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Biomedical subjects

R Zamora

Publications and source records attributed to R Zamora.

At least 37 records · Page 2Linked to original sources
PubMed1999

Determination of epsilon-N-pyrrolylnorleucine in fresh food products.

epsilon-N-Pyrrolylnorleucine was determined in different fresh food products to study its presence as a normal component of food proteins. Twenty-two different products were screened: cod, cuttlefish, salmon, sardine, trout, beef, chicken, pork, broad bean, broccoli, chickpea, garlic, green pea, lentil, mushroom, soybean, spinach, sunflower, almond, hazelnut, peanut, and walnut. Foods were homogenized, their proteins were precipitated with trichloroacetic acid and hydrolyzed with 2 N NaOH for 20 h, and the epsilon-N-pyrrolylnorleucine content was determined by capillary electrophoresis. The epsilon-N-pyrrolylnorleucine, which was identified by HPLC/MS in sardine muscle hydrolysate, ranged in the 22 foods analyzed from 0.24 to 6.36 micromol/g. This concentration was correlated with the protein content of the food (r = 0.687, p = 0.00041). In addition, the epsilon-N-pyrrolylnorleucine/lysine ratio was found to be a function of the lipid, iron, and protein contents of the food (r = 0.881, p < 0.0001) and was directly correlated with lipid and iron contents and inversely correlated with the protein content. These results are in agreement with the oxidative stress origin proposed for epsilon-N-pyrrolylnorleucine and suggest that the epsilon-N-pyrrolylnorleucine/lysine ratio is a characteristic of each food. In addition, epsilon-N-pyrrolylnorleucine seemed to be a normal component of many fresh food products, in which it may be acting as a natural antioxidant.

Animals↗
PubMed1999

Modification of histidine residues by 4,5-epoxy-2-alkenals.

The reactions of 4,5(E)-epoxy-2(E)-heptenal with 4-methylimidazole and N(alpha)-acetyl-L-histidine methyl ester were studied to characterize the adducts produced in the modification of histidine residues by epoxyalkenals and to develop a methodology for the determination of these adducts in protein hydrolysates. The reaction products, which were isolated and characterized, resulted in the Michael adducts produced in the addition of one of the imidazolic nitrogens to the carbon-carbon double bond of the epoxyalkenal. Only some of the theoretical isomers were produced. Thus, in the reaction with 4-methylimidazole, the main product was 4, 5-epoxy-3-(4-methylimidazol-1-yl)heptanol (88%), although the formation of 4,5-epoxy-3-(5-methylimidazol-1-yl)heptanol (12%) was also observed. On the other hand, the reaction with N(alpha)-acetyl-L-histidine methyl ester produced exclusively N(alpha)-acetyl-1-[1'-(1' ',2' '-epoxybutyl)-3'-hydroxypropyl]-L-histidine methyl ester. This last compound was used to develop a procedure for the determination of 4, 5(E)-epoxy-2(E)-heptenal-histidine adducts in protein hydrolysates. When this procedure was applied to the analysis of bovine serum albumin treated with 0.01-10 mM 4,5(E)-epoxy-2(E)-heptenal, the formation of the adduct was observed and its concentration increased with the concentration of the aldehyde and the incubation time, and was parallel to the histidine losses observed in the protein after acid hydrolysis as well as to the formation of protein carbonyls. In addition, the number of histidine residues lost in the protein was very similar to the number of adduct residues produced, suggesting that the addition reaction is the major mechanism for histidine losses suffered by proteins following their reaction with epoxyalkenals.

Aldehydes↗
PubMed1998

A spectrophotometric method for the determination of proteins damaged by oxidized lipids.

