Biomedical subjects

José L F C Lima

Publications and source records attributed to José L F C Lima.

At least 19 recordsLinked to original sources

PubMed2007

New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen.

In the present study, a new fluorescence microplate screening assay for evaluating scavenging activity against singlet oxygen (1O2) was implemented. The chemical generation of 1O2 was promoted using the thermodissociable endoperoxide of disodium 3,3'-(1,4-naphthalene)bispropionate (NDPO2). The detection of 1O2 was achieved using dihydrorhodamine 123 (DHR), a nonfluorescent molecule that is oxidizable to the fluorescent form rhodamine 123 (RH). The combined use of a 1O2-selective generator and a highly sensitive probe (DHR) was then successfully applied to perform a screening assay of the 1O2 scavenging activities of ascorbic acid, penicillamine, cysteine, N-acetylcysteine (NAC), methionine, reduced glutathione (GSH), dihydrolipoic acid, lipoic acid, and sodium azide. All of these antioxidants exhibited concentration-dependent 1O2 scavenging capacities. They could be ranked according to observed activity: ascorbic acid>cysteine>penicillamine>dihydrolipoic acid>GSH>NAC>sodium azide>lipoic acid (IC50 values of 3.0+/-0.2, 8.0+/-0.7, 10.9+/-0.8, 25.2+/-4.5, 57.4+/-5.9, 138+/-13, 1124+/-128, 2775+/-359 microM, mean+/-SEM, respectively)>methionine (35% of scavenging effect at 10 mM). In conclusion, the use of NDPO2 as a selective generator for 1O2 and its fluorescence detection by the highly sensitive probe DHR is shown to be a reliable and resourceful analytical alternative means to implement a microplate screening assay for scavenging activity against 1O2.

Antioxidants↗

PubMed2006

Automatic method for the determination of Folin-Ciocalteu reducing capacity in food products.

In the present work, an automatic flow procedure based on multi-syringe flow injection analysis was developed for the assessment of Folin-Ciocalteu reagent (FCR) reducing capacity in several types of food products using gallic acid as the standard. Different strategies for mixing of sample and reagent were tested (continuous flow of FCR, merging zones, and intercalated zones approaches); lower reagent consumption and higher determination throughput were attained for the merging zones approach (100 microL of sample+100 microL of FCR). The application of the proposed method to compounds with known antioxidant activity (both phenolic and nonphenolic) and to samples (wines, beers, teas, soft drinks, and fruit juices) provided results similar to those obtained by the conventional batch method. The detection limit was 0.6 mg L-1, and the determination frequency was about 12 h-1. Good repeatability was attained (RSD<1.3%, n=10).

Antioxidants↗

PubMed2006

Kinetic enzymatic determination of glycerol in wine and beer using a sequential injection system with spectrophotometric detection.

A sequential injection system for the automatic determination of glycerol in wine and beer was developed. The method is based on the rate of formation of NADH from the reaction of glycerol and NAD+ catalyzed by the enzyme glycerol dehydrogenase in solution. The determination of glycerol was performed between 0.3 and 3.0 mmol L(-1) (0.028 and 0.276 g L(-1)), and good repeatability was attained (rsd < 3.6%, n = 5) for all samples tested. The determination rate was 54 h(-1), the reagent consumption was only 0.75 micromol of NAD+ and 5.4 ng of enzyme per assay, and the waste production was 2.12 mL per assay. Results obtained for samples were in agreement with those obtained with the batch enzymatic method.

Beer↗

PubMed2006

Exploiting gas diffusion for non-invasive sampling in flow analysis: determination of ethanol in alcoholic beverages.

A tubular gas diffusion PTFE membrane is exploited for non-invasive sampling in flow analysis, aiming to develop an improved spectrophotometric determination of ethanol in alcoholic beverages. The probe is immersed into the sample, allowing ethanol to diffuse through the membrane. It is collected into the acceptor stream (acidic dichromate solution), leading to formation of Cr(III), monitored at 600 nm. The analytical curve is linear up to 50% (v/v) ethanol, baseline drift is < 0.005 absorbance during four working-hours, and sample throughput is 30 h(-1), meaning 0.6 mmol K2Cr2O7 per determination. Results are precise (r.s.d.< 2%) and in agreement with an official procedure.

Alcoholic Beverages↗

PubMed2006

Antioxidant activity of beta-blockers: an effect mediated by scavenging reactive oxygen and nitrogen species?

