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J L Zweier

Publications and source records attributed to J L Zweier.

At least 145 records · Page 8Linked to original sources
PubMed1988

Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues.

Oxygen free radicals have been demonstrated to be important mediators of postischemic reperfusion injury in a broad variety of tissues; however, the cellular source of free radical generation is still unknown. In this study, electron paramagnetic resonance measurements with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrate that bovine endothelial cells subjected to anoxia and reoxygenation become potent generators of superoxide and hydroxyl free radicals. A prominent DMPO-OH signal aN = aH = 14.9 G is observed on reoxygenation after 45 min of anoxic incubation. Quantitative measurements of this free radical generation and the time course of radical generation are performed. Both superoxide dismutase and catalase totally abolish this radical signal, suggesting that O2 is sequentially reduced from O2-. to H2O2 to OH.. Addition of ethanol resulted in trapping of the ethoxy radical, further confirming the generation of OH.. Endothelial radical generation was shown to cause cell death, as evidenced by trypan blue uptake. Radical generation was partially inhibited and partially scavenged by the xanthine oxidase inhibitor allopurinol. Marked inhibition of radical generation was observed with the potent xanthine oxidase inhibitor oxypurinol. These studies demonstrate that endothelial cells subjected to anoxia and reoxygenation, conditions observed in ischemic and reperfused tissues, generate a burst of superoxide-derived hydroxyl free radicals that in turn cause cell injury and cell death. Most of this free radical generation appears to be from the enzyme xanthine oxidase. Thus, endothelial cell free radical generation may be a central mechanism of cellular injury in postischemic tissues.

Animals↗
PubMed1988

Electron paramagnetic resonance measurements of free radicals in the intact beating heart: a technique for detection and characterization of free radicals in whole biological tissues.

Free radicals have been hypothesized to be important mediators of disease in a variety of organs and tissues. Electron paramagnetic resonance (EPR) spectroscopy can be applied to directly measure free radicals; however, it has not been possible to measure important biological radicals in situ because conventional spectrometer designs are not suitable for the performance of measurements on whole organs or tissues. We report the development of an EPR spectrometer designed for optimum performance in measuring free radicals in intact biological organs or tissues. This spectrometer consists of a 1- to 2-GHz microwave bridge with the source locked to the resonant frequency of a recessed gap loop-gap resonator. With this spectrometer, radical concentrations as low as 0.4 microM can be measured. Isolated beating hearts were studied in which simultaneous real time measurements of free radicals and cardiac contractile function were performed. This in vivo EPR technique was applied to study the kinetics of free radical uptake and metabolism in normally perfused and globally ischemic hearts. In addition, we show that this technique can be used to noninvasively measure tissue oxygen consumption. Thus, it is demonstrated that EPR spectroscopy can be applied to directly measure in vivo free radical metabolism and tissue oxygen consumption. This technique offers great promise in the study of in vivo free radical generation and the effects of this radical generation on whole biological tissues.

Animals↗
PubMed1987

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart.

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.

Adenine Nucleotides↗
PubMed1987

Direct measurement of free radical generation following reperfusion of ischemic myocardium.

Electron paramagnetic resonance spectroscopy was used to directly measure free radical generation in perfused rabbit hearts. Hearts were freeze-clamped at 77 degrees K during control perfusion, after 10 min of normothermic global ischemia (no coronary flow), or following post-ischemic reperfusion with oxygenated perfusate. The spectra of these hearts exhibited three different signals with different power saturation and temperature stability: signal A was isotropic with g = 2.004; signal B was anisotropic with axial symmetry with g parallel = 2.033 and g perpendicular = 2.005; signal C was an isotropic triplet with g = 2.000 and hyperfine splitting an = 24 G (1 G = 0.1 mT). The g values, linewidth, power saturation, and temperature stability of signal A are identical to those of a carbon-centered semiquinone, whereas those of signal B are similar to alkyl peroxyl or superoxide oxygen-centered free radicals; signal C is most likely a nitrogen-centered free radical. In the control heart samples signal A predominated, whereas in ischemic hearts signal A decreased in intensity, and signals B and C became more intense; with reperfusion all three signals markedly increased. Free radical concentrations derived from the intensities of the B and C signals peaked 10 sec after initiation of reflow. At this time the oxygen-centered free radical concentration derived from the intensity of signal B was increased over six times the concentration measured in control hearts and over two times the concentration measured in ischemic hearts. Hypoxic reperfusion did not increase any of the free radical signals over the levels observed during ischemia. These experiments directly demonstrate that reactive oxygen-centered free radicals are generated in hearts during ischemia and that a burst of oxygen radical generation occurs within moments of reperfusion.

