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Biomedical subjects

J W Eaton

Publications and source records attributed to J W Eaton.

At least 55 records · Page 3Linked to original sources
PubMed1995

Thalassaemic erythrocytes: cellular suicide arising from iron and glutathione-dependent oxidation reactions?

Both beta-thalassaemic red blood cells and normal red blood cells (RBC) artificially loaded with unpaired alpha-haemoglobin chains exhibit increased amounts of membrane-bound haem and iron. In the model beta-thalassaemic RBC the amount of free haem and iron was as much as 20 times that which could have been contributed by the entrapped alpha-haemoglobin chains alone. This excess haem/iron arises from destabilization of haemoglobin via reactions between ferric iron (Fe3+), initially contributed by the unpaired alpha chains, and cytoplasmic constituents, primarily reduced glutathione (GSH). Indeed, in the presence of Fe3+ (100 microM) addition of even small amounts of GSH (0.5 mM) to dilute RBC haemolysates (0.15 mg haemoglobin/dl) greatly accelerated methaemoglobin formation. In contrast, lysates from GSH-depleted RBC demonstrated a significantly reduced rate of iron-mediated haemoglobin oxidation which was reversible by addition of GSH. The initiation, and subsequent propagation, of Fe(3+)-mediated haemoglobin oxidation was significantly inhibited by iron chelators. Finally, Fe(3+)-driven haemoglobin oxidation was synergized by low amounts of H2O2, an oxidant spontaneously generated in thalassaemic RBC. To summarize, the release of small amounts of free iron from unpaired alpha-haemoglobin chains in the beta-thalassaemic RBC can initiate self-amplifying redox reactions which simultaneously deplete cellular reducing potential (e.g. GSH), oxidize additional haemoglobin, and accelerate the red cell destruction.

Cell Death↗
PubMed1994

Heme and the vasculature: an oxidative hazard that induces antioxidant defenses in the endothelium.

Heme proteins transport oxygen and facilitate redox reactions. Heme, however, may be dangerous, especially when free in biologic systems. For example, iron released from hemoglobin-derived heme can catalyze oxidative injury to neuronal cell membranes and may be a factor in post-traumatic damage to the central nervous system. We have shown that heme catalyzes the oxidation of low density lipoproteins which can damage vascular endothelial cells. The endothelium is susceptible to damage by oxidants generated by activated phagocytes, and this has been invoked as an important mechanism in a number of pathologies including the Adulte Respiratory Distress Syndrome (ARDS), acute tubular necrosis, reperfusion injury and atherosclerosis. Because of its highly hydrophobic nature, heme readily intercalates into endothelial membranes and potentiates oxidant-mediated damage. This injury is dependent on the iron content of heme and is completely blocked when concomitant hemopexin is added. Ferrohemoglobin, when added to cultured endothelial cells, is without deleterious effects, but if oxidized to ferrihemoglobin (methemoglobin), it greatly amplifies oxidant damage. Methemoglobin, but not ferrohemoglobin, releases its hemes which can then be incorporated into endothelial cells. Cultured endothelial cells, when exposed to methemoglobin but not ferrohemoglobin, cytochrome c or metmyoglobin, potentiate this oxidant injury. Stabilization of the methemoglobin by cyanide, haptoglobin or capture of the heme by hemopexin abrogates this effect. Paradoxically, more prolonged exposure of endothelium to heme or methemoglobin renders them remarkably resistant to oxidant challenge. Endothelium defends itself from heme by induction of the heme degrading enzyme heme oxygenase and the concomitant production of large amounts of the iron binding protein ferritin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Albumin binding surfaces for biomaterials.

The surfaces of medical devices may promote both coagulation and infections caused by adherent microorganisms. In the case of polymeric elastomers, these iatrogenic effects are likely intermediated by absorbed host proteins that spontaneously bind to the device surface, promoting both bacterial adherence and thrombotic events. We earlier attempted to produce biomaterial surfaces that would selectively bind host albumin because albumin-coated surfaces were known to diminish both coagulation and bacterial adherence. To this end, an albumin-binding high molecular weight dextran:Cibacron blue adduct was bulk incorporated into polyetherurethane (Keogh et al., J Biomed Mater Res 1992;26:441). The modified material bound albumin selectively and reversibly and showed evidence of enhanced biocompatibility. However, approximately 30% of the surface of this material was evidently unmodified and still capable of exerting the above adverse effects. In the present work, we have covalently surface-modified polyetherurethane with sequential additions of acrylamide, amino-propylmethacrylamide, dextran, and Cibacron blue. This derivatized polyurethane preferentially and reversibly binds albumin, even from complex mixtures of proteins such as plasma. Furthermore, this material inhibits the clotting of nonanticoagulated whole human blood (for > 16 hours at room temperature), perhaps by virtue of binding and activation of antithrombin III by the sulfonic acid residues on the surface-immobilized Cibacron blue. Finally, such surfaces, especially when bearing bound albumin, diminish the adherence of Staphylococcus epidermidis, a pathogen frequently associated with device-centered infections. We conclude that similar albumin-affinity surfaces may hold promise for the development of more biocompatible materials for implantation and blood contact applications.

