Oxidation Of Homovanillic Acid Research Articles

The role of 3,4-dihydroxyphenylacetic acid adsorption in the oxidation of homovanillic acid at a glassy carbon rotating disc electrode

Homovanillic acid (HVA) is an important biomarker for neurological disorders. Traditional methods for detection of this compound are chromatographic methods coupled with fluorescence, mass spectrometric, or electrochemical detectors. However, voltammetric methods can be useful because of their low cost, easy automation, portability, and sensitivity. 3,4-Dihydroxyphenylacetic acid (DOPAC) is formed during electrochemical oxidation of HVA. The formation of this compound could interfere with the quantification of HVA using electrochemical methods. This work aims at describing the behavior of the intermediate species generated in the oxidation of HVA using cyclic voltammetry (CV), linear staircase voltammetry (LSV), and electrochemical impedance spectroscopy (EIS) with a glassy carbon rotating disc electrode (GC RDE). The importance and desirability of detailed investigation of surface phenomena connected with electrochemical oxidation used for electroanalytical purposes are thus clearly demonstrated. The EIS measurements indicate that the intermediate DOPAC formed by the oxidation of HVA can adsorb on the surface of a glassy carbon electrode (GCE). For the static GCE, a semi-finite linear diffusion was observed and for the GC RDE, a finite-length diffusion was verified. In the case of the semi-finite linear diffusion, EIS measurements revealed that the resistance to charge transfer decreases from 100 to 50Ω, and the capacitance of the double layer increases from 1.44 to 1.66μF with increasing concentration of HVA. However, LSV results showed that Levich equations for electrochemical reduction of K3[Fe(CN)6] probe in basic medium are ilim=−0.157 ω1/2−0.006 (R2=0.9999) in the absence of HVA (i.e. without DOPAC adsorption at GCE) and ilim=−0.154 ω1/2−0.021 (R2=0.9998) in the presence of HVA (i.e. with DOPAC adsorption at GCE). The small difference in these two equations proves a negligible influence of DOPAC adsorption on the quantification of HVA using stationary methods.

Pentamines as Substrate for Human Spermine Oxidase

Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO.

Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite

Elevated levels of arsenite, the trivalent form of arsenic, in drinking water correlates with increased vascular disease and vessel remodeling. Previous studies from this laboratory demonstrated that environmentally relevant concentrations of arsenite caused oxidant-dependent increases in nuclear transcription factor levels in cultured porcine vascular endothelial cells. The current studies characterized the reactive species generated in these cells exposed to levels of arsenite that initiate cell signaling. These exposures did not deplete 5′-triphosphate, nor did they affect basal or bradykinin-stimulated intracellular free Ca 2+ levels, indicating that they were not lethal. Electron paramagnetic resonance (EPR) spectroscopy, including spin trapping with carboxy-PTIO (cPTIO), demonstrated that 5 μM or less of arsenite did not increase NO levels over a 30-min period relative to NO release stimulated by bradykinin. However, these same levels of arsenite rapidly increased both oxygen consumption and superoxide formation, as measured by EPR oximetry and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), respectively. Pretreatment of the cells with DPI, apocynin, or superoxide dismutase abolished arsenite-stimulated DMPO-OH adduct formation. Finally arsenite increased extracellular accumulation of H 2O 2, measured as oxidation of homovanillic acid, with the same time and dose dependence, as seen for superoxide formation. These data suggest that superoxide and H 2O 2 are the predominant reactive species produced by endothelial cells after arsenite exposures that stimulate cell signaling and activate transcription factors.

A fluorometric rate assay of hydrogen peroxide using immobilized peroxidase with a fibre-optic detector

Abstract Horseradish peroxidase is immobilized in polyurethane membranes and employed in conjunction with a fibre-optic probe to establish an enzyme reactor/sensor-based rate assay for hydrogen peroxidase. The fluorescence of the dimer formed by the enzyme-catalysed oxidation of homovanillic acid is utilized as the analytical signal. In the presence of 1×10−3 mol l−1 homovanillic acid, H2O2 can be detected between 5×10−8 and 5×10−5 mol l−1. Up to 30 determinations can be performed per hour. The relative standard deviation for 1.6×10−6 mol l−1 H2O2 is 5% (n=7). The half life of the sensor system is five days. Measurements can be carried out with reasonable sensitivity for a period of at least nine days.