Animals↗
PubMed1987

Improvement of postischemic myocardial function and metabolism induced by administration of deferoxamine at the time of reflow: the role of iron in the pathogenesis of reperfusion injury.

Reperfusion of ischemic myocardium has been postulated to result in a specific oxygen radical-mediated component of tissue injury. In a previous study we demonstrated improved recovery of ventricular function and metabolism when the superoxide radical scavenger superoxide dismutase was administered at the time of postischemic reflow. Studies in vitro, have suggested that superoxide toxicity might be mediated via the generation of more reactive hydroxyl radicals in an iron-catalyzed reaction. The present study was designed to test the hypothesis that myocardial reperfusion injury might be reduced by administration of the iron chelator deferoxamine at the time of reflow, most likely by preventing hydroxyl radical formation. Sixteen isolated Langendorff rabbit hearts, perfused within the bore of a superconducting magnet, were subjected to 30 min of normothermic (37 degrees C) total global ischemia followed by 45 min of reperfusion. At reflow eight treated hearts received a 10 ml bolus containing 50 mumol of deferoxamine followed by an infusion of 11 mumol/min for the first 15 min of reflow. The hearts were then perfused with standard perfusate for an additional 30 min. Eight untreated control hearts received a similar bolus of perfusate followed by 45 min of standard reperfusion. Serial 5 min 31P nuclear magnetic resonance spectra were recorded. Myocardial phosphocreatine (PCr) content fell to 5% to 7% of control during ischemia in both groups of hearts. Deferoxamine-treated hearts recovered 99 +/- 10% of control PCr content, while untreated hearts recovered 60 +/- 16% (p less than .05). Intracellular pH fell to 5.9 during ischemia in both groups, before showing more rapid and complete recovery in treated hearts (p less than .01). Recovery of developed pressure reached 70 +/- 6% of control in treated hearts compared with 35 +/- 10% in untreated hearts (p less than .05). Iron content of the perfusate was 7 microM, and by electron paramagnetic resonance spectroscopy was in the form of Fe3+-EDTA complexes. In the effluent of treated hearts iron was in the form of Fe3+-deferoxamine chelates. In summary, administration of the iron chelator deferoxamine at the time of postischemic reflow results in greater recovery of myocardial function and energy metabolism, which supports the hypothesis that iron plays an important role in the pathogenesis of reperfusion injury.

Adenosine Triphosphate↗
PubMed1987

Recombinant superoxide dismutase reduces oxygen free radical concentrations in reperfused myocardium.