Anticoagulants↗
PubMed1994

Free fatty acids enhance hypochlorous acid production by activated neutrophils.

Activated polymorphonuclear neutrophils (PMNs) may contribute to the genesis of chronic obstructive lung disease in long-term cigarette smokers. However, it is not presently known which elements in smoke are important in triggering this progressive pulmonary damage or in affecting the activities of inflammatory cells such as PMNS. We earlier found substances in organic concentrates of cigarette smoke that bound ferrous iron and transferred the metal into organic phases. These substances were later identified as saturated free fatty acids, predominantly palmitic and stearic acids (16:0 and 18:0). We now report investigations of the effects of fatty acids on the oxidative metabolism of PMNs. In accord with most earlier reports, we find that saturated fatty acids have little direct effect on PMN oxidative metabolism. However, micromolar amounts of free fatty acids will more than double production of hypochlorous acid (HOCl) by PMNs stimulated with small amounts of phorbol myristate acetate. Similar fatty acid-mediated increases in HOCl production also occur when PMNs are stimulated with 1,2-dioctanoyl-sn-glycerol and 1-oleoyl-2-acetyl-sn-glycerol (also thought to be agonists of protein kinase C) but not when cells are stimulated with the calcium ionophore A23187, the formylated tripeptide f-met-leu-phe, or opsonized zymosan. Fatty acid-mediated enhancement of PMN HOCl production evidently arises from increased release of myeloperoxidase from stimulated PMNs. Furthermore, in the presence of free fatty acids, stimulated PMNs are much more cytotoxic toward cultured mink lung epithelial cells, a toxicity that is blocked by scavengers of HOCl. These results suggest that the relatively large amounts of free fatty acids present in tobacco smoke may act to amplify PMN-mediated oxidative damage to the lungs of smokers.

Adult↗
PubMed1993

Fibrin(ogen) mediates acute inflammatory responses to biomaterials.

Although "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and chronic inflammatory responses. Early interactions between implants and inflammatory cells are probably mediated by a layer of host proteins on the material surface. To evaluate the importance of this protein layer, we studied acute inflammatory responses of mice to samples of polyester terephthalate film (PET) that were implanted intraperitoneally for short periods. Material preincubated with albumin is "passivated," accumulating very few adherent neutrophils or macrophages, whereas uncoated or plasma-coated PET attracts large numbers of phagocytes. Neither IgG adsorption nor surface complement activation is necessary for this acute inflammation; phagocyte accumulation on uncoated implants is normal in hypogammaglobulinemic mice and in severely hypocomplementemic mice. Rather, spontaneous adsorption of fibrinogen appears to be critical: (a) PET coated with serum or hypofibrinogenemic plasma attracts as few phagocytes as does albumin-coated material; (b) in contrast, PET preincubated with serum or hypofibrinogenemic plasma containing physiologic amounts of fibrinogen elicits "normal" phagocyte recruitment; (c) most importantly, hypofibrinogenemic mice do not mount an inflammatory response to implanted PET unless the material is coated with fibrinogen or the animals are injected with fibrinogen before implantation. Thus, spontaneous adsorption of fibrinogen appears to initiate the acute inflammatory response to an implanted polymer, suggesting an interesting nexus between two major iatrogenic effects of biomaterials: clotting and inflammation.

Ancrod↗
PubMed1993

Endothelial-cell heme uptake from heme proteins: induction of sensitization and desensitization to oxidant damage.