The therapeutic effects of beta-blockers are normally explained by their capacity to block the beta-adrenoceptors, however, some of the beneficial cardiovascular effects shown by this group of compounds have already been associated with the antioxidant properties that some of them seem to possess. The beta-blockers atenolol, labetalol, metoprolol, pindolol, propranolol, sotalol, timolol, and carvedilol were tested for their putative scavenging activity for ROS (O(2)(-), H(2)O(2), HO(.), HOCl, and ROO(.)) and RNS ((.)NO and ONOO(-)). Some of the studied compounds are effective ROS and/or RNS scavengers, these effects being possibly useful in preventing oxidative damage verified in hypertension as well as in other cardiovascular diseases that frequently emerge in association with oxidative stress.

Adrenergic beta-Antagonists↗

PubMed2006

Fluidized beds in flow analysis: use with ion-exchange separation for spectrophotometric determination of zinc in plant digests.

A novel strategy for utilization of solid reagents in flow analysis is proposed. Establishment of diffuse and reproducible geometry enables the solid particles to be maintained in constant floating, reflux, and circulating motion inside a mini-chamber. This is efficiently accomplished with pulsed flows, a characteristic of multi-pumping flow systems. Drawbacks inherent in solid-phase packed columns, for example backpressure, preferential pathways, swelling, etc., and some limitations inherent in immobilized reagents are minimised. Spectrophotometric determination of zinc in plants was selected as an application of the technique. Dowex 1-X8 anionic resin was kept freely inside a mini-chamber. Zinc chloro-complexes were adsorbed on the moving particles and derivatization with zincon was performed after elution. Analytical figures of merit and the potential and limitations of the approach are discussed.

Chromatography, Ion Exchange↗

PubMed2006

Use of fluorescence probes for detection of reactive nitrogen species: a review.

The biological and toxicological effects that have been attributed to reactive nitrogen species (RNS) are increasingly stirring the scientific inquisitiveness about the molecular mechanisms involved. However, RNS present some characteristics that complicate their detection, namely their short lifetime and the normal presence of a variety of endogenous compounds capable of reacting with these reactive species, when the studies are performed in biological matrices. The development of methodologies capable of circumvent these difficulties is thus of fundamental importance. Fluorescence probes are particularly important due to their high sensibility and usefulness in temporal and spatial monitoring of RNS, particularly in microanalysis conditions in biological media akin to cells or tissues. In the present review is given an account of the fluorescence probes that have been used for detection of nitric oxide (*NO), peroxynitrite anion (ONOO-), as well as of some of its derivatives in biological and nonbiological media.

Animals↗

PubMed2006

Antioxidant profile of dihydroxy- and trihydroxyphenolic acids--a structure-activity relationship study.

Eight structurally similar dihydroxy and trihydroxyphenolic acids (protocatechuic acid, 3,4-dihydroxyphenylacetic acid, hydrocaffeic acid, caffeic acid, gallic acid, 3,4,5-trihydroxyphenylacetic acid, 3-(3,4,5-trihydroxyphenyl)propanoic acid and 3-(3,4,5-trihydroxyphenyl)propenoic acid) were examined for their total antioxidant capacity (TAC). Furthermore, their ability to scavenge peroxyl radicals, generated by AAPH in liposomes, was determined. The antioxidant/pro-oxidant activity of the compounds was screened using the 2'-deoxyguanosine assay. All compounds behave as radical scavengers, with 3,4,5-trihydroxyphenylacetic acid being the most potent. Nevertheless, in the lipid peroxidation assay an inverse ranking order was observed, 3,4-dihydroxyphenylacetic acid being the most effective compound. All the dihydroxylated compounds showed a pro-oxidant behaviour leading to an increase of 50% in 8-OH-dG induction. From the structure-antioxidant activity relationship studies performed it may be concluded that the number of phenolic groups and the type of the alkyl spacer between the carboxylic acid and the aromatic ring strongly influence the antioxidant activity.

Antioxidants↗

PubMed2006

Interactions between oxicams and membrane bilayers: an explanation for their different COX selectivity.

