Rolando Budini

Determination of superoxide dismutase in erythrocytes by a chemiluminescent assay.

A highly rapid chemiluminescent assay for the determination of superoxide dismutase (SOD) activity in erythrocytes was developed. The inhibition of the luminescent emission caused by the decrease of generated superoxide anions was measured. The aim of this work was to verify the application of a non amplified luminol SOD luminescent assay (CLM) in erythrocytes starting from an amplified method already used for the determination of XOD activity in milk (CLME). Both the assays had a detection limit of 3x10(-2)+/-7x10(-3) U/ml of SOD at 2sigma level, and a linear range of activity from 5.2 to 0.03 U/ml of SOD. The imprecision of assays (repeatability) presented coefficients of variations ranging from 3.1 to 7.9% for the CLME method and from 0.6 to 17.7% for CLM method. Both luminescent techniques were compared using a spectrophotometric kit, that had a detection limit of 0.3 U/ml, and showed good agreement.

Bioluminescent flow sensor for D-(-)-lactate

Abstract Previously, a bioluminescent flow sensor was developed for the determination of the content of l -lactate in biological fluids (serum, plasma, ventricular fluid) by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. Based on a similar scheme, a sensor has now been developed for d -(−)-lactate. The co-immobilization of alanine aminotransferase (ALT) with d -LDH improved the lactate transformation to 40–60%. The response was linear from 1 to 100 μmol 1 −1 at 25 °C for the LDH-ALT reactor. The intra- and inter-assay relative standard deviations were less than 75% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and applications to d -(−)-lactate determination in serum, milk and microorganism extracts are reported together with those for l -(+)-lactate.

Plasma antioxidant capacity determination: comparative evaluation of chemiluminescent and spectrophotometric assays.

All aerobic organisms have developed different mechanisms for neutralising the free radicals, mostly produced by the monoelectronic reduction of O(2), and preventing the severe damages these can provoke. The efficiency of these mechanisms can be assessed, in different matrices, by a simple and direct chemiluminescent assay (CL) based on luminol oxidation catalysed by horseradish peroxidase. Light emission is mediated by the production of free radicals and it is inhibited after a sample addition in a way that is directly proportional to the sample total content of molecules displaying antioxidant activity. The performances of this chemiluminescent assay were compared with those of two spectrophotometric methods already applied in clinical practice. First spectrophotometric method measures, like CL assay, the total antioxidant capacity, whereas the second one determines free thiol groups content. The chemiluminescent assay has a linearity interval between 0.60 and 9.46 mumol l(-1) of Trolox (y=34.91x+3.10; r=0.999; n=5) with an imprecision, expressed as CV, of 3.8% and an inaccuracy, expressed as percentage recovery, of 109%. The first spectrophotometric method, based on the same reference standard, the Trolox molecule, has a linearity interval between 0.2 and 2.5 mmol l(-1) of Trolox (y=-0.01x+4.54; r=0.95; n=5); the thiol groups assay has a linearity interval between 0.1 and 1 mmol l(-1) of l-cysteine (y=1.68x-47.09; r=0.998; n=5). Different clinical samples of plasma from healthy individuals, obese subjects and patients with liver diseases were tested. Interesting correlations were obtained among the three methods, but no significant correlations emerged between antioxidant capacity and clinical parameters. Significant differences were there only between men and women among obese subjects and between drinkers and non-drinkers among liver disease patients.

Microdetermination of indole-3-acetic acid in vegetable extracts by high-performance liquid chromatography with fluorescence detection

Abstract A HPLC assay method for the quantitation of indole-3-acetic acid in vegetable extracts is described, using fluorescence detection. The method, which is based on extraction in the presence of poly(vinylpyrrolidone), preliminary cleanup of the extract using Sep Pak C18, and separation on a silica column, has been optimized for the determination of IAA in grape berries. The procedure described is determined to be accurate, sensitive, and reliable over time. The detection limit for IAA is about 0.1 ng.