Iron-derived reactive oxygen species are implicated in the pathogenesis of various vascular disorders including atherosclerosis, vasculitis, and reperfusion injury. The present studies examine whether heme, when liganded to physiologically relevant proteins as in hemoglobin, can provide potentially damaging iron to intact endothelium. We demonstrate that reduced ferrohemoglobin, while relatively innocuous to cultured endothelial cells, when oxidized to ferrihemoglobin (methemoglobin), greatly amplifies oxidant (H2O2)-mediated endothelial-cell injury. Drawing upon our previous observation that free heme similarly primes endothelium for oxidant damage, we posited that methemoglobin, but not ferrohemoglobin, releases its hemes that can then be incorporated into endothelial cells. In support, cultured endothelial cells exposed to methemoglobin--in contrast to exposure to ferrohemoglobin, cytochrome c, or metmyoglobin--rapidly increased their heme oxygenase mRNA and enzyme activity, thereby supporting heme uptake; ferritin production was also markedly increased after such exposure, thus attesting to eventual incorporation of Fe. These cellular methemoglobin effects were inhibited by the heme-scavenging protein hemopexin and by haptoglobin or cyanide, agents that strengthen the liganding between heme and globin. If the endothelium is exposed to methemoglobin for a more prolonged period (16 hr), it accumulates large amounts of ferritin; concomitantly, and presumably associated with iron sequestration by this protein, the endothelium converts from hypersusceptible to hyperresistant to oxidative damage. We conclude that when oxidation of hemoglobin facilitates release of its heme groups, catalytically active iron is provided to neighboring tissue environments. The effect of this relinquished heme on the vasculature is determined both by extracellular factors--i.e., plasma proteins, such as haptoglobin and hemopexin--as well as intracellular factors, including heme oxygenase and ferritin. Acutely, if both extra- and intracellular defenses are overwhelmed, cellular toxicity arises; chronically, when ferritin is induced, resistance to oxidative injury may supervene.

Cells, Cultured↗
PubMed1993

Suppression of colonic cancer by dietary phytic acid.

Large differences exist between human populations in the frequency of colonic cancer. Epidemiological evidence indicates that these differences are strongly influenced by country of residence, and a negative correlation has been found between the fiber content of the diet and frequency of colonic cancer. This has prompted the hypothesis that high-fiber diets are in some way protective. However, reanalysis of the dietary data provides equally strong support for the hypothesis that the protective element may be phytic acid (inositol hexaphosphate). This heat- and acid-stable substance is present in high concentration in many food items, including cereal grains, nuts, and seeds. Phytic acid forms chelates with various metals and suppresses damaging iron-catalyzed redox reactions. Inasmuch as colonic bacteria have been shown to produce oxygen radicals in appreciable amounts, dietary phytic acid might suppress oxidant damage to intestinal epithelium and neighboring cells. Indeed, rapidly accumulating data from animal models indicate that dietary supplementation with phytic acid may provide substantial protection against experimentally induced colonic cancer. Should further investigations yield additional support for this hypothesis, purposeful amplification of dietary phytic acid content would represent a simple method for reducing the risk of colonic carcinogenesis.

Colonic Neoplasms↗
PubMed1993

Degradation of biomaterials by phagocyte-derived oxidants.

Polymers used in implantable devices, although relatively unreactive, may degrade in vivo through unknown mechanisms. For example, polyetherurethane elastomers used as cardiac pacemaker lead insulation have developed surface defects after implantation. This phenomenon, termed "environmental stress cracking," requires intimate contact between polymer and host phagocytic cells, suggesting that phagocyte-generated oxidants might be involved. Indeed, brief exposure of polyetherurethane to activated human neutrophils, hypochlorous acid, or peroxynitrite produces modifications of the polymer similar to those found in vivo. Damage to the polymer appears to arise predominantly from oxidation of the urethane-aliphatic ester and aliphatic ether groups. There are substantial increases in the solid phase surface oxygen content of samples treated with hypochlorous acid, peroxynitrite or activated human neutrophils, resembling those observed in explanted polyetherurethane. Furthermore, both explanted and hypochlorous acid-treated polyetherurethane show marked reductions in polymer molecular weight. Interestingly, hypochlorous acid and peroxynitrite appear to attack polyetherurethane at different sites. Hypochlorous acid or activated neutrophils cause decreases in the urethane-aliphatic ester stretch peak relative to the aliphatic ether stretch peak (as determined by infrared spectroscopy) whereas peroxynitrite causes selective loss of the aliphatic ether. In vivo degradation may involve both hypohalous and nitric oxide-based oxidants because, after long-term implantation, both stretch peaks are diminished. These results suggest that in vivo destruction of implanted polyetherurethane involves attack by phagocyte-derived oxidants.

Animals↗
PubMed1993

Hemoglobin catabolism and host-parasite heme balance in chloroquine-sensitive and chloroquine-resistant Plasmodium berghei infections.

