Biomedical subjects

E A Lissi

Publications and source records attributed to E A Lissi.

At least 19 recordsLinked to original sources

PubMed2001

Evaluation of total reactive antioxidant potential (TRAP) of tissue homogenates and their cytosols.

This study evaluates the possibility of obtaining total reactive antioxidant potential (TRAP) indexes in homogenates and their cytosolic fractions by a procedure based on the quenching of luminol luminescence induced by the thermolysis of 2,2'-azo-bis(2-amidinopropane). Measurements were performed in rat brain, liver, kidney, and heart homogenates. TRAP indexes can be easily determined both in homogenates and their cytosolic fractions. The results obtained indicate that heart homogenates are the least and liver homogenates the most protected of the systems considered. Glutathione is the measured antioxidant that contributes the most to TRAP values, while uric acid makes a significant contribution only in liver. A calculation of theoretical TRAP values from the measured concentrations of the main antioxidants (glutathione, uric acid, ascorbic acid, and alpha-tocopherol) for the different homogenates shows that, in most tissues (liver, brain, and kidney), nearly 50% of the experimentally determined TRAP values are not accounted for. This difference is mainly due to the contribution of proteins to the measured TRAP.

Animals↗

PubMed2001

Kinetics of peroxidation of linoleic acid incorporated into DPPC vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride.

In a previous work [Chem. Phys. Lipids 2000 104, 49], we have derived the following rate law for the oxidation of lipids in compartmentalized systems: R(T)=(k(1)/k(t))(0.5) k(p) [In](0.5) c(0.5) [LH], where, R(T) is the total rate of oxidation, k(1) is the rate constant for the production of free radicals, k(t) and k(p) are the intra-particle rate constants for the termination and propagation sets, respectively, [In] is the concentration of a water-soluble initiator, c is the concentration of particles, and [LH] is the intra-particle concentration of oxidable lipid. In the present work, we have investigated on the applicability of the proposed kinetic rate law for a system where it takes place the oxidation of a reactive lipid incorporated into an inert matrix. With this purpose, we have measured the rate of oxidation of linoleic acid incorporated into dipalmitoylphosphatidylcholine vesicles initiated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) dihydrochloride as a function of the initiator, particles, and intra-particle LH concentrations. The experimentally determined kinetic orders obtained were 0.54+/-0.02, 0.48+/-0.05 and 0.83+/-0.04 for the dependence of the oxidation rate with initiator, particles, and LH intra-particle concentrations, respectively, in agreement with those theoretically predicted. The lower value obtained for the kinetic order in LH is attributed to a change in k(t) with the increase in oxidable lipid intra-particle concentration. The main point to be emphazised from the results here obtained is that the kinetic rate law for the oxidation of lipids in compartmentalized systems can be significantly different than that observed when to the oxidation takes place in homogeneous solution.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Free-radical-induced inactivation of lysozyme and carbonyl residue generation in protein are not necessarily associated.

The 2,2'-azobis(2-amidinopropane) (AAPH)-induced inactivation and oxidative modification of lysozyme, as determined by the loss of tryptophan-associated fluorescence (TAF) and the increase in dinitrophenylhydrazine-reactive carbonyl groups (CO), were studied in the absence and in the presence of antioxidants. AAPH induced a progressive inactivation of the enzyme and a parallel decrease of its TAF. Both changes were closely correlated (R2 = 0.97); however, the inactivation was only partially associated with an increase in CO. The latter reached maximal values at times half those needed to attain maximal losses in both lysozyme activity and TAF. A stoichiometric comparison reveals that whereas over 74% of the enzyme molecules had lost their activity, only 5% exhibited an increment in CO. CO formation was affected differentially by boldine and trolox. Both antioxidants fully protected against the early inactivation and loss of TAF; however, the increase in CO was completely unaffected by trolox. Exposure of lysozyme to Fe3+/ascorbate induced no loss of activity or TAF, but it led to an accumulation of CO similar to that induced by AAPH. Results indicate that CO formation and lysozyme inactivation are two mechanistically dissociable events and that changes in the former parameter can perfectly occur in the absence of changes in the latter.

Amidines↗

PubMed2000

Acid phosphatase reaction with peroxyl radicals: inactivation mechanism and behavior of the partially modified ensemble.

Acid phosphatase (AP) is readily inactivated when exposed to the free radicals generated in the pyrolysis of 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions. On average, a large number of tryptophan groups are modified by each protein molecule that loses its catalytic activity. In spite of this, the enzyme inactivation takes place without induction times, a result that indicates either that damage is progressive or that damage of a critical target is needed to inactivate the enzyme (all-or-nothing mechanism). A Lineweaver-Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that after exposure of the native protein ensemble to the free radical source there are partially damaged molecules whose affinity for the substrate is widely different from that of the native molecules. On the other hand, the partially damaged ensemble shows a normal Michaelis-Menten behavior when the activity is measured at pH 7.0, with only a reduced value of V(M), relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation studies of the native and pretreated proteins support this proposal.

