxygen Radical Absorbance Capacity (ORAC) method to measure antioxidant activity in vitro. However, the use of these solubility enhancers may alter fluorescent readings, which has led to contradictory results being described in the literature. The low specificity of these oligosaccharides and their controlled release effect can result in cyclodextrins forming inclusion complexes with other reagents in the assay, changing the kinetics. In this study, the cause of cyclodextrins' interference in the ORAC method is evaluated through a physicochemical and computational approach. Cyclodextrins showed a clear increase in the fluorescent signal both in the presence and absence of the antioxidant oxyresveratrol, the precise effect being dependent on the type and concentration of cyclodextrin. Although the glucidic nature of cyclodextrins could play a minimal role in this effect, it seems that the main cause was the encapsulation of other substrates in the reaction, fluorescein and AAPH., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Current HPLC methods for analyzing cAMP Phosphodiesterase activity (PDE) use salts, limiting the life of the columns. For this reason, we have developed an improved "ion pairing agent free" method, using a simple 150 mm C18-hydro column at 30 °C and two phases: (a) water with 0.1% acetic acid and (b) 85/15 w/w MeOH/tetrahydrofuran with 0.1% acetic acid. Using this method the peaks for cAMP and AMP were obtained with good resolution (R ≈ 1.35) and sensitivity (5·10 -9 mols) in only 15 min. Moreover, the method was applied to the GMP/cGMP pair obtaining the same sensitivity and resolution (R ≈ 1.38). The precision and accuracy were tested and the method was verified with a Type IV Phosphodiesterase reaction, which produced AMP from cAMP. The method is cleaner and less aggressive, and represents an interesting alternative to currently used methods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A new methodology, based on high resolution liquid chromatography with light scatterin detector, is applied to analyze Hydroxypropyl-beta-Cyclodextrin (HPβCD) in urine samples of a child affected by Niemann-Pick Type C disease. The treatment not only stopped disease progression, but has also increased the life expectancy and quality of our patient. The pharmacokinetic of HPβCD in the patient was studied with a 92.8% of HPβCD recovered. At 88 h, no HPβCD was found in the urine. During the treatment, HPβCD has not shown toxicity. Before application of the new treatment, injections were given every two weeks but, we have demonstrated that this can be increased to every four days., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An improved method based on the p-nitrophenyl long chain esters method is proposed for measuring lipase hydrolytic activity in aqueous media. Using ethylene glycol as co-solvent for hydrophobic p-nitrophenyl substrates in aqueous buffer, lipase activity is measured by following the release of p-nitrophenol. This fast and easy to handle method improves the solubility of both substrate and product, and also the stability of the substrate. It avoids the use of solvents such as ethanol or propanol, permits the comparison of all the p-nitrophenol acyl ester substrates and allows the influence of ions like Ca +2 to be studied, while avoiding turbidity in the reaction medium., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hydrogen peroxide triggers a redox cycle between methemoglobin and ferrylhemoglobin, leading to protein inactivation and oxygen evolution. In the present paper, the catalase-like oxygen production by human methemoglobin in the presence of H(2)O(2) was kinetically characterized with a Clark-type electrode. Progress curves showed a pseudo-steady state in the first minutes of the reaction, while double-reciprocal plots were upwardly concave, indicating positive co-operativity dependent upon protein concentration, which is a very unusual kinetic behavior. Addition of superoxide radical scavengers slightly increased activity, suggesting that most oxygen was produced biocatalytically. By considering all the experimental data obtained, a possible mechanism was proposed, including: (a) competition between the one-electron and the two-electron reductions of the oxoferryl free radical species of hemoglobin, giving rise to ferrylhemoglobin and methemoglobin, respectively; (b) competition between the superoxide-dependent inactivation of the protein and its reduction back to the met state. Computer simulations of that model have been performed by numerically integrating the differential equations set describing the mechanism, which was seen to yield