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Biomedical subjects

T R Green

Publications and source records attributed to T R Green.

At least 19 recordsLinked to original sources
PubMed1992

Serial ECGs are less accurate than serial CK-MB results for emergency department diagnosis of myocardial infarction.

HYPOTHESIS: Serial creatine kinase-MB (CK-MB) levels provide more accurate predictive information regarding myocardial infarction than serial ECGs in emergency department patients with chest discomfort and no ST-segment elevation on the initial ECG. DESIGN: Prospective, observational study. SETTING: University hospital and university-affiliated Veterans Affairs Medical Center EDs. PARTICIPANTS: Two hundred sixty-one patients 30 years or older with chest discomfort warranting an ECG and consenting to observation. Exclusions included hemodynamic or rhythm instability and ST-segment elevation of 0.1 mV or more in two or more electrically contiguous leads at presentation. MEASUREMENTS: ECGs were obtained at presentation and three to four hours after presentation. Significant serial ECG changes sought on comparison of initial and three- to four-hour ECGs were 0.05 mV or more ST elevation or depression, Q-wave development, or T-wave inversion changes in two or more electrically contiguous leads. CK-MB levels were obtained at presentation and hourly for three hours (positive level, 8 or more ng/mL). Myocardial infarction was determined by record review and was based on independent CK-MB measurements. RESULTS: Twenty-eight (11%) patients were diagnosed with a myocardial infarction. Thirty-eight (15%) patients had a serial ECG change. Eleven of the myocardial infarction patients (39%) had a serial ECG change compared with 27 (12%) of the non-myocardial infarction patients (P < .001). Sensitivities and specificities of a serial ECG change versus serial CK-MBs for myocardial infarction were 39% versus 68% (sensitivity) and 88% versus 95% (specificity), respectively. Serial CK-MBs were more accurate than a serial ECG change for predicting myocardial infarction (P < .03). CONCLUSION: Serial changes in ECGs during a three- to four-hour interval were associated with the diagnosis of myocardial infarction but were infrequent and less accurate than serial CK-MB levels obtained for the same interval.

Adult↗
PubMed1991

Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils.

The subcellular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotaurine and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50:1. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiring cells by exposure to opsonized zymosan. These results prompted in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypotaurine competes with 5,5'-dimethyl-1-pyrroline N-oxide) (DMPO) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.

Antioxidants↗
PubMed1991

Do CK-MB results affect chest pain decision making in the emergency department?

STUDY OBJECTIVE: To analyze the effect of creatine kinase isoenzyme (CK-MB) results on decision making in the evaluation of emergency department patients with chest pain. DESIGN: Prospective, controlled observational study of clinical decision making. SETTING: EDs of two teaching hospitals, a Veterans Affairs medical center, and a medical school university hospital. TYPE OF PARTICIPANTS: Patients more than 30 years old complaining of chest discomfort warranting an ECG. Excluded were hemodynamically unstable patients and patients with ECG evidence of an acute myocardial infarction (AMI). INTERVENTIONS: After the initial assessment including ECG but not CK-MB data, physicians answered questions regarding estimated probability of AMI and disposition plans. CK-MB levels were drawn every hour for as long as three hours (from one to four CK-MBs) with results readily available. Physicians could admit their patients into the hospital at any time. At disposition after reviewing a second ECG and all available CK-MB data, the physicians answered the same questions and rank ordered the contribution to disposition of the following six factors: initial and serial clinical evaluations, initial and serial ECGs, and initial and serial CK-MB enzymes, respectively. The absolute log likelihood ratio test measured the contribution of CK-MB to decision certainty. MEASUREMENTS AND MAIN RESULTS: Three hundred seventy-six patients were studied: 29 (7.7%) with AMI and 347 without AMI (nonAMI). Physicians indicated that CK-MB levels were useful for more than one third of study patients. When considered useful, CK-MB results strengthened the impression of AMI in AMI patients and decreased the impression of AMI for nonAMI patients; CK-MB also correlated with the perceived need for cardiac care unit admission in AMI patients and with a reduced need to admit nonAMI patients. The use of CK-MB results did not significantly increase ED release rates. CONCLUSION: The rapid availability of serial CK-MB results appears to affect decision making in one third of ED patients with chest pain and nondiagnostic ECGs. CK-MB levels appear to complement clinical evaluation of the ED chest pain patient in a manner analogous to the ECG.