The Ehrlich reaction was optimized to determine pyrrolized proteins produced as a consequence of lipid peroxidation and oxidative stress. The procedure consisted of the treatment of the modified protein with p-(dimethylamino)benzaldehyde at a controlled acidity and temperature, and the determination of adducts produced against the blank obtained in the absence of the reagent. The extinction coefficient of Ehrlich adducts was calculated by using epsilon-N-pyrrolylnorleucine (Pnl) as standard and was 35,000 M-1 cm-1. The response was linear and reproducible within the range 0. 16-20 microM Pnl. The assay was applied to determination of pyrrole content in bovine serum albumin, bovine alpha-globulins, bovine gamma-globulins, and mixtures of them, incubated overnight with 1 mM of 4,5(E)-epoxy-2(E)-heptenal, obtaining results similar to those from determination of Pnl by capillary electrophoresis after basic hydrolysis of the protein. The method was also applied to pyrrole determination in bovine plasma proteins either incubated with epoxyalkenals, hydroxyalkenals, lipid hydroperoxides, and secondary products of lipid peroxidation, or oxidized with Fe3+/ascorbate. All these treatments produced pyrrolization of plasma proteins and all Ehrlich adducts gave very similar absorbance spectra with the exception of that produced in the treatment with hydroxyalkenals. The above results suggest that protein pyrrolization is a normal consequence of the lipid peroxidation process and of oxidative stress, and that Ehrlich adducts may be valid to determine this pyrrolization.

Aldehydes↗
PubMed1998

The role of prostaglandin E2 and nitric oxide in cell death in J774 murine macrophages.

We investigated the role of prostaglandin E2 (PGE2) and its interactions with nitric oxide (NO) on cell death and NO-mediated cytotoxicity in the murine macrophage cell line J774. Stimulation of the J774 cells with lipopolysaccharide together with interferon-gamma resulted in a dose-dependent cytotoxicity and production of PGE2 and NO, measured as nitrite. Our results showed a linear correlation between PGE2 release and cytotoxicity. The cyclooxygenase (COX) inhibitor indomethacin completely inhibited PGE2 biosynthesis, without affecting NO production or cell death. This supports previous reports suggesting that overproduction of endogenous PGE2 is mainly the consequence of cell death and does not cause it. In contrast, the NO synthase inhibitor N(omega)-monomethyl-L-arginine (L-NMMA) gave a significant, though incomplete suppression of NO release and cell death. This points to the presence of other cytotoxic factors besides NO. To evaluate the toxic effect solely due to NO, macrophages were exposed to the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Incubation with SNAP also resulted in a concentration-dependent cell injury and PGE2 production. When exogenously added, PGE2 protected against SNAP-mediated cytotoxicity and simultaneously increased PGE2 release into the medium, without inducing COX-2. The cytoprotection and the stimulation of PGE2 release were both reversed by indomethacin. In conclusion, PGE2 biosynthesis may represent a mechanism by which inflammatory macrophages protect themselves against the cytotoxic effects of NO.

Animals↗
PubMed1998

Intermediate-term results of Phase I Food and Drug Administration Trials of buttoned device occlusion of secundum atrial septal defects.

OBJECTIVES: This study was conducted to evaluate the intermediate-term results of the multi-institutional U.S. trial of the buttoned device for transcatheter closure of atrial septal defects (ASDs). BACKGROUND: The trial was conducted in three centers (University of Arizona, University of Michigan and University of Wisconsin) under a Food and Drug Administration (FDA)-approved clinical trial with investigational device exemption. Only short-term follow-up is known. METHODS: All 46 patients who had successful implantation of the device were prospectively followed up. Patients were evaluated at 1, 6 and 12 months after device occlusion and yearly thereafter. RESULTS: This cohort was followed up from 51 to 68 months (mean 60.8, median 62). Patient ages ranged from 1 to 62 years (median 4); weights ranged from 10 to 105 kg (median 18); and stretched ASD sizes were 14 +/- 4 mm (left to right shunts) and 10 +/- 3 mm (right to left shunts). Of the 46 patients, 45 (98%) had effective occlusion of their ASD, and 34 (74%) had complete ASD closure. The incidence of residual shunts decreased from 65% (30 of 46 patients) at 1 month after device placement to 27% (12 of 45 patients) at last follow-up. All residual shunts were quantitated as trivial. Only two patients (4%) required reintervention for significant residual defects. There were no cases of endocarditis or thromboembolism in 224 patient-years of follow-up. CONCLUSIONS: In up to 5.5 years of follow-up, the buttoned device provided effective closure in 98% of patients in whom the device was successfully implanted. The incidence of residual shunts decreased during follow-up, and no instances of endocarditis or thromboembolism were observed.

Actuarial Analysis↗
PubMed1997

Feed-back inhibition of oxidative stress by oxidized lipid/amino acid reaction products.