Enzymatic Methods for the Quantification of Polyamines Using Plant Amine Oxidases

Summary Maize polyamine oxidase, which catalyses specifically the oxidation of spermidine and spermine, and diamine oxidase from Euphorbia characias latex, which reacts only with diamines such as putrescine and cadaverine, have been utilised for developing an enzymatic test for the quantification of di- and polyamines. The enzymatic test was performed exploiting an end-point-assay method, either (1) spectrophotometrically, following the formation of a red-coloured adduct of Δ1-pyrroline with ninhydrin reagent, or (2) polarographically, estimating by a Clark electrode the O2 depletion, or (3) spectrofluorimetrically, following the formation of H2O2 by the peroxidase-dependent oxidation of homovanillic acid to a fluorescent product. These methods have a sensitivity ranging between 2 nmoles (fluorimetric method) and 10 nmoles (ninhydrin and polarographic method), and can be applied to the determination of polyamines in crude plant and animal perchloric acid extracts. The polarographic assay was exploited for the determination of acid-soluble polyamines in a perchloric extract of pea or maize stems and mouse liver or brain. The results obtained were compared with those found by thinlayer chromatography.

Spectrofluorimetric determination of hydrogen peroxide based on the catalytic effect of peroxidase-like manganese tetrakis(sulphophenyl) porphyrin on the oxidation of homovanillic acid

Traces of hydrogen peroxide (8.5 × 10 −8–2.5 × 10 −6 mol/l) and, indirectly, glucose (3–44 × 10 −6 mol/l) can be determined by the fluorescence reaction between homovanillic acid and hydrogen peroxide. Mn-TPPS 4 is found to have very similar catalytic properties to horse peroxidase.

Mimesis of peroxidase by Mn-TMPyP in the catalytic fluorescence reaction of the homovanillic acid-hydrogen peroxide system

The fluorescence produced by the catalytic effect of the manganese (III)-tetrakis-(N-methylpyridinium)porphyrin complex (Mn-TMPyP) on the oxidation of homovanillic acid by hydrogen peroxide has been studied. The reaction product fluoresces at 424 nm (with excitation at 316 nm). Traces of hydrogen peroxide (1.3 × 10−7−2.4 × 10−6 M) and glucose (1.5–5.0 μg/ml) can be determined with good accuracy and reproducibility. The characteristics of the mimetic enzyme Mn-TMPyP have been compared with those of horseradish peroxidase.

22] Measurement of hydrogen peroxide production by phagocytes using homovanillic acid and horseradish peroxidase

This chapter describes a method for the determination of hydrogen peroxide (H 2 O 2 ) produced by neutrophils, monocytes, and macrophages. The assay is based on the oxidation of homovanillic acid (HVA) into a fluorescent dimer, which is catalyzed by horseradish peroxidase and depends on the H 2 O 2 generated by the cells. Human neutrophils are purified from anticoagulated blood, suspended in isotonic NaCl (10 7 -10 8 cells/ml), and kept at 6–10° C until use. Mononuclear phagocytes may be used as a suspension or as monolayers in small plastic culture wells. In both cases, the medium is Hanks' balanced salt solution (HBSS). Mouse bone marrow-derived macrophages are obtained by proliferation in Teflon bag cultures, which are harvested from the bags, centrifuged at 200 g for 10 minutes at room temperature, and resuspended at the desired density in medium 199 containing 15% heat-inactivated horse serum and 2 m M glutamine for seeding culture plates or in HBSS for direct use. Human monocytes are purified from buffy coats of donor blood by centrifugal elutriation and used as a suspension in HBSS or as adherent cultures in flat-bottom 96-well microtiter plates.