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R C Murphy

Publications and source records attributed to R C Murphy.

At least 145 records · Page 8Linked to original sources
PubMed1987

Metabolism of platelet-activating factor in isolated perfused rat lung.

The administration of platelet-activating factor (PAF) into the airway system of the lung is known to cause profound effects, yet little is known about the metabolism of this active lipid mediator. 3H-Labeled PAF administered into the airway of isolated rat lungs was rapidly and extensively metabolized. The tissue retained 96% of the administered radiolabel while the perfusate contained 4%. Characterization of the tissue retained lipid indicated metabolism into lyso-PAF (3.3%), phosphatidylcholine (82.3%), neutral lipid (1.7%) and intact PAF (10.2%). Analysis of tissue phosphatidylcholine by mass spectrometric techniques revealed metabolism of PAF to 1-0-hexadecyl-2-arachidonoyl-GPC, which represented 20-23% of the administered radiolabeled hexadecyl-PAF. These findings support the hypothesis that a relationship between PAF and arachidonate metabolism exists at the intact organ level. Autoradiographic analysis of the cellular distribution of the radiolabeled PAF metabolites in the lung tissue indicated labeling of two cell types, the alveolar type II cell and the nonciliated bronchiolar epithelial cell (Clara cell).

Animals↗
PubMed1987

Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry.

Two physicochemical methods have been developed for the quantitative analysis of lyso-platelet activating factor (lyso-PAF) based on gas-liquid chromatography-mass spectrometry (GLC/MS) and fast atom bombardment-mass spectrometry (FAB/MS) using stable isotope dilution. After addition of deuterated internal standards, lyso-PAF produced from neutrophils was purified by silicic acid chromatography and thin-layer chromatography (TLC). The GLC/MS assay employed phospholipase C or hydrofluoric acid for hydrolysis of the phosphocholine moiety to yield ether monoglycerides. Condensation of monoglycerides with acetone yielded the 1-O-alkyl-2,3-isopropylidene glycerol which could be analyzed by GLC/MS. The ions corresponding to M-15 fragments for both the labeled and unlabeled derivatives were monitored in a selected ion recording mode. Standard curves were found to be linear over the range tested (10-2000 ng) with a limit of detection found to be below 200 pg injected on column. For the FAB/MS assay, the unmodified lyso-PAF was well suited for direct analysis; however, the limit of detection (S/N greater than 3) using a glycerol matrix was found to be 5 ng placed on the probe tip. It was found that human neutrophils contain approximately 300 pg/10(6) cells which increased 2-3-fold during the 5-min period following challenge with 1.9 microM calcium ionophore, A23187. Two molecular species of lyso-PAF were identified as hexadecyl and octadecyl ethers at sn-1 with the octadecyl molecular species of lyso-PAF predominating in abundance after stimulation.

Calcimycin↗
PubMed1987

omega-Hydroxylation of 15-hydroxyeicosatetraenoic acid by lung microsomes from pregnant rabbits.

15-Hydroxyeicosatetraenoic acid (15-HETE) was converted by lung microsomes from pregnant rabbits to a polar metabolite that was identified by mass spectrometry as the 15,20-dihydroxyeicosatetraenoic acid. The formation of the 20- or omega-hydroxylated product was NADPH dependent, with a specific activity of 1.87 +/- 0.53 nmol/min/mg of microsomal protein. Other hydroxylated derivatives of eicosatetraenoic acid that possessed hydroxy groups at the 5- and 12-carbon atoms were not metabolized by the lung microsomes. This hydroxylation of 15-HETE was observed in lung microsomes of pregnant rabbits and only minor amounts were formed by nonpregnant rabbits. The specific activity for 15-HETE omega-hydroxylation was similar to the value obtained for prostaglandin E1 (1.48 +/- 0.33 nmol/min/mg). It is known that rabbit lungs possess a cytochrome P-450 that is induced during pregnancy and catalyzes the 20-hydroxylation of prostaglandins. The addition of the antibody to cytochrome P-450 prostaglandin omega-hydroxylase or prostaglandin E1, a substrate of this enzyme, resulted in potent inhibition of 15-HETE omega-hydroxylation, providing strong evidence that a common cytochrome P-450 catalyzes the omega-hydroxylation of both prostaglandins and 15-HETE.

Animals↗
PubMed1986

Remodeling of arachidonate-containing phosphoglycerides within the human neutrophil.