Meloxicam was launched as a major new NSAID for the treatment of arthritis following extensive published research confirming its selectivity for COX-2. Several studies proposed possible explanations for its effectiveness and superior safety profile. The proposed theories included chemical structural relationships between meloxicam and other effective NSAIDs with low gastrointestinal toxic effects. However, other oxicams have similar chemical groups, but despite this, are not considered COX-2 selective drugs and exhibit less gastric tolerance. Hence, the aim of this work was to investigate the interactions between oxicams and biomembrane models as it could influence their resorption from the upper gastrointestinal tract and may affect their local gastromucosal tolerability. The partition of oxicams within membranes was determined by calculating their partition coefficients between liposomes and water. Moreover, their location within the bilayer was determined by fluorescence quenching. Finally, zeta-potential measurements were made to complete the information about the binding behaviour of the oxicams and steady-state anisotropy measurements were made to determine their induced perturbation in membrane structure. These studies proved that, in spite of structural similarities, oxicams present different interactions with membranes making possible a virtual division of the class in two groups. Tenoxicam and piroxicam known as COX-1 inhibitors demonstrated higher partition capacity in liposomes/water systems together with a smaller ability to change the membrane fluidity and surface potential. In contrast lornoxicam and meloxicam, which demonstrated activity against COX-2, have revealed smaller partition capacity in liposomes/water systems together with a higher ability to change the membrane fluidity and surface potential.

Anisotropy↗

PubMed2005

Fluorescence probes used for detection of reactive oxygen species.

Endogenously produced pro-oxidant reactive species are essential to life, being involved in several biological functions. However, when overproduced (e.g. due to exogenous stimulation), or when the levels of antioxidants become severely depleted, these reactive species become highly harmful, causing oxidative stress through the oxidation of biomolecules, leading to cellular damage that may become irreversible and cause cell death. The scientific research in the field of reactive oxygen species (ROS) associated biological functions and/or deleterious effects is continuously requiring new sensitive and specific tools in order to enable a deeper insight on its action mechanisms. However, reactive species present some characteristics that make them difficult to detect, namely their very short lifetime and the variety of antioxidants existing in vivo, capable of capturing these reactive species. It is, therefore, essential to develop methodologies capable of overcoming this type of obstacles. Fluorescent probes are excellent sensors of ROS due to their high sensitivity, simplicity in data collection, and high spatial resolution in microscopic imaging techniques. Hence, the main goal of the present paper is to review the fluorescence methodologies that have been used for detecting ROS in biological and non-biological media.

Animals↗

PubMed2005

Inhibition of human neutrophil oxidative burst by pyrazolone derivatives.

The risk of agranulocytosis associated with the use of pyrazolone drugs at therapeutical doses and for short periods of time has been considered to be very low. However, little or no attention at all has been devoted to the possible hindrance of neutrophil burst and scavenging of neutrophil-generated reactive oxygen species (ROS) by these compounds. Such an effect could be beneficial in the case of overactivation of neutrophils but could also be highly detrimental if the number of circulating neutrophils is already decreased. Thus, the aim of the present study was to evaluate the putative inhibitory effect of the pyrazolones dipyrone, aminopyrine, isopropylantipyrine, and antipyrine against human neutrophil burst and their scavenging activity against O2.-, H2O2, HO., ROO., and HOCl. The obtained results showed that dipyrone and aminopyrine prevent phorbol-12-myristate-13-acetate-induced neutrophil burst with high efficiency, while isopropylantipyrine had little effect and antipyrine had no effect at all. Dipyrone and aminopyrine were highly potent scavengers of HO. and HOCl, while, in accordance with the neutrophil burst results, isopropylantipyrine had little effect and antipyrine had no effect at all against these two ROS. None of the studied pyrazolones was capable of scavenging O2.- or H2O2, while dipyrone was shown to be the most reactive against ROO..

Aminopyrine↗

PubMed2005

Structure-property studies on the antioxidant activity of flavonoids present in diet.