Three oxidized lipid/amino acid reaction products (OLAARPs): 1-methyl-4-pentyl-1,4-dihydropyridine-3,5-dicarbaldehyde, 1-(5-amino-1-carboxypentyl)pyrrole, and N-(carbobenzyloxy)-1(3)-[1-(formylmethyl)hexyl]-l-histidine dihydrate, were prepared and tested for antioxidative activity in a microsomal system in order to investigate the effect that OLAARP formation may be playing in the oxidative stress process. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 microM Cu2+, 1 mM Fe3+/5 mM ascorbate, or 1 mM Cu2+/10 mM H2O2, and the compound to be tested as antioxidant added at 50 microM. At different periods of time, samples were tested for lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, which was evaluated by the formation of protein carbonyls and amino acid analysis. The three OLAARPs and butylated hydroxytoluene significantly (p < 0.05) protected against lipid peroxidation and protein damage for the three systems assayed. On the contrary, neither the amino acids used in the preparation of OLAARPs nor alpha-tocopherol, mannitol, aminoguanidine, or 4, 5-dihydroxy-1,3-benzenedisulfonic acid exhibited this constant protection. Because OLAARPs were produced at inhibitory levels during microsomal lipid peroxidation, these results suggest that OLAARP formation may be an antioxidative defense mechanism by which oxidative stress is feed-back-inhibited, delaying the damage caused by reactive oxygen species.

Alanine↗
PubMed1997

The protective role of thiols against nitric oxide-mediated cytotoxicity in murine macrophage J774 cells.

Nitric oxide (NO) plays an important role in the cytotoxic activity of macrophages towards tumour cells and microbial pathogens. We investigated whether alteration of intracellular thiol levels modulates the cytotoxic effects of different NO donors and lipopolysaccharide-induced NO in the murine macrophage cell lin J774A.1. The NO-releasing compound S-nitroso-N-acetylpenicillamine caused a significant concentration-dependent loss of viability of the macrophages only under glucose-limiting conditions. The cytotoxic effect of S-nitroso-N-acetylpenicillamine was prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO). Depletion of total glutathione before exposure to S-nitroso-N-acetylpenicillamine further decrease cell viability while pretreatment with N-acetylcysteine was protective. Comparing equimolar concentrations of various NO donors including S-nitrosoglutathione, S-nitrosocysteine and 3-morpholino-sydnonimine hydrochloride, cytotoxicity appeared to be related to the relative stability of the test compound. Both the order of stability and the order of potency for cell killing was S-nitrosoglutathione > S-nitroso-N-acetylpenicillamine > S-nitrosocysteine = 3-morpholino-sydnonimine hydrochloride. Stimulation of the macrophages with lipopolysaccharide and interferon-gamma resulted in dose-dependent cell injury and NO production. Glutathione depletion prior to stimulation considerably decreased macrophage viability as well as the NO production. In contrast to the protective effect on S-nitroso-N-acetylpenicillamine-mediated injury, pretreatment with N-acetylcysteine did not influence the lipopolysaccharide-mediated cytotoxicity. These results demonstrate that (a) reduction in the availability of glucose and intracellular glutathione renders the cells more vulnerable to the cytotoxic effects of NO donors, (b) in this model of cytotoxicity, long-lived NO donors were more cytotoxic than short-lived NO donors, (c) the differential effects of N-acetylcysteine on S-nitroso-N-acetylpenicillamine-induced and bacterial lipopolysaccharide-mediated cytotoxicity support the existence of other toxic species different from NO or NO-related compounds with a potent cytotoxic activity in immunostimulated macrophages, and (d) other non-protein thiols like N-acetylcysteine may substitute for glutathione as a major component of the cellular antioxidant defense system.

Acetylcysteine↗
PubMed1995

Oxidative release of nitric oxide accounts for guanylyl cyclase stimulating, vasodilator and anti-platelet activity of Piloty's acid: a comparison with Angeli's salt.