Initial incorporation and subsequent remodeling of 16 phosphoglyceride molecular species containing arachidonate in the human neutrophil have been studied. Neutrophils were pulse-labeled with [3H]arachidonic acid (AA) for 5 min, then phospholipids were analyzed either at this time point or after a subsequent 120-min incubation. [3H]AA was found to be incorporated into phosphoglycerides phosphatidylinositol (PI) greater than phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) by 5 min. Incorporation of [3H]AA was not related to pool size, but reflected an increase in phosphoglyceride turnover. Following the 120-min incubation, only PE gained a significant amount of labeled arachidonate. Specific activity analysis revealed that PI contained the highest labeled/unlabeled ratio at both 5 min and 120 min. After the initial 5-min pulse, the majority of [3H]arachidonate was incorporated into 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-PC, -PE, and -PI showing no preference for fatty acyl chains at the sn-1 position. However, [3H]AA was remodeled into 1-alkyl-acyl-and 1-alk-1-enyl-acyl-sn-glycero-3-PC and -PE molecular species in those neutrophils incubated for the additional 120 min. Specific activities of [3H]AA within all diacyl molecular species were initially higher relative to those alkyl-acyl and alk-1-enyl-acyl molecular species, but for PC and PE became more uniform as label shifted into ether and plasmalogen pools during the additional 120-min incubation. In contrast, the specific activity of 1-stearoyl-2-arachidonoyl-sn-glycero-3-PI remained constant throughout the 120-min incubation.

Arachidonic Acid↗
PubMed1986

Metabolism of leukotriene B4 in isolated rat hepatocytes. Identification of a novel 18-carboxy-19,20-dinor leukotriene B4 metabolite.

Isolated rat heptocytes were found to metabolize leukotriene B4 (LTB4) to a number of products which could be separated by reverse phase high performance liquid chromatography (HPLC). After incubation of LTB4 with hepatocytes for 15 min, the known omega-oxidized metabolites, 20-hydroxy- and 20-carboxy-LTB4, were identified by HPLC retention time and gas chromatography-mass spectrometry. An early fraction corresponding to 15% of the initial LTB4 was structurally characterized as a novel metabolite, 18-carboxy-19,20-dinor-LTB4, by ultraviolet spectroscopy and gas chromatography-mass spectrometry of the derivatized and derivatized, reduced metabolite. The short HPLC retention time of this metabolite was consistent with its reduced lipophilicity. An additional minor metabolite was tentatively identified as 3-hydroxy-LTB4. These two novel metabolites provide evidence for beta-oxidation as an important route of hepatic biotransformation of LTB4 and 20-hydroxy-LTB4.

Chemical Phenomena↗
PubMed1986

Hydrogen peroxide induced pulmonary vasoconstriction in isolated rat lungs is attenuated by U60,257, a leucotriene synthesis blocker.

Reactive oxygen metabolites cause pulmonary vasoconstriction and activate arachidonic acid metabolism. We proposed that hydrogen peroxide, generated enzymatically in an insolated rat lung model, would cause vasoconstriction which was independent of circulating cells, but dependent on activation of the arachidonic acid cascade. Although hydrogen peroxide caused an increase in lung effluent thromboxane B2 concentration, indomethacin did not inhibit hydrogen peroxide induced vasoconstriction. In order to test the hypothesis that hydrogen peroxide activates the 5-lipoxygenase pathway, lung effluents were analyzed for 5-hydroxy-eicosatetranoic acid (5-HETE) using a sensitive, highly specific mass spectrometer technique. Glucose oxidase increased the 5-HEFE effluent concentrations and this was prevented by U60,257. We therefore conclude that hydrogen peroxide stimulates the 5-lipoxygenase pathway and that substances derived from this pathway are at least in part responsible for the hydrogen peroxide induced vasoconstriction in isolated rat lungs.

Animals↗
PubMed1986

Fast atom bombardment analysis of arachidonic acid-containing phosphatidylcholine molecular species.

Fast atom bombardment (FAB) mass spectrometry was evaluated as a means for the characterization of molecular species of glycerophosphocholines (GPC) from HL60 cells. Previous reports of phospholipid molecular species analysis have suggested that several inherent problems with FAB could limit its analytical usefulness in such an application. The GPC-related secondary ions produced by the FAB experiment were found to be dependent on the matrix employed. Triethanolamine was found to minimize mass-related discrimination in ion emission when mixtures were studied; and furthermore, this matrix maintained 60% maximal ion current after 12 minutes as compared to a glycerol matrix which diminished to 10% maximal ion current. Using triethanolamine, the major GPC species in HL60 cells prelabeled with (2H8) arachidonic acid were found to be 16:0a16:1, 16:0e/18:1, 16:0a/18:1, 18:1a/18:1 and 18:0a/18:1. It was possible to identify the minor GPC species containing arachidonic acid only after partial purification by reverse-phase high-performance liquid chromatography. Comparison of the 2H8/2H0 enrichment data estimated by FAB with data obtained by gas chromatographic/mass spectral analysis of arachidonic acid following GPC hydrolysis revealed that the precision of FAB was less precise than gas chromatography/mass spectrometry. Yet the FAB technique did allow the observation of one unexpected molecular species (18:1a/20:4) due to the fact that the GPC was not degraded to simpler species prior to analysis. In this respect, the two strategies of molecular species analysis complement each other.