Catabolism of host hemoglobin by the malaria parasite liberates required amino acid precursors, but is also releases large amounts of potentially toxic heme that accumulates in parasite food vacuoles during intra-erythrocytic development. The schizonticidal drug chloroquine binds to free heme with high affinity and is concentrated in parasite food vacuoles. To better understand the disposition of heme within the host-parasite complex, we studied the balance of hemoglobin and heme in Plasmodium berghei-infected reticulocytes in the rat and compared this process in chloroquine-sensitive (CS) and chloroquine-resistant (CR) parasites. We found that CS P. berghei parasites have 1.5-fold more heme than CR parasites isolated from rats, and that CS P. berghei-infected reticulocytes accumulate more chloroquine than CR P. berghei-infected reticulocytes. Despite these differences in parasite heme content, the decrease in host cell hemoglobin content and the rate of free amino acid generation within the host-parasite complex is similar in CS and CR P. berghei-infected rat reticulocytes. The heme content of the infected reticulocyte-parasite complex decreases with increasing parasitemia but to a lesser extent than expected for the decrease in hemoglobin. Furthermore, the decrease in host-parasite heme is accelerated in the CR P. berghei infection compared with the CS P. berghei infection. Therefore, hemoglobin catabolism by malaria parasites is associated with the overall loss of heme from the host-parasite complex and with variable deposition of heme within parasites.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

Inflammatory responses to implanted polymeric biomaterials: role of surface-adsorbed immunoglobulin G.

In many cases, evidently inert and nontoxic biomaterials may trigger procoagulant and inflammatory responses. Because most polymeric biomaterials accumulate a surface layer of protein immediately after implantation, these adverse reactions may stem from secondary interactions between the host and this surface layer of adsorbed proteins. Using polyester terephthalate (the polymer from which both Dacron and Mylar are produced) as a model, we have explored the hypothesis that surface-adsorbed immunoglobulin might mediate subsequent inflammatory responses. We find, as have others, that immunoglobulin G (IgG) does spontaneously adsorb to polymer surfaces, both in vitro and in vivo. Furthermore, polymer implants precoated with IgG do activate human polymorphonuclear neutrophils in vitro and also attract substantial numbers of phagocytes (especially polymorphonuclear neutrophils and macrophages) when implanted in mice. However, when implants are placed in mice having a form of severe combined immunodeficiency (and, consequently, almost undetectable levels of serum IgG), a near-normal influx of phagocytic cells ensues. Thus, spontaneously-adsorbed surface IgG does not appear to be a necessary precedent to inflammatory responses directed against implanted biomaterials.

Adsorption↗
PubMed1992

Ferritin: a cytoprotective antioxidant strategem of endothelium.

Phagocyte-mediated oxidant damage to vascular endothelium is likely involved in various vasculopathies including atherosclerosis and pulmonary leak syndromes such as adult respiratory distress syndrome. We have shown that heme, a hydrophobic iron chelate, is rapidly incorporated into endothelial cells where, after as little as 1 h, it markedly aggravates cytotoxicity engendered by polymorphonuclear leukocyte oxidants or hydrogen peroxide (H2O2). In contrast, however, if cultured endothelial cells are briefly pulsed with heme and then allowed to incubate for a prolonged period (16 h), the cells become highly resistant to oxidant-mediated injury and to the accumulation of endothelial lipid peroxidation products. This protection is associated with the induction within 4 h of mRNAs for both heme oxygenase and ferritin. After 16 h heme oxygenase and ferritin have increased approximately 50-fold and 10-fold, respectively. Differential induction of these proteins determined that ferritin is probably the ultimate cytoprotectant. Ferritin inhibits oxidant-mediated cytolysis in direct relation to its intracellular concentration. Apoferritin, when added to cultured endothelial cells, is taken up in a dose-responsive manner and appears as cytoplasmic granules by immunofluorescence; in a similar dose-responsive manner, added apoferritin protects endothelial cells from oxidant-mediated cytolysis. Conversely, a site-directed mutant of ferritin (heavy chain Glu62----Lys; His65----Gly) which lacks ferroxidase activity and is deficient in iron sequestering capacity, is completely ineffectual as a cytoprotectant. We conclude that endothelium and perhaps other cell types may be protected from oxidant damage through the iron sequestrant, ferritin.

Animals↗
PubMed1992

Multicellular oxidant defense in unicellular organisms.