The decomposition of benzenesulphohydroxamic acid (Piloty's acid; PA) and some of its derivatives has been reported to yield nitroxyl ions (NO-), a species with potent vasodilator properties. In a previous study we demonstrated that the oxidative breakdown of PA results in the formation of nitric oxide (NO) and suggested that NO rather than NO- may account for its vasorelaxant properties. Using isolated aortic rings in organ baths, we now show that high concentrations of cysteine potentiate the vasorelaxant response to PA, whereas responses to Angeli's salt (AS), a known generator of NO-, were almost completely inhibited. These different behaviours of PA and AS are mirrored by their distinct chemistries. By using HPLC it was shown that, at physiological pH and in the absence of oxidizing conditions, PA is a relatively stable compound. Direct chemical determination of NO, stimulation of soluble guanylyl cyclase, and measurement of platelet aggregation under various experimental conditions confirmed the requirement for oxidation to release NO from PA, and quite weak oxidants were found to be sufficient to promote this reaction. In contrast, at pH 7.4 AS decomposed rapidly to yield nitrite (NO2-) and NO-, bu did not produce NO on reaction with dioxygen (O2) or hydrogen peroxide (H2O2). Thus sulphohydroxamic acids are a new class of thiol-independent NO-donors that generate NO rather than NO- under physiological conditions.

Animals↗
PubMed1995

Linoleic acid oxidation in the presence of amino compounds produces pyrroles by carbonyl amine reactions.

The reactions of 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-LOOH) and its degradation product 4,5(E)-epoxy-2(E)-decenal with butylamine and lysine were studied to determine whether pyrrole derivatives isolated in model reactions were produced in complex systems involving hydroperoxides. Incubated reaction mixtures were studied by gas chromatography coupled with mass spectrometry or high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS), and some compounds were isolated by column chromatography or semipreparative HPLC, and identified by 1H- and 13C-nuclear magnetic resonance spectroscopy and MS. The reaction of epoxyalkenals with amino groups produced two types of pyrrole derivatives: 1-substituted 2-(1'-hydroxyalkyl)pyrroles and 1-substituted pyrroles. 1-Substituted 2-(1'-hydroxyalkyl)pyrroles were responsible for the development of color and fluorescence by a polymerization reaction, which implied the formation of dipyrrylmethanes and dipyrrylmethenes. 1-Substituted pyrroles were final products in these reactions and their determination might be used as an index of oxidative stress. The above reactions were also observed between 13-LOOH and amino compounds, and suggested that the pyrrole polymerization mechanism plays a role in the fluorescence observed by reaction of hydroperoxides and amino groups.

Aldehydes↗
PubMed1995

Determination of lysine modification product epsilon-N-pyrrolylnorleucine in hydrolyzed proteins and trout muscle microsomes by micellar electrokinetic capillary chromatography.

epsilon-N-Pyrrolylnorleucine (Pnl) is a product of the reaction between the lipid peroxidation product 4,5(E)-epoxy-2(E)-heptenal (EH) and the epsilon-amino group of lysine. Because Pnl might also be produced in proteins, a specific method to determine this compound in protein hydrolysates has been developed. Homoarginine, added as the internal standard, and Pnl are derivatized with diethyl ethoxymethylenemalonate and analyzed by micellar electrokinetic capillary chromatography. The method also analyzes lysine and arginine, and these analyses were useful in determining losses of these amino acids after treatment with EH. The lowest concentration of Pnl detected with acceptable reproducibility is 5 nmol/mL, and the coefficient of variation was determined from four standard curves assayed on separate days. Detector response was linear for samples containing 1.6 to 74 nmol/mL of Pnl. The assay was applied in investigations of Pnl production in bovine serum albumin (BSA) and trout muscle microsomes treated with EH. When BSA was incubated overnight with 30 mM EH, 76% of lysine residues were modified, and a part of these residues were detected as Pnl (12%). Pnl formation was also detected when trout muscle microsomes were incubated for three hours with 1 or 10 mM EH. These results show that Pnl is produced in vitro in proteins treated with the lipid peroxidation product EH, and suggest that Pnl might also be constituent of in vivo damaged proteins by their reaction with oxidized lipids.

Aldehydes↗
PubMed1995

Semilunar valve switch in aortic insufficiency.

The use of the Ross procedure in young patients is gaining wider acceptance. The need for a foreign pulmonary valve that will require replacement, however, is a serious drawback. To circumvent this problem, we reimplanted the native aortic valve in the pulmonary position in four patients (ages 12, 15, 15 and 17 years old) operated on utilizing the Ross procedure for aortic insufficiency. One patient had congenital and three isolated rheumatic aortic insufficiency. The root replacement technique with coronary artery reimplantation was used. All patients did well initially with marked reduction of left ventricular dilatation and good function of the reimplanted native aortic valve. One patient, however, died a month later from rupture of a false aneurysm that developed at the pulmonary autograft to ascending aorta anastomosis. We feel that the use of the native aortic valve in the pulmonary position makes the Ross procedure more attractive and potentially curative. The diseased aortic valve works well in the pulmonary position because of lower pulmonary artery pressure and resistance.