Acid Phosphatase↗

PubMed2000

Kinetics of phycocyanine bilin groups destruction by peroxyl radicals.

Bilin groups in c-phycocyanine are readily bleached by peroxyl radicals produced in the thermolysis of 2, 2'-azobis(2-amidinopropane). From an evaluation of the bilin groups destroyed per radical that interacts with the protein, it is concluded that the bilin moiety is the main target of the radicals. Kinetic expressions are derived that allows an estimation of the substrate reactivity from the analysis of the rate of bilin group modification as a function of the protein concentration. From this analysis it is concluded that micromolar concentrations of c-phycocyanine are able to reduce the steady state concentration of the peroxyl radicals by one half, indicating a high antioxidant activity for this compound. This conclusion is confirmed by measuring the capacity of the protein to protect 1-naphthol from modification by peroxyl radicals. The results obtained show that the bilin groups have, on a molar basis, an antioxidant activity similar to that of potent antioxidants such as catechin.

Amidines↗

PubMed2000

Kinetics of lipid peroxidation in compartmentalized systems initiated by a water-soluble free radical source.

Kinetic rate laws arising from theoretical expectations for the oxidation of lipids initiated by water-soluble free radicals in compartmentalized systems under different experimental conditions are deduced. In particular, the predictions for the kinetic reaction orders in: (a) intra-particle oxidizable compound concentration (at fixed number of particles and particle size), alpha; (b) number of particles or analytical lipid concentration (at fixed intra-particle concentration and particle size), beta and (c) initiator, gamma, are obtained. The reaction orders beta and gamma are determined by the fraction of initiator derived radicals captured by the particles (f) and the mean number of chain carrying radicals per particle ( ) when the system reaches the steady state condition. Predicted orders in initiator range from 0 ( = 0.5) to 0.5 (f-->1; > > 1), while the order in number of particles ranges between 0.5 (f-->1; > > 1) and 1. These predictions are tested by measuring the kinetic law for the oxidation of SUV's egg yolk phosphatidylcholine vesicles initiated by the thermal decomposition of ABAP. The results indicate that, under the conditions employed, beta = 0.68 +/- 0.05 and gamma = 0.46 +/- 0.04. These values are close to those expected for a system in which > > 1 and the efficiency of capture is relatively high. This last condition is confirmed by estimating the efficiency of capture from a comparison of induction times elicited by similar concentrations of Trolox and alpha-tocopherol.

Amidines↗

PubMed1999

Kinetics and mechanism of St I modification by peroxyl radicals.

St I is a toxin present in the Caribbean Sea anemone Stichodactyla helianthus which is highly hemolytic in the nanomolar concentration range. Exposure of the toxin to free radicals produced in the pyrolysis of 2,2'-azobis(2-amidinopropane) hydrochloride leads to a progressive loss of hemolytic activity. This loss of hemolytic activity is accompanied by extensive modification of tryptophan residues. On the average, three tryptophan residues are modified by each inactivated toxin. The loss of hemolytic activity of St I takes place without significant changes in the protein structure, as evidenced by the similarity of the fluorescence and CD spectra of native and modified proteins. Also, the native and modified ensembles present a similar resistance to their denaturation by guanidinium chloride. The hemolytic behavior and the performance of the toxin at the single-channel level when incorporated to black lipid membranes suggest that the modified ensemble can be considered as composed of inactive toxins and active toxins whose behavior is similar to that of the native proteins. These results, together with the lack of induction time in the activity loss, suggest that the fall of hemolytic activity takes place by an all-or-nothing inactivation mechanism in which the molecules become inactive when a critical amino acid residue is modified.

Amino Acids↗

PubMed1999

Total antioxidant potential of resinous exudates from Heliotropium species, and a comparison of the ABTS and DPPH methods.

Total reactive antioxidant potential (TRAP) of resinous exudates from Heliotropium species was evaluated by measuring the bleaching of stable free radicals. The antioxidant capacity of the resinous exudates in Trolox equivalents, evaluated from the bleaching of ABTS derived radical cations, ranged from 2.0 M (H. huascoense) to 5.2 M (H. stenophyllum), indicating a very high concentration of phenolic compounds. Considerably smaller values were obtained by measuring the bleaching of DPPH radicals. The ratio between the values obtained employing ABTS derived radicals and DPPH, ranged from 37 (H. megalanthum) to 4.5 (H. chenopodiaceum variety typica). The magnitude of the difference can be considered as an indication of the relative reactivity of the antioxidants present in the exudates. Similar ratios were observed when stoichiometric coefficients were evaluated for representative purified flavonoids obtained from the resinous exudates.