It has been proposed that oxygen free radicals mediate damage that occurs during postischemic reperfusion. Recombinant human superoxide dismutase (r-h-SOD) has been shown to be effective at reducing reperfusion injury, but it is not known if this infused enzyme actually reduces oxygen free radical concentrations in the myocardial tissue. Electron paramagnetic resonance spectroscopy was used to directly measure the effect of r-h-SOD on free radical concentrations in the postischemic heart. Hearts were freeze clamped at 77 degrees K after 10 min of normothermic global ischemia followed by 10 s of reflow with control perfusate (n = 7) or perfusate containing 60,000 U r-h-SOD (n = 7). The spectra of these hearts exhibited three different signals: signal A isotropic, g = 2.004, identical to the carbon-centered ubiquinone free radical; signal B anisotropic with axial symmetry, g parallel = 2.033, g perpendicular = 2.005, identical to the oxygen-centered alkyl peroxyl free radical; and the signal C an isotropic triplet, g parallel = 2.000, an = 24 G, similar to a nitrogen-centered free radical such as a peroxyl amine. With r-h-SOD administration the concentration of the oxygen free radical, signal B, was reduced 49% from 6.8 +/- 0.3 microM to 3.5 +/- 0.3 microM (P less than 0.01) and the concentration of the nitrogen free radical, signal C, was reduced 38% from 3.4 +/- 0.3 to 2.1 +/- 0.3 microM (P less than 0.01). The concentration of the carbon-centered free radical, signal A, however, was increased 51% from 3.3 +/- 0.2 to 5.0 +/- 0.2 microM (P less than 0.01). Identical reperfusion with peroxide-inactivated r-h-SOD did not alter the concentrations of free radicals indicating that the specific enzymatic activity of r-h-SOD is required to decrease the concentrations of reactive oxygen free radicals. Additional measurements performed varying the duration of reflow demonstrate a burst of oxygen free radical generation peaking at 10 s of reperfusion. r-h-SOD entirely abolished this burst. These studies demonstrate that superoxide-derived free radicals are generated during postischemic reperfusion and suggest that the beneficial effect of r-h-SOD is due to its specific enzymatic scavenging of superoxide free radicals.

Animals↗
PubMed1986

Magnetic resonance studies of fredericamycin A: evidence for O2-dependent free-radical formation.

Fredericamycin A, a newly described potent antitumor antibiotic, exhibits unusual spectroscopic and physical properties. The drug shows a striking color change from red to blue on exposure to O2, with the appearance of an optical absorption band at 675 nm; on addition of acid these changes are readily reversed. 1H and 13C NMR spectra of fredericamycin A show that the resonances from the quininoid half of the molecule disappear after exposure to O2 but reappear on acidification in parallel with the observed optical spectral shift. These unusual NMR data are explained by electron spin resonance studies which demonstrate that fredericamycin A spontaneously forms an oxidized free radical with electron transfer to O2. The observed hyperfine structure of this radical is consistent with one-electron oxidation of the quininoid group. After fredericamycin A is exposed to O2, an EPR signal is observed with axial symmetry with temperature and power saturation behavior suggestive of .O2-. Spin-trapping EPR studies demonstrate that the drug reduces O2 to .O2- and H2O2 to .OH. This spontaneous mechanism of O2 reduction with the generation of oxidized drug free radicals and reduced oxygen free radicals is unprecedented among anticancer drugs, suggesting that fredericamycin A could be the forerunner of a new class of anticancer drug.

Antibiotics, Antineoplastic↗
PubMed1986

Mechanism of early contractile failure during hypoxia in intact ferret heart: evidence for modulation of maximal Ca2+-activated force by inorganic phosphate.

We tested the hypothesis that accumulation of H+ or inorganic phosphate (Pi) is responsible for the early contractile failure of hypoxia by measuring maximal Ca2+-activated pressure and 31P nuclear magnetic resonance spectra in Langendorff-perfused ferret hearts at 30 degrees C. Maximal Ca2+-activated pressure was identified by the saturation of pressure with respect to [Ca2+]o observed during tetani as [Ca2+]o was increased to 15 mM in HEPES-buffered, 100% O2-bubbled perfusate and during hypoxia induced by bubbling with room air or with 100% N2. Tetani were produced by pacing at 8-12 Hz following exposure to ryanodine (1-5 microM), an inhibitor of Ca2+ release from the sarcoplasmic reticulum, and were elicited once a minute to measure maximal Ca2+-activated pressure during acquisition of nuclear magnetic resonance spectra. An inverse correlation was observed between [Pi] and maximal Ca2+-activated pressure (r = -0.87 mean, n = 12), with an average decline of 8.6% in pressure per 1 mumol/g wet wt. increase in [Pi]. Intracellular pH (pHi) showed no significant correlation with maximal Ca2+-activated pressure (r = 0.49 mean, n = 12). Two other protocols, pacing at variable rates and gated measurements at two different times during the tetanus, were also used to correlate [Pi], pHi, and maximal Ca2+-activated pressure. These protocols confirmed the highly significant correlation between [Pi] and maximal Ca2+-activated pressure, as well as the lack of correlation with pHi. Acidosis induced by NH4Cl (20 mM) or by bubbling with 95% O2/5% CO2 was associated with less than 20% depression of maximal Ca2+-activated pressure in the pHi range down to 6.8, but much greater depression at lower pHi. The data are consistent with depression of maximal Ca2+-activated force during the early phase of hypoxia by Pi but not by H+.