predictions of the kinetic parameters variation consistently with the kinetic behavior experimentally observed. We suggest that the catalase-like activity of methemoglobin must predominantly be a biocatalytic reaction that protects the protein against H(2)O(2)-induced suicide inactivation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this work, a reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of pterostilbene in food samples. The novel method is based on the addition of cyclodextrins (CDs) to the mobile phase where the complexation of pterostilbene by CDs is carried out. In order to select the most suitable conditions for the RP-HPLC method, the effect of several physico-chemical parameters on the complexation of pterostilbene by CDs was studied. Our results show that the addition of 12 mM HP-β-CD to a 50:50 (v/v) methanol:water mobile phase at 25°C and pH 7.0 significantly improves the main analytical parameters. In addition, it was seen that pterostilbene forms a 1:1 complex with HP-β-CD, showing an apparent complexation constant of 251±13 M(-1). Finally, in order to study the validity of the proposed method, blueberries were analyzed and the concentration of pterostilbene has been determined., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The enzymatic peroxidation of pterostilbene, a strong antifungal belonging to the stilbene family, by peroxidase (POX), is reported for the first time as a model of phytoalexin detoxification carried out by the enzymatic pool of pathogens. Kinetic characterization of the pterostilbene oxidation reaction pointed to an optimum pH of 7.0, at which value the thermal stability of POX was studied. Moreover, the data showed that pterostilbene inhibits POX activity at high concentrations of substrate. Several kinetic parameters, including V(max), K(m) and K(I), were calculated and values of 0.16ΔAbs min(-1), 14.61 μM, and 31.41 μM were reported. To understand the possible physiological role of this reaction in the phytoalexin detoxification process, the products of pterostilbene oxidation were identified using HPLC-MS and a radical-radical coupling reaction mechanism was proposed. Three main products with a high molecular weight and pronounced hydrophobicity were identified: pterostilbene cis dehydromer, pterostilbene trans dehydromer and pterostilbene open dimer. The dimeric structures of these molecules indicate that the pterostilbene oxidation reaction took place at the 4'-OH position of the hydroxystilbenic moieties and the three above mentioned dimeric products were found, due to the ability of electron-delocalized radicals to couple at various sites. Finally, the capacity of cyclodextrins (CDs) as starch model molecules in plants to complex both the substrate and the products of the oxidation reaction was evaluated. The inhibition process of POX activity was modified at high pterostilbene concentrations due to sequestering of the substrate reaction and to the different affinity of the reaction products for CDs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Knowledge of the complexation process of oxyresveratrol with beta-cyclodextrin (beta-CD) under different physicochemical conditions is essential if this potent antioxidant compound is to be used successfully in both food and pharmaceutical industries as ingredient of functional foods or nutraceuticals, despite its poor stability and bioavailability. In this paper, the complexation of oxyresveratrol with natural CDs was investigated for first time using RP-HPLC and mobile phases to which alpha-, beta-, and gamma-CD were added. Among natural CDs, the interaction of oxyresveratrol with beta-CD was more efficient than with alpha- and gamma-CD. The decrease in the retention times with increasing concentrations of beta-CD (0-4 mM) showed that the formation constants (KF) of the oxyresveratrol/beta-CD complexes were strongly dependent on both the water-methanol proportion and the temperature of the mobile phase employed. However, oxyresveratrol formed complexes with beta-CD with a 1:1 stoichiometry in all the physicochemical conditions tested. Moreover, to obtain information about the mechanism of the oxyresveratrol affinity for beta-CD, the thermodynamic parameters DeltaG degrees, DeltaH degrees and DeltaS degrees were obtained. Finally, to gain information on the effect of the structure of different compounds belonging to the stilbenoids family on the KF values, the complexation of other molecules, resveratrol, pterostilbene and pinosylvin, was studied and compared with the results obtained for the oxyresveratrol/beta-CD complexes., (Copyright 2010 Elsevier B.V. All rights reserved.)