Antioxidants↗

PubMed1999

Total reactive antioxidant potential in human saliva of smokers and non-smokers.

Uric acid is the most important non-enzymatic antioxidant present in human saliva. There is a great variability among individuals, both in salivary uric acid content and saliva total reactive antioxidant potential (TRAP). The uric acid present in saliva correlates with plasma uric acid, suggesting that the former is imported from plasma. There are not statistical differences between uric acid or TRAP values in saliva of smokers and non-smokers. Also, smoking a cigarette does not modify the levels of antioxidants present in saliva.

Adult↗

PubMed1998

Synthesis of a new neoglycolipid (AgH-1) and its effect upon the properties of dipalmitoylphosphatidylcholine: cholesterol liposomes.

A new neoglycolipid (AgH-1) bearing carbohydrate units that mimics the antigenic determinant of the O-blood group was synthesized and the effect of its incorporation in dipalmitoylphosphatidylcholine (DPPC): cholesterol liposomes was evaluated. The results obtained show that AgH-1 is readily incorporated into DPPC:cholesterol liposomes. The conditions leading to the optimal incorporation are the result of a compromise between incorporation efficiency and incorporation extent. The presence of AgH-1 produces liposomes of smaller size, with only small changes in the properties of the bilayer. However, the data obtained employing diphenylhexatriene and laurodan as fluorescence probes and merocyanine 540 as optical probe suggest that AgH-1 incorporation leads to a small rigidization of the liposomes at temperatures lower than ca. 42 degrees C.

Blood Group Antigens↗

PubMed1998

The role of ionic strength on the enhancement of the hemolytic activity of sticholysin I, a cytolysin from Stichodactyla helianthus.

Sticholysin I (St I) is a potent cytolytic polypeptide purified from the Caribbean sea anemone Stichodactyla helianthus. The hemolytic activity of sticholysin is potentiated by its preincubation at high ionic strengths. In the present work the mechanism of the potentiating action of the medium ionic strength on the toxin hemolytic capacity is investigated. It is suggested that preincubation with high saline concentration induces a transition of St I to a more relaxed conformation that facilitates the lytic process.

Animals↗

PubMed1998

Modification of sticholysin II hemolytic activity by free radicals.

Sticholysin II is a highly hemolytic toxin present in the caribbean sea anemone Stichodactyla helianthus. Pre-incubation of St II with 2,2'-azobis(2-amidinopropane), a source of peroxyl radicals in air saturated solution, readily reduces its hemolytic activity. Analysis of the amino acids present in the protein after its modification shows that only tryptophan groups are significantly modified by the free radicals. According to this, the loss of hemolytic activity correlates with the loss of the protein intrinsic fluorescence. The results indicate that, at high toxin concentrations, nearly a tryptophan residue and 0.2 toxin molecules are inactivated by each radical introduced into the system. Association of St II to multilamellar liposomes (egg yolk phosphatidyl choline:sphingomyelin 1:1) increases the toxin intrinsic fluorescence, indicating a more hydrophobic average environment of the five tryptophan groups of the protein. In agreement with this, incorporation of St II to the liposomes reduces the rate of fluorescence loss during its modification by free radicals, particularly at long incubation times. These results are explained in terms of two populations of tryptophans that are quenched at different rates by acrylamide and whose rates of inactivation by free radicals are also different.

Acrylamide↗

PubMed1997

In situ rat brain and liver spontaneous chemiluminescence after acute ethanol intake.

The influence of acute ethanol administration on the oxidative stress status of rat brain and liver was assessed by in situ spontaneous organ chemiluminescence (CL). Brain and liver CL was significantly increased after acute ethanol administration to fed rats, a response that is time-dependent and evidenced at doses higher than 1 g/kg. Ethanol-induced CL development is faster in liver compared with brain probably due to the greater ethanol metabolic capacity of the liver, whereas the net enhancement in brain light emission at 3 h after ethanol treatment is higher than that of the liver, which could reflect the greater susceptibility of brain to oxidative stress. The effect of ethanol on brain and liver CL seems to be mediated by acetaldehyde, due to its abolishment by the alcohol dehydrogenase inhibitor 4-methylpyrazole and exacerbation by the aldehyde dehydrogenase inhibitor disulfiram. In brain, these findings were observed in the absence of changes in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase. However, the content of brain glutathione was significantly decreased by 31%, by ethanol, thus establishing an enhanced oxidative stress in this tissue.

Alcohol Deterrents↗

PubMed1997

Protein degradation in red cells exposed to 2,2'-azo-bis(2-amidinopropane) derived radicals.