Adult↗
PubMed1990

Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils.

Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate. Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions. Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex. Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns. Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex. When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.

Carrier Proteins↗
PubMed1988

Purification of the solubilized NADPH:O2 oxidoreductase of human neutrophils. Isolation of its catalytically inactive cytochrome b and flavoprotein redox centers.

The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.

Cell Membrane↗
PubMed1987

A reassessment of the product specificity of the NADPH:O2 oxidoreductase of human neutrophils.

Native ferricytochrome c, but not acetylated ferricytochrome c, stimulates the flow of electron equivalents passing through the neutrophil NADPH:O2 oxidoreductase complex. At 28 mM it increases NADPH oxidase activity by 157 +/- 15% (n = 5) over that measured in its absence. Enhanced activity is predominantly seen in oxidoreductase-rich 27,000 X g membrane preparations obtained from phorbol myristate acetate activated cells. Superoxide formation is also enhanced. Although some of the stimulatory activity seen with addition of native ferricytochrome c to oxidoreductase-rich membrane suspensions might have been explained in terms of mitochondrial contamination, this was ruled out. Comparable membrane preparations from resting cells were devoid of NADPH oxidase activity. Azide, a well-known inhibitor of the electron transport chain, did not block the enhancing effect of native ferricytochrome c. These results indicate that native ferricytochrome c is not a suitable scavenger of superoxide in quantitating the product specificity of the oxidoreductase since it amplifies the apparent rate of superoxide formation with respect to measured rates of NADPH oxidation conducted in its absence. By using acetylated ferricytochrome c in place of native ferricytochrome c in quantitating the product specificity of the oxidoreductase we show that no more than 70% of the electron equivalents donated by NADPH to the oxidoreductase are involved in superoxide formation. The remaining 30% of the electron equivalents given up by NADPH to the oxidoreductase appear to be involved in direct formation of hydrogen peroxide.

Acetylation↗
PubMed1986

The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2.

The ratio of superoxide production to oxidation of NADPH affected by the NADPH:O2 oxidoreductase of human neutrophils is strongly influenced by pH, NADPH substrate concentration, aging of the enzyme, or its exposure to excess deoxycholate. Freshly prepared enzyme exhibited a Km for NADPH of 52 microM as determined by assaying NADPH oxidase activity, or approximately 33 microM by measurement of superoxide formation. In the range of 100-150 microM NADPH at pH 7.6 and in the presence of 0.06% deoxycholate, the univalent flux of electron equivalents given up by NADPH to O2 was 99%. Following storage of the oxidoreductase overnight on ice, its Km for NADPH rose to 125 microM as determined by monitoring oxidation of NADPH but was unaltered when measured in terms of superoxide production. Concomitantly, its capacity to affect univalent reduction of O2 fell approximately 20-30% under the same assay conditions. Univalent flux rates of less than 40% were observed with exposure of the enzyme to concentrations of deoxycholate in excess of 0.1% or to pH values below 6.0 or above 8.0. The capacity of the enzyme to carry out univalent reduction fell with increasing NADPH concentrations in a manner resembling that previously reported with increasing concentrations of xanthine in the case of xanthine oxidase (Fridovich, I. (1970) J. Biol. Chem. 245, 4053-4057). The reduced form of the neutrophil oxidoreductase, like xanthine oxidase, thus appears to be capable of conducting both 1- and 2-electron transfer steps in reducing O2. Its capacity to affect univalent reduction of O2 depends upon the concentration of electron donor (NADPH) supplied as well as conditions of storage and assay of the native enzyme.

Deoxycholic Acid↗
PubMed1986

Diagnostic value of creatine kinase and creatine kinase MB isoenzyme in chronic hemodialysis patients: a longitudinal study.