Although catalase is thought to be a major defense against hydrogen peroxide (H2O2), the catalase activity within individual Escherichia coli fails to protect against exogenous H2O2. Contrary to earlier reports, we find that dilute suspensions of wild-type and catalase-deficient E. coli are identical in their sensitivity to H2O2, perhaps because even wild-type, catalase-positive E. coli cannot maintain an internal/external concentration gradient of this highly diffusible oxidant. However, concentrated suspensions or colonies of catalase-positive E. coli do preferentially survive H2O2 challenge and can even cross-protect adjacent catalase-deficient organisms. Furthermore, high-density catalase-positive--but not catalase-negative--E. coli can survive and multiply in the presence of competitive, peroxide-generating streptococci. These observations support the concept that bacterial catalase may defend colonial, but not individual, E. coli against environmental H2O2. Group protection by the activity of enzymes that mitigate oxidative stress may have been a driving force in the evolution of multicellular organisms.

Biological Evolution↗
PubMed1992

Albumin-binding surfaces for implantable devices.

Surfaces of implantable and blood contact-devices accumulate adsorbed and denatured proteins. This anomalous layer of proteins may help trigger unwanted events such as activation of coagulation systems and, perhaps, chronic inflammation. Because, in many experimental systems, the purposeful coating of surfaces with albumin will biologically "passivate" materials, we have attempted to develop polymers which, when exposed to blood or body fluids, will spontaneously, selectively, and reversibly adsorb host albumin. We report here a novel derivatization technique for increasing the albumin affinity of implantable polyetherurethane (PU). The technique is based on the incorporation of high-molecular-weight dextran to which the albumin-binding dye Cibacron Blue is covalently attached. Somewhat surprisingly, the amounts of human albumin adsorbed by Blue Dextran-modified and unmodified PU are quite similar. There are, however, important differences. First, the binding of albumin to derivatized PU is specific and not readily blocked by proteins in albumin-depleted human serum. Second, the majority of albumin associated with derivatized PU appears to be reversibly bound. Third, the binding of albumin to derivatized PU evidently is mediated primarily through ligand-specific binding of the protein to the albumin-binding dextran-dye conjugate. We conclude that it is possible to produce implantable polymers having surfaces which display albumin-binding dyes that selectively and reversibly bind albumin. Materials with this property, when implanted or exposed to blood, should form an infinitely renewable coating of albumin derived from physiologic fluids. This surface modification strategy may spawn a new generation of implantable materials with improved biologic compatibility.

Adsorption↗
PubMed1991

Spirohydantoin inhibitors of aldose reductase inhibit iron- and copper-catalysed ascorbate oxidation in vitro.

Transition metal-catalysed oxidations have been implicated in the complications of diabetes. We report here that some experimental inhibitors of the enzyme aldose reductase (implicated in diabetes mellitus via its ability to catalyse glucose reduction to sorbitol) are also potent inhibitors of transition metal-catalysed ascorbate oxidation. The inhibition appears to be dependent upon the presence of a spirohydantoin group. It is conceivable that the copper- and iron-binding capacity of these compounds may contribute to some of their observed biological effects and may provide a starting point for a new generation of experimental drugs for the treatment of diabetes mellitus.

Aldehyde Reductase↗
PubMed1991

A spectrophotometric assay for chlorine-containing compounds.

Determinations of hypochlorous acid and chloramine compounds are important in a number of areas. Several techniques are now available for such analyses, but most require unstable reagents and/or multiple steps in the analytical procedure. We have developed a simple, one-step spectrophotometric assay for reactive chlorine-containing compounds involving the oxidation of ascorbic acid by hypochlorous acid or chloramines. There is no interference from other nonhalide oxidants such as hydrogen peroxide or hypothiocyanous acid. Because small amounts of ascorbic acid will not damage biological materials, this method also allows continuous measurements of the generation of chlorine-containing compounds by activated neutrophils. This simple assay permits precise analysis of as little as 1 nmol of HOCl.

Ascorbic Acid↗
PubMed1991

Is the lens canned?

The ocular lens somehow remains pellucid despite bombardment by ultraviolet radiation and endogenous hydrogen peroxide (present in the humoral fluids which bathe this tissue). The lens and adjacent aqueous and vitreous humors contain exceptionally high concentrations of reducing substances, particularly ascorbic acid, thought to be important in lenticular oxidant defense. However, in the presence of traces of transition metals, or when exposed to ultraviolet radiation, ascorbic acid readily reacts with oxygen, yielding hydrogen peroxide, and damaging lens crystallins. We propose the alternative hypothesis that the real antioxidant function of ascorbic acid, particularly that in the aqueous and vitreous humors, may be effecting the conversion of oxygen to water. Because the lens lacks a blood supply, coupled reactions of ascorbic acid with oxygen in the humoral fluid spaces should produce a metabolically sustained anaerobiosis. If so, nature may have preinvented the process of canning, wherein food (or in this case, the lens) is preserved by a combination of sterility and anoxia.

Animals↗