The screening of natural flavonoids for their bioactivity as antioxidants is usually carried out by determinination of their profile as chain-breaking antioxidants, by the evaluation of their direct free radical-scavenging activity as hydrogen- or electron-donating compounds. Since this may not be the only mechanism underlying the antioxidant activity it is important to check the ability of these compounds to act as chelators of transition metal ions. Accordingly, in the present study the acidity constants of catechin and taxifolin, as well as the formation constants of the corresponding copper (II) complexes, were investigated by potentiometry and/or spectrophotometry. Moreover, a detailed quantitative examination of the coordination species formed is presented. In addition, the partition coefficients of both catechin and taxifolin in a biomimetic system (micelles) were determined, since these properties may also contribute to the antioxidant behavior of this type of compound. The log P values determined depend on the electrostatic interactions of the compounds with the differently charged micelles (the highest values were obtained for zwitterionic and cationic micelles). The prooxidant behavior of the compounds was assessed through the oxidation of 2'-deoxyguanosine, induced by a Fenton reaction, catalyzed by copper. The data obtained reveal that the flavonoids under study did not present prooxidant activity, in this particular system. The results obtained are evidence of a clear difference among the pKa, the complexation properties, and the lipophilicity of the flavonoids studied, which can partially explain their distinct antioxidant activity. The most stable geometries of the free compounds were determined by theoretical (ab initio) methods, in order to properly account for the electron correlation effects which occur in these systems, thus allowing a better interpretation of the experimental data.

Antioxidants↗

PubMed2005

Evaluation of the total antioxidant capacity by using a multipumping flow system with chemiluminescent detection.

An automated flow-based procedure for assessment of total antioxidant capacity was developed. It involved a multipumping flow system, a recent approach to flow analysis, and exploited the ability of selected compounds to inhibit the chemiluminescence reactions of luminol or lucigenin with hydrogen peroxide. The system included several discretely actuated solenoid micropumps as the only active components of the flow manifold. This enabled the reproducible insertion and efficient mixing of very low volumes of sample and reagents as well as the transportation of the sample zone toward a flow-through luminometer, where the chemiluminometric response was monitored. With luminol as the chemiluminogenic reagent, linearity of the analytical curves was noted up to 3.2x10(-4), 1.1x10(-3), and 8.8x10(-8) molL-1 for Trolox, ascorbic acid, and resveratrol, respectively. With lucigenin, linear calibration plots up to 2x10(-5) molL-1 of Trolox and 5.7x10(-5)molL-1 of ascorbic acid were obtained. As favorable analytical figures of merit, the measurement precision (RSD typically between 0.2 and 2.0%, n=10), low operational costs, low reagent consumption, sampling rate (160 and 70 h-1), and versatility should be highlighted. The proposed system can be used in distinct analytical circumstances without requiring physical reconfiguration.

Antioxidants↗

PubMed2005

Pindolol is a potent scavenger of reactive nitrogen species.

Pindolol is an indolic drug that has been shown to enhance and/or accelerate selective serotonin specific reuptake inhibitors (SSRI)-induced antidepressant (AD) effect, even though the respective mechanism is still unclear. It has been demonstrated that inhibition of nitric oxide (*NO) synthesis in CNS produces anxiolytic and AD-like behavioural effects in a variety of animal paradigms. On the other hand, sustained high levels of *NO may be deleterious to CNS, predominantly due to the formation of peroxynitrite anion (ONOO-), which is generated via reaction of *NO with superoxide radical (O2*-). Therefore, the purpose of the present study was to characterize the putative pindolol scavenging effect on *NO, ONOO-, and O2*-, using in vitro non-cellular systems. The obtained results clearly show that pindolol is a potent scavenger of *NO (IC50 of 449+/-33 microM) and ONOO- (IC50 of 131+/-24 microM). Additionally, the scavenging effect of pindolol increased almost 8 times in the presence of 25 mM NaHCO3 (IC50 of 17+/-3 microM), which indicates that pindolol efficiently scavenges reactive species that are produced from the ONOO-/CO2 reaction such as the nitrogen dioxide radical (*NO2) and the carbonate radical anion (CO3*-). These effects may contribute for the reduction of SSRI antidepressant latency that has been attributed to pindolol and may also constitute an additional value for this drug when depression is associated with pro-oxidant neurodegenerative diseases.

Antidepressive Agents, Second-Generation↗

PubMed2005

Effects of diclofenac on EPC liposome membrane properties.