Serial measurements of total creatine kinase (CK) and the MB isoenzyme of CK (CK-MB) were performed on 18 stable hemodialysis patients over a 36 month interval in a longitudinal study designed to examine CK and CK-MB variability. Total CK was measured by the DuPont ACA pack procedure. CK-MB was quantitated by the ACA CK-MB ion exchange pack procedure, and by electrophoresis. The mean CK level was 242 +/- 179 IU/l (+/- SD). The mean CK-MB level was approximately 8% of the total CK. Seventy-two percent of the patients had at least one elevated CK level during the course of this study. Depending upon the method of analysis, 88 to 100% of the patients had at least one elevated CK-MB level. Marked and sporadic variations in both total CK and CK-MB levels were apparent, but were not related to the assay. Patients receiving long-term intramuscular androgens had higher CK, but not CK-MB, than did hemodialysis patients not receiving these agents. Discontinuation of the intramuscular androgens for four weeks led to a fall in CK in only one of four patients. Therefore there appears to be a biological effect of exogenous androgens which may play a role in elevating CK in hemodialysis patients.

Adult↗
PubMed1985

Myeloperoxidase oxidation of sulfur-centered and benzoic acid hydroxyl radical scavengers.

Myeloperoxidase (MPO) oxidizes sulfur-centered and benzoate hydroxyl radical scavengers through formation of HOCl. Sulfur-centered hydroxyl radical scavengers compete with benzoate as antioxidants of HOCl. We conclude from these observations that competition experiments between benzoate and sulfur-centered hydroxyl radical scavengers are not sufficiently specific to infer participation of hydroxyl radicals in oxidative reactions mediated by neutrophils because of the unique action of MPO in affecting oxidation of the test radical scavengers.

Benzoates↗
PubMed1985

Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils.

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.

Chromatography, Gel↗
PubMed1985

Detection of NADPH diaphorase activity associated with human neutrophil NADPH-O2 oxidoreductase activity.

At approximately equimolar concentrations (approximately 70 microM), and in the presence of excess catalase and superoxide dismutase, DCIP, ferricytochrome c and ferricyanide abstracted 21, 6 and 61%, respectively, of the electron equivalents given up by NADPH to the NADPH-O2 oxidoreductase complex derived from phorbol myristate acetate-stimulated human neutrophils. With a 10-fold increase in ferricyanide, all of the electron equivalents given up by NADPH to the oxidoreductase complex were shunted to ferricyanide concomitant with complete inhibition of NADPH-dependent O2 consumption. These results substantiate the existence of intrinsic diaphorase activity associated with the superoxide generating NADPH-O2 oxidoreductase of human neutrophils.

Ferricyanides↗
PubMed1984

Myeloperoxidase-dependent fluorescein chlorination by stimulated neutrophils.

Hypochlorous acid (HOC1) rapidly chlorinates fluorescein compounds forming, sequentially, the corresponding 4'-chlorofluorescein and 4',5'-dichlorofluoresceins. Chlorination by cell-free myeloperoxidase-catalyzed chloride peroxidation systems gives rise to these compounds as well as variable amounts of isomeric compounds chlorinated in the 2'- and 2',7'-positions. The fluorescence intensity of the dianionic form of the dye is partially quenched upon chlorination, and its proton equilibrium constants are shifted to more acidic values. Fluorescein covalently bound to zymosan (5-isothiocyanatofluorescein-zymosan) also formed these products when the unopsonized particles were incubated with phorbol myristate acetate- or N-formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils. This reaction was associated with a fall in fluorescence intensity, which was not observed when cells from individuals with chronic granulomatous disease or myeloperoxidase deficiency were used or when azide or catalase were added to the reaction medium. Fluorescent changes accompanying phagocytosis of serum-opsonized 5-isothiocyanatofluorescein-zymosan were also consistent with chlorination of the label; the changes were shown to be myeloperoxidase-dependent by use of myeloperoxidase-deficient or azide-treated cells. Oxidative bleaching of the structurally similar sulfonphthalein dyes by HOCl also occurs at rates which parallel the dye basicities. Results are discussed in relation to the use of fluoresceinated particles and sulfonphthalein dyes in the measurement of intraphagosomal acidification.