Animals↗
PubMed1985

Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen.

The anticancer drug adriamycin binds iron and these complexes cycle to reduce molecular oxygen (Zweier, J. L. (1984) J. Biol. Chem. 259, 6056-6058). Optical absorption, EPR, and Mössbauer spectroscopic data are correlated with polarographic O2 consumption and chemical Fe2+ extraction measurements in order to characterize each step in this cycle. Fe3+ binds to adriamycin at physiologic pH forming a complex with an optical absorbance maximum at 600 nm. EPR signals at g = 4.2 and g = 2.01, and a doublet Mössbauer spectrum with isomer shift delta = 0.57 mm/s and quadrupole splitting delta EQ = 0.74 mm/s are observed indicating that the Fe3+ bound to adriamycin is high spin S = 5/2. Under anaerobic conditions the absorbance maximum at 600 nm decreases with an exponential decay constant = 0.77 h-1, and the EPR and Mössbauer spectra of Fe3+-adriamycin similarly decrease as the Fe3+ is reduced to EPR silent Fe2+. The Fe2+-adriamycin complex which is formed exhibits a Mössbauer spectrum with delta = 1.18 mm/s and delta EQ = 1.82 mm/s indicative of high spin Fe2+. As the EPR spectra of Fe3+-adriamycin decrease on reduction of the Fe3+ to Fe2+ a signal of the oxidized adriamycin free radical appears at g = 2.004 with line width of 8 G. On exposure to O2 the absorption maximum at 600 nm, the Fe3+ EPR, and the Fe3+ Mössbauer spectra all return. Polarographic measurements demonstrate that O2 is consumed and that H2O2 is formed. Addition of high affinity Fe2+ chelators block O2 consumption indicating that Fe2+ formation is essential for O2 reduction. This cycle of iron-mediated O2 reduction can explain the formation of the reactive reduced oxygen and adriamycin radicals which are thought to mediate the biological activity of adriamycin.

Catalase↗
PubMed1985

Iron-mediated formation of an oxidized adriamycin free radical.

Electron paramagnetic resonance studies are reported which demonstrate that the reduction of Fe3+ to Fe2+ by adriamycin results in the formation of an oxidized adriamycin free radical with an EPR signal at g = 2.004. A transient iron-adriamycin free radical complex is also observed at g = 2.34. The free radical is quantitated and its aerobic stability is determined. Observation of the oxidized adriamycin free radical signal confirms that adriamycin donates an electron to the bound Fe3+. In the presence of glutathione the drug-mediated reduction of Fe3+ to Fe2+ is bypassed, and the oxidized adriamycin radical signal is not observed. The oxidized adriamycin radicals and reduced oxygen radicals which are formed are two different mediators, whose relative concentrations could modulate the therapeutic and toxic effects of adriamycin.

Doxorubicin↗
PubMed1984

Reduction of O2 by iron-adriamycin.

Electron paramagnetic resonance studies of the Fe2+- and Fe3+-adriamycin complexes are reported which demonstrate iron-mediated reduction of O2 by adriamycin. Under anaerobic conditions, Fe2+ binds to adriamycin, giving rise to an EPR-silent Fe2+-adriamycin complex. On addition of O2, the Fe2+ is oxidized to Fe3+ and a spectrum of Fe3+-adriamycin is seen. Under anaerobic conditions, the signal of Fe3+-adriamycin decreases as a function of time as the Fe3+ bound to adriamycin is reduced to Fe2+, and a transient spectrum of iron bound to oxidized adriamycin is observed. On addition of O2, the EPR signal of Fe3+-adriamycin returns as Fe2+ is oxidized back to Fe3+ with electron transfer to O2. This cycle of iron-catalyzed O2 reduction may be the mechanism of adriamycin's antitumor potency and some of its toxic side effects.