A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.

Betaxanthins are natural water-soluble yellow pigments present in plants of the order Caryophyllales. The native fluorescence of these compounds is extensively characterized in this work, with study of the fluorescent properties of 14 different betaxanthins. All the species showed a similar behavior, with excitation maxima between 463 and 475 nm and emission maxima between 506 and 515 nm. Thus, betaxanthins absorb light corresponding to the blue color and emit visible green light. Similarities in excitation and emission spectra point to the responsibility of betalamic acid in fluorescence. The influence of the amine moiety is discussed. For the first time fluorescent properties of betaxanthins are applied to the detection of these pigments after separation by high-performance liquid chromatography. Wavelengths used were 460 nm for excitation and 510 nm for emission, which were suitable for detecting the native fluorescence of all the pigments assayed. Calibration was performed in each case and it exhibited linearity within the range considered, at least 20 microM. The lowest detection limit was 100 nM, corresponding to betaxanthins derived from methionine sulfoxide and leucine. Fluorescence detection was applied to the quantification of betaxanthins present in Carpobrotus acinaciformis. The present work opens up new possibilities for the analysis of betaxanthins by improving existing protocols through fluorescence detection.

A kinetic study of acetaminophen oxidation by tyrosinase in the presence of a physiological nucleophilic agent such as the amino acid L-proline is performed in the present paper. The o-quinone product of the catalytic activity, 4-acetamido-o-benzoquinone, becomes unstable through the chemical addition of L-proline, in competition with the nucleophilic addition of hydroxide ion from water. In both cases, the catechol intermediate, 3(')-hydroxyacetaminophen, is generated, as can be demonstrated by liquid chromatography. When the effect of the presence of the nucleophilic agent on the time course of the enzymatic reaction was kinetically analyzed, it was seen to decrease the duration of the lag period and increase the steady-state rate. Rate constants for the reaction of 4-acetamido-o-benzoquinone with water and L-proline were also determined. The results obtained in this paper open a new possibility to acetaminophen toxicity, that has been attributed hitherto to its corresponding p-quinone, N-acetyl-p-benzoquinone imine.

A method for the quantitative determination of acetaminophen by measuring the kinetics of its oxidation by the enzyme tyrosinase in the presence of the nucleophilic agent 3-methyl-2-benzothiazolone hydrazone (Besthorn's hydrazone) is described. The hydrazone traps the o-quinone product of the catalytic activity, 4-acetamido-o-benzoquinone, thus increasing the level of the catecholic intermediate, 3'-hydroxyacetaminophen, in the pathway, as was shown by liquid chromatography. This reduces the time necessary for the steady state to be reached and increases the steady state oxidation rate, reaching activations of 124 times at an initial concentration of 3mM hydrazone. Despite being rapid and simple the method is thorough and provides a linear range of quantification of the drug from 10 nmol/ml to 1.4 micromol/ml. The assay can be performed in continuous or discontinuous form, meaning that it can be used with the immobilized enzyme for the design of specific biosensors.

3,4-Dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium-4-methylbenzene sulfonate (MDL 73,404) is a cardioselective water-soluble quaternary ammonium analogue of Vitamin E which is synthesized to augment the antioxidant defence in situations of free radical injury such as myocardial infarction/reperfusion. Its oxidation by any peroxidative enzyme has not been studied kinetically. This paper describes its enzymatic oxidation by horseradish peroxidase (HRP). The activity was followed spectrophotometrically at 255nm, and the experimental results were simulated using the program "KINETIC 3.1" for Windows 3.x. The MDL 73,404 was oxidized by horseradish peroxidase in the presence of H2O2 to its corresponding MDL 73,404 quinone. During this oxidation, the horseradish peroxidase showed an unexpectedly slow kinetic response with time, which contrast with the linear product accumulation curve measured with 2,2'-azino-bis-(3-estilbenzotiazol-6-sulfonic acid) (ABTS). This response was dependent on the respective concentrations of enzyme, MDL 73,404 and H2O2. However, when the enzyme was incubated with H2O2, the slow kinetic response disappeared and a lag period was observed. Furthermore, when p-coumaric acid (PCA) was added, the activity increased and the slow kinetic response became a straight line. In order to explain this anomalous behaviour, a kinetic model has been proposed and its differential equations simulated. From the correlation between experimental and simulated results it is concluded that MDL 73,404 can act as a slow response substrate for peroxidase, probably due to the presence of a quaternary ammonium side chain that confers on it a slow capacity to convert compound III into ferriperoxidase.