In this work the interaction of a non-steroidal anti-inflammatory drug (NSAID), diclofenac, with egg yolk phosphatidylcoline (EPC) liposomes, used as cell-membrane models, was quantified by determination of the partition coefficient. The liposome/aqueous phase partition coefficient was determined by derivative spectrophotometry, fluorescence quenching, and measurement of zeta-potential. Theoretical models based on simple partition of the diclofenac between two different media, were used to fit the experimental data, enabling the determination of K(p). The three techniques used yielded similar results. The effects of the interaction on the membrane's characteristics were further evaluated, either by studying membrane potential changes or by effects on membrane fluidity. The liposome membrane potential and the size and size-homogeneity of liposomes were measured by light scattering. The effects of diclofenac on the internal viscosity or fluidity of the membrane were determined by use of spectroscopic probes-a series of n-(9-anthroyloxy) fatty acids in which the carboxyl terminal group is located at the interfacial region of the membrane and the fluorescent anthracene group is attached at different positions along the fatty acid chain. The location of the diclofenac on the membrane was also evaluated, by fluorescence quenching using the same series of fluorescent probes. Because the fluorescent anthracene group is attached at different positions along the fatty acid chain, it is possible to label at a graded series of depths in the bilayer. The interactions between the drug and the probe are a means of predicting the location of the drug on the membrane.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2005

Chemiluminometric determination of propranolol in an automated multicommutated flow system.

In this work, a fully automated analytical methodology for the chemiluminometric determination of propranolol hydrochloride in pharmaceutical preparations is proposed. The developed procedure was based on the oxidation of propranolol by potassium permanganate in acidic medium and was implemented in a multicommutated flow system. The combined automated actuation of syringe pumps and two-way solenoid valves, under computer control, assured a high versatility in terms of sample and reagent manipulation and an effective run-time control of all analytical parameters, including a time-based insertion that enabled a significant solutions saving and the exploitation of different strategies for sample/reagent mixing. The calibration graph was linear over the range 20-150 mg l(-1) of propranolol hydrochloride with a relative standard deviation lower than 1.6% (n = 7). The results were in agreement with those obtained by the reference procedure with a relative deviation between -3.8 and 3.7% and a sampling rate of about 27 samples h(-1).

Autoanalysis↗

PubMed2005

Effect of anti-inflammatory drugs on splenocyte membrane fluidity.

In this study, fluorescence anisotropy measurements were performed using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene to investigate the effects on membrane fluidity resulting from the interaction between nonsteroidal anti-inflammatory drugs (NSAIDs)-indomethacin, diclofenac, piroxicam, tenoxicam, indoprofen, clonixin, and etodolac-and mouse splenocyte membranes. This study was performed in splenocyte membranes because most of the fluidity studies have been performed in membrane models; thus, clear correlations of the pharmacological action of drugs with molecular effects at the cellular membrane level were lacking. Besides providing a basis for studying the molecular mechanism of pharmacological action of NSAIDs, this research provides a data analysis of steady-state anisotropy measurements, taking into account that the probe itself strongly influences the data given that this problem is usually overlooked. Results show that the anti-inflammatory drugs indomethacin, diclofenac, piroxicam, and tenoxicam increase the membrane fluidity in a concentration-dependent manner. Their order of effectiveness reflected in their respective IC50 values (concentration of each NSAID required to increase the fluidizing effect ratio by 50%) is as follows: tenoxicam>piroxicam>indomethacin>clonixin. For the other drugs, the perturbation in membrane fluidity is not evident under these circumstances.

Animals↗

PubMed2005

A catalytic multi-pumping flow system for the chemiluminometric determination of metformin.

A multi-pumping flow system for the chemiluminometric determination of the hypoglycaemic drug metformin was implemented. The developed methodology was based on the metformin-induced inhibition (metformin acts as a Cu(II) scavenger) of the catalytic effect of Cu(II) ions on the chemiluminescent reaction between luminol and hydrogen peroxide. The flow manifold configuration was based on the utilisation of multiple solenoid-actuated micro-pumps that were simultaneously accountable for sample/reagent introduction and reaction zone formation/propulsion, thus resulting in a fully automated, simple and highly selective multi-pumping flow system. A versatile sample manipulation allowed the establishment of distinct sampling strategies with low reagent consumption. The characteristic pulsed flow ensured an effective sample/reagent mixing leading to a better and faster reaction zone homogenisation and thus improved analytical signals. Linear calibration plots were obtained for metformin hydrochloride concentrations ranging from 5 to 15 mg L(-1) with a relative standard deviation lower than 2.0% (n=5). Detection limit was 0.94 mg L(-1), and the sampling rate was about 95 determinations per hour. The developed methodology was applied to the analysis of pharmaceutical formulations and the obtained results were in agreement with those furnished by the reference method with relative percentage deviations of lower than 1.5%.

Catalysis↗