COMPARISON OF ANALYTICAL METHODS IN DETERMINING TOTAL ANTIOXIDANT CAPACITY IN RED WINE

ABSTRACT Total antioxidant capacity was measured in red wine samples by two analytical methods: chemiluminescent (CL) assay based on the luminol/peroxidase system and a spectrophotometric (SP) method based on the use of crocin. The imprecision, expressed as coefficient of variation (CV) for each method, was similar (1.2%), whereas the detection limit (LOD) was 35 and 0.3 µmol of Trolox for SP and CL methods, respectively. In addition, the chemiluminescent method has the advantage of being simple, rapid, sensitive and inexpensive. Different samples of red wine were analysed and both methods showed that the antioxidant capacity is related to the total polyphenol content and varies over time depending on storage conditions.

A simple and continous assay for decarboxylase activity using a carbon dioxide-selective electrode

Abstract A carbon dioxide electrode has been used for kinetic studies of decarboxylase activity. The methodology used requires only a sensitive pH meter operating in the millivolt mode with the electrode. The CO2 release may be followed continuously. Compared to other assay procedures the method outlined offers the advantages of rapidity, continuity, specificity, and sufficient precision. Techniques for the calibration of the electrode response are discussed. The applicability of this assay was appraised by determining the Km of lysine decarboxylase. The value obtained showed good agreement with that reported in literature. The procedure might be useful for the study of enzyme-inhibitor interactions.

Enzymatic spectrophotometric determination of nitrites in beer

ABSTRACT A colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10−8 – 10−2 M range with a detection limit of 10−8 M and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NiR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10−5 – 10−2 M) and a higher detection limit (10−5 M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.

Bioluminescent flow sensor for L-phenylalanine determination in serum

A highly sensitive and rapid bioluminescent flow sensor was developed for the determination of the content of L-phenylalanine (Phe) in serum by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized phenylalanine dehydrogenase (PheDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. The L-PheDHs extracted from Bacillus badius, Bacillus sphaericus and Rhodococcus sp. M 4 were investigated and the performances of the three immobilized L-PheDHs were analysed. The B. badius reactor was found to give higher transformation rate and better sensitivity; the response was linear from 1 to 100 microM at 25 degrees , with a detection limit of 10 pmoles (0.5 microM). The intra- and inter-assay coefficients of variation were less than 5% and recoveries ranged from 90 to 101%. The results agreed well with those obtained with a chromatographic method for the Phe determination in serum and with the normal reference values.

External GSSG enhances intracellular glutathione level in isolated cardiac myocytes.

The addition of external GSSG at concentrations in the range 50-500 microM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. On the contrary, external GSH at the above same indicated concentrations did not change the cell glutathione pool. The pretreatment of the cells with diethylamaleate depleted the myocytes of glutathione and enhanced the GSSG-induced replenishment effect on GSH level. On the contrary, the addition of GSH did not increase the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase the intracellular glutathione pool, probably through the reduction of the imported GSSG to GSH.

Involvement of superoxide radicals on adrenochrome formation stimulated by arachidonic acid in bovine heart sarcolemmal vesicles

Highly purified sarcolemmal membranes prepared from bovine heart muscle produced superoxide radicals, especially when incubated with NADPH or NADH, as revealed by the oxidation of adrenaline to adrenochrome. The reaction was inhibited by superoxide dismutase or by heat denaturation of the sarcolemmal vesicles. Less evident was the inhibitory effect shown by catalase, while mannitol, deferoxamine or dicumarol were uneffective. The formation of adrenochrome was an oxygen-dependent reaction with a Km for adrenaline of 8-10 microM. Moreover, the reaction was inhibited by preincubating the sarcolemmal membranes with propranolol, while the alpha-antagonist phentolamine was without effect. Adrenaline oxidation was unaffected by the presence of exogenous linolenic acid or methylarachidonic acid, while arachidonic acid, with a Km for this reaction of 175 microM, showed a marked stimulatory effect. This activation was suppressed by superoxide dismutase, catalase and NaCN, while mannitol was without effect. Moreover, the reaction was blocked by the cyclooxygenase inhibitor indomethacin, differently from the lipooxygenase inhibitor nordihydroguaiaretic acid. Also, the incubation of the sarcolemmal vesicles with phospholipase A2 and calcium produced a stimulation of adrenochrome formation which was partially suppressed by albumin. In the experiments using arachidonic acid or phospholipase A2, the addition of indomethacin blocked the adrenaline oxidation. These results indicate that arachidonic acid accentuated the heart sarcolemmal adrenochrome formation presumably by participating in the cyclooxygenase reaction.