Chromatography, High Pressure Liquid↗
PubMed1983

Delineation of the catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils.

Four catalytic components of the NADPH-dependent O2- generating oxidoreductase of human neutrophils have been identified. DCIP reductase, cytochrome c reductase and a chromophore 450-455 reductase are present in phorbol myristate acetate stimulated neutrophils and absent in resting cells and phorbol myristate acetate stimulated chronic granulomatous disease cells. Quinol dehydrogenase activity has also been demonstrated in activated and resting cells. Furthermore, a chromophore absorbing in the reduced state at 450-455 nm participates in superoxide production. This chromophore is reduced by NADPH or duroquinol and is missing in cell lysates derived from a patient with chronic granulomatous disease.

Cytochrome Reductases↗
PubMed1983

The O2- generating oxidoreductase of human neutrophils: evidence of an obligatory requirement for calcium and magnesium for expression of catalytic activity.

NADPH-dependent O2- generating oxidoreductase activity recovered from cell lysates of phorbol myristate acetate-stimulated human neutrophils exhibits dependence on Ca+2 and Mg+2 for full expression of its catalytic activity. O2- generating activity was completely abolished by exposure of the oxidoreductase to EDTA, then reconstituted by exposure of the enzyme to Ca+2 and Mg+2 in excess of the EDTA concentration used to block catalytic activity. The oxidoreductase responded maximally to either 0.25 mM Ca+2 or 0.80 mM Mg+2. The pH optimum of the oxidoreductase exposed to Ca+2 and Mg+2 is between pH 7.0 and 7.6. The molar ratio of NADPH oxidation to O2- production determined at pH 7.6 in the presence of Ca+2 and Mg+2 is 0.49, indicating 1 mole of NADPH oxidized per 2 moles of O2- formed. Particulate fractions recovered from cell lysates of resting neutrophils exhibited no oxidoreductase activity under the same conditions.

Calcium↗
PubMed1982

"Outcome-counting'-Significance tests from incomplete predictions of order.

Experiments testing models usually predict that some treatment effects will be greater than others, but they do not as a rule feel able to predict the exact ordering of treatment effects. A useful significance test for such incomplete order predictions can be obtained by comparing the number of possible outcomes (orderings) with the number that satisfy the predictions. The difficulty is to count the outcomes satisfying the predictions, which can be a lengthy task if there are many treatments. A straightforward method of outcome-counting is derived from simple algebra and examples of its use are given.

Humans↗
PubMed1981

Intrinsic dichlorophenolindophenol reductase activity associated with the superoxide-generating oxidoreductase of human granulocytes.

NADPH-dependent dichlorophenolindophenol (DCIP) reductase activity cosediments with NADPH-dependent O(2-)-generating activity in subcellular particulate fractions of zymosan-stimulated human polymorphonuclear leukocytes (PMN's). Subcellular fractions derived from unstimulated PMN's were devoid of both activities, as were fractions from zymosan-stimulated PMN's of a patient known to have chronic granulomatous disease. NADPH-dependent DCIP reduction associated with the oxidoreductase-rich subcellular fractions was unaffected by addition of excess superoxide dismutase sufficient to abolish all traces of O2- production as measured by conversion of ferricytochrome c to its ferrous form. Moreover, DCIP inhibited NADPH-dependent production of O2- in subcellular fractions derived from normal donors. In contrast to the subcellular studies, whole cell suspensions were ineffective in reducing extracellular DCIP despite their capacity to generate O2-, albeit at a lesser rate, in the presence of this electron-accepting dye. These results demonstrate that DCIP reductase activity is associated with the oxidoreductase complex and suggest that it is located on the inner side of the PMN's plasma membrane. The stability of the oxidoreductase complex is markedly improved by storage in glycerol. Both overall O(2-)-generating activity and DCIP reductase activity exhibit a similar pH optimum of 7.0. The Km of the oxidoreductase complex for DCIP is 33 micro M.

Granulocytes↗