Anaerobiosis↗
PubMed1983

Electron paramagnetic resonance measurement of the distance between the metal binding sites of transferrin.

The distance between the two metal binding sites of human serum transferrin is calculated from measurements made of the paramagnetic broadening of the EPR spectrum of Cu2+ bound at the NH2-terminal (B) site from Fe3+ bound at the COOH-terminal (A) site. Complexes of monoferric(A)-monocupric(B) transferrin, monocupric(B) transferrin, and monogallium(A)-monocupric(B) transferrin were prepared under identical conditions. Computer simulation and direct measurement of the EPR spectra from these complexes demonstrate a paramagnetic broadening of 0.6 +/- 0.1 G in the line width of the Cu2+ spectrum due to the paramagnetic Fe3+ bound at the other site. The distance between the two metal binding sites is calculated to be 41.6 +/- 2.8 A.

Binding Sites↗
PubMed1982

Electron spin echo studies of the copper complexes of conalbumin.

Electron spin echo envelope spectroscopy was used to probe the two metal binding sites of Cu(II)-conalbumin. The echo envelope spectrum of Cu(II)-conalbumin-oxalate, with metal ion at either one or both of the binding sites, contains lines arising from the interaction of the electron spin of Cu(II) with bound imidazole, demonstrating histidine ligation to the metal ion. The 13C superhyperfine interaction of bound [13C]oxalate, obtained from the ratio of the electron spin echo envelopes of Cu(II)-conalbumin-[13C]oxalate to that of Cu(II)-conalbumin-[12C]oxalate, is about twice the free precession frequency and indicates a contact interaction between 13C and Cu(II). This study indicates that oxalate is directly coordinated to the metal ion. Over the pH range 7.0 to 10.0, where Cu(II)-conalbumin binds carbonate as an associated anion, the echo envelope spectrum indicates that at least one imidazole ligand is coordinated to Cu(II). Below pH 6.0 and above pH 11.0, imidazole coordination is not observed.

Binding Sites↗
PubMed1980

Genetic differences in phenytoin pharmacokinetics. In vivo clearance and in vitro metabolism among inbred strains of mice.

Plasma phenytoin elimination rates were examined among twelve inbred strains of mice. Two populations are identified--the 'fast metabolizers' (BALB/cN, C57BL/6N, C57BL/6J, AKR/N, AKR/J and C3H/HeN) having almost exactly twice as rapid an elimination rate as the 'slow metabolizers' (CL/FR, CBA/J, DBA/2N, STAR/N, SJL/N, DBA/2J and RF/N). The difference in elimination rate between C57BL/6J and DBA/2J cannot be accounted for by dissimilarities in volume of distribution. The phenytoin elimination rate in the (C57BL/6J)(DBA/2J)F1 heterozygote is expressed as an additive trait. A good correlation exists between phenytoin elimination rates in vivo and phenytoin metabolism by liver microsomes in vitro, as determined by a newly described assay using high-performance liquid chromatography. 3-Methylcholanthrene pretreatment does not enhance phenytoin elimination or metabolism. The cytochrome P-450-mediated monoxygenase metabolism of phenytoin is not associated with the Ah locus or with coat color among progeny of the (C57BL/6N)(DBA/2N) F1 x DBA/2N backcross. Phenobarbital pretreatment enhances phenytoin elimination and metabolism in both a fast metabolizer (C57BL/6N) and a slow metabolizer (DBA/2N) strain. Phenobarbital pretreatment probably also induces non-P-450 enzymes, such as those which form the phenytoin dihydrodiol and the glucuronide and glutathione conjugates, in addition to inducing one or more forms of P-450 that oxygenate phenytoin. These data probably reflect allelic differences in a structural gene encoding for one (or more) form(s) of control cytochrome P-450 that metabolizes phenytoin, rather than allelic differences in a regulatory gene. The marked sensitivity of inbred mouse strains CL/FR and A/J and the marked resistance of STAR/N, Swiss-Webster, and C57BL/6 to phenytoin-induced cleft lip and/or palate cannot be explained by genetic differences in phenytoin elimination rates or liver microsomal metabolism in vitro, as measured by the methods described in this report.

Animals↗