Adolescent↗
PubMed1995

Human and rat macrophages mediate fungistatic activity against Rhizopus species differently: in vitro and ex vivo studies.

Both rat alveolar macrophages and a human macrophages cell line with characteristics of human tissue (e.g., alveolar) macrophages (THP-1) were found to inhibit the germination of Rhizopus spores. However, the conditions under which fungistatic activity occurs are different for these two cell types. The inhibition of Rhizopus spore germination by rat alveolar macrophages requires the activation of macrophages and the presence of serum and L-arginine. During rat alveolar macrophage-mediated fungistatic activity, L-arginine is oxidized to nitric. Human macrophage-mediated fungistatic activity is similar to that mediated by rat macrophages in terms of the serum requirement, but it does not require L-arginine. Human macrophages did not produce any nitrite detectable by the colorimetric assay. Their ability to inhibit germination was enhanced by the combination of endotoxin and gamma interferon. The inhibition of Rhizopus spore germination by rat alveolar macrophages is thus mediated by the generation of nitric oxide, whereas the mechanism of similar inhibition by human macrophages remains poorly understood. Serum samples from diabetic rats as well as from patients with diabetes or uremia decreased the inhibitory effect of macrophages on spore germination. Dialysis of the serum samples against a buffered salt solution antagonized this phenomenon, indicating that a low-molecular-weight factor in the sera of patients with diabetes or uremia may modulate local antifungal defense mechanisms. The absence of L-arginine-dependent nitrogen oxidation in human macrophages, compared with its presence in rat alveolar macrophages, under conditions during which fungistatic activity occurs suggests that this phenomenon is species specific.

Animals↗
PubMed1995

In vitro production of long chain pyrrole fatty esters from carbonyl-amine reactions.

The reaction of lipoxygenase pathway products methyl 9,10(Z)-epoxy-13-oxo-11(E)-octadecenoate (13-MZEO) and methyl 12,13(Z)-epoxy-9-oxo-10(E)-octadecenoate (9-MZEO), with butylamine and lysine was studied in order to investigate whether some oxidized fatty acids are able to react with amino groups, analogously to volatile aldehydes, and, therefore, contribute to the overall protein damage produced during oxidative stress. The reaction mixtures were incubated at room temperature or 37 degrees C; the products were fractionated by semipreparative high performance liquid chromatography, and characterized by 1H and 13C nuclear magnetic resonance spectroscopy and mass spectrometry. The products identified were methyl 9-hydroxy-9-(5-pentyl-N-alkylpyrrole-2-)nonanoate and N-alkyl-2-pentylpyrrole for the 13-MZEO, and methyl 8-[5-(1'-hydroxyhexyl)-N-alkyl-pyrrole-2-]octanoate and methyl 8-(N-alkylpyrrole-2-)octanoate for the 9-MZEO. Formation of these pairs of analogs suggested that compounds were produced by one mechanism with the loss of the 5-substituent as a short-chain aldehyde, which was identified for the 13-MZEO. A reaction mechanism that explains the formation of all these compounds is proposed and the biological significance of these findings in relation to low density lipoprotein oxidation is discussed. Although these results do not demonstrate the formation of long-chain pyrrole fatty acids in vivo, they suggest that these pyrroles could be produced as an ultimate step in the lipid peroxidation process.

Amines↗
PubMed1995

[Production of an acid extract of rice straw].

The chemical composition of rice straw was determined by means of standard analytical procedures. The material showed an adequate content of potentially assimilable carbohydrates for the growth of microorganisms. The optimum result of the rice straw treatment corresponds to a particle size of 60 mesh mixed with 5% H2SO4 in a weight: volume relation of 1:10 and submitted to a temperature of 121 degrees C. Under these conditions a rice straw's acid extract was obtained, containing 20 g/lt of total sugars and 15 g/lt of reducing sugars. This content of sugars is enough to support the growth of microorganisms in aerobic conditions.

Candida↗