Extensive proteolysis is observed when red blood cells are exposed to free radicals produced in the thermolysis of 2,2'-azo-bis(2-amidinopropane). It is evaluated that nearly one amino terminal group is produced by each free radical introduced into the system. These groups are considered to arise mainly from band 3 fragmentation due to the action of red cell proteinases. Protein fragmentation takes place prior to significant hemolysis or lipid peroxidation, as evaluated by thiobarbituric acid-reactive substances measurements.

Amidines↗

PubMed1996

Surface changes induced by osmotic shrinkage on large unilamellar vesicles.

Osmotic shrinkage changes the surface properties of dipalmitoylphosphatidylcholine large unilamellar vesicles depending on the phase state of the bilayer. In the gel state, shrinkage produces an increase in the adsorption of hydrophobic dyes, such as Merocyanine 540 (MC540) monomers, toluidine and anilinonaphthalene sulfonic acid (TNS, ANS). In the fluid state, shrinkage does not affect the bilayer surface when gradients between the inner and the outer compartments below 0.2-0.25 M NaCl (higher concentration outside) are applied. Larger differences in concentrations produce an increase in packing as inferred from the desorption of the MC monomers. Kinetic experiments show that the surface changes correlate with the volume decrease produced by the water extrusion from the vesicle interior. It is interpreted that the decrease of water content compels the vesicles to a state in which defects at the membrane surface are likely to occur when the bilayer is in the gel state.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Sod and catalase inactivation by singlet oxygen and peroxyl radicals.

Both superoxide dismutase and catalase are readily deactivated by singlet oxygen and by the radicals produced in the pyrolysis of 2,2'-azo-bis-(2-amidinpropano) under aerobic conditions. The rate constant for the loss of enzymatic activity induced by singlet oxygen are 3.9 x 10(7) and 2.5 x 10(7) M-1 sec-1 for SOD and catalase, respectively. The similarity between these values implies that in systems where SOD and catalase are exposed to similar singlet oxygen concentrations, it can be expected a parallel inactivation of both enzymes. The inactivation of both enzymes by the radicals produced by 2,2'-azo-bis-(2-amidinopropane) pyrolysis under aerobic conditions follows a first-order kinetics at low enzyme concentrations and a zero-order kinetics at higher concentrations. Although at low enzymatic concentrations the rate of inactivation of both enzymes is similar, this results from a compensation of effects because there are wide differences in the reactivity of both enzymes towards peroxyalkyl radicals. Catalase is considerably more reactive, but a large number of protein/radical reactive interactions are needed to inactivate one enzyme. On the other hand, the reactivity of SOD is smaller, but the average enzyme activity decreases by nearly 20% in each SOD/radical reactive interaction.

Animals↗

PubMed1996

The reaction between ABTS radical cation and antioxidants and its use to evaluate the antioxidant status of serum samples.

The 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation can be generated by incubation of ABTS and 2,2'-azo-bis(2- amidinopropane) at 45 degrees C. The ABTS radical cation is stable for several minutes at room temperature and reacts quantitatively and instantaneously with several antioxidants, such as Trolox, ascorbic acid, uric acid, cysteine, glutathione and bilirubin. In contrast, the ABTS radical cation reacts slowly with albumin. When serum is added to a solution of the ABTS radical cation, the bleaching of the radical follows biphasic kinetics, with a fast decay followed by a slow decay that takes place within several minutes. The fast decay is primarily due to uric acid, while the slow decay is related to the protein content of the sample. We propose that this procedure can provide an independent and simultaneous evaluation of the low molecular weight and protein antioxidants present in biological samples such as serum.

Antioxidants↗

PubMed1995

Structure-antioxidative activity relationships in benzylisoquinoline alkaloids.

The antioxidative properties of the aporphines boldine, glaucine and apomorphine, and of the benzyltetrahydroisoquinolines (+/-)-coclaurine and (+/-)-norarmepavine were compared in the brain homogenate autoxidation model. The IC50 values found lay in the 16-20 microM range for the aporphines and were 131.7 microM, and 79.3 microM for coclaurine and norarmepavine, respectively. These results indicate that the antioxidative capacity (AC) of these compounds is related to the presence of the biphenyl system rather than phenol groups. The non-phenolic glaucine inhibited the 2,2'-azobis-(2-amidinopropane)(AAP)-induced inactivation of lysozyme with an IC50 value of 12 microM, while the corresponding values for the phenolic coclaurine and norarmepavine were 10 and 20 microM, respectively. N-Methylation of glaucine to its quaternary ammonium reduced its protective effect by two-thirds. This result suggests that a benzylic hydrogen neighbouring a nitrogen lone electron pair may be the key to the protective effect of non-phenolic aporphines.

Animals↗