Arachidonic Acid↗
PubMed1986

The effect of microbial contamination on musculocutaneous and random flaps.

Although musculocutaneous flaps have been shown to have the ability to withstand a greater inoculum of bacteria than random flaps, it has not been shown that the musculocutaneous flap has any greater ability to decrease the bacterial population in a contaminated wound. In this series of experiments, granulating wounds were developed containing 10(4), 10(5), or 10(6) bacteria per gram of tissue. These contaminated wounds were then covered either with musculocutaneous flaps, random flaps, or left uncovered as a control. In the heavily contaminated wounds containing 10(6) bacteria per gram of tissue, neither type of flap was able to prevent bacterial proliferation and all flaps dehisced. In the minimally contaminated wounds containing 10(4) bacteria per gram of tissue, both the musculocutaneous and random flaps achieved wound healing and decreased the bacterial level in the wound. However, in the intermediate group containing 10(5) bacteria per gram of tissue, musculocutaneous flaps lowered the bacterial count and allowed wound closure, whereas the random flaps did not control the bacterial growth and failed. Therefore, in the moderately contaminated wound, musculocutaneous flaps are advantageous and can decrease bacterial counts and obtain successful closure when random flaps cannot.

Animals↗
PubMed1986

Analysis of leukotriene B4 in human lung lavage by HPLC and mass spectrometry.

Although leukotrienes are believed to mediate symptoms of human lung disease, there is little direct evidence of their existence in the lung. This is due to the difficulty in obtaining lung samples, the small amounts of leukotrienes typically present in such samples and the problems associated with purifying and analyzing leukotrienes in complex biological samples. In this study, lung lavagates were collected and analyzed for leukotrienes. The methods in this analysis included solid phase extraction using a C-18 reverse phase cartridge followed by HPLC using a new photodiode array detector which provides full UV spectra of eluting compounds. Lung lavage fluid from a patient with chronic pulmonary disease contained a compound with a UV spectra of LTB4 which was found to elute with synthetic [3H]-LTB4. This compound was confirmed as LTB4 using gas chromatography/mass spectrometry in the negative ion-chemical ionization mode. The inclusion of oxygen-18 LTB4 as an internal standard allowed approximate quantitation of the amount of LTB4 present in this 5 ml lung lavagate as 40-50 ng.

Chromatography, High Pressure Liquid↗
PubMed1986

Analysis of 6-keto PGF1 alpha, 5-HETE, and LTC4 in rat lung: comparison of GM/MS, RIA, and EIA.

The recent availability of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the lung extract assayed for 6-keto-PGF1 alpha, 5-HETE and LTC4. By EIA lung tissue was found to contain large quantities of 6-keto-PGF1 alpha after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF1 alpha amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF1 alpha, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Pak) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher values obtained by RIA. LTC4 was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC4 levels were identical in unpurified lung extract and after Sep-Pak purification, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproducible results in lung tissue, although LTC4 and 5-HETE must be purified prior to analysis.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Characterization of the arachidonate-containing molecular species of phosphoglycerides in the human neutrophil.

Neutrophils were labeled with high specific activity, [3H]-arachidonate and separated into their arachidonate-containing phosphoglycerides using a slight modification of the normal phase and reverse phase HPLC described by Patton et al. The individual molecular species of PC, PE and PI containing arachidonate were characterized by fast atom bombardment/mass spectrometry techniques. Eight major arachidonate-containing molecular species of PC, six major arachidonate-containing molecular species of PE and two major arachidonate-containing molecular species of PI could be found. With the structures of the phosphoglycerides eluting from the reverse phase HPLC column known, general trends in the HPLC elution behavior of 1-0-alkyl, 1-alk-1-enyl and 1-acyl-2-arachidonoyl phosphoglycerides could be observed.

Arachidonic Acid↗
PubMed1986

Combined prostacyclin and thromboxane synthetase inhibitor UK 38485 in flap survival.

Thromboxane, a prostanoid derivative, is a central mediator of the progressive dermal ischemia seen in the distal dying flap. Prostacyclin; a vasoactive prostanoid derivative, has been found to enhance ischemic flap survival. This study examines the effects of prostacyclin and UK 38485 (specific thromboxane synthetase inhibitor), separately and combined, in axial flap survival in the pig. Each increased flap survival over control flaps; their combined use demonstrated an even greater flap survival (p less than 0.005).

Animals↗
PubMed1986

On the conversion of verapamil to carboxy verapamil.

The reaction of verapamil with concentrated sulfuric acid was studied. It was found, in disagreement with a published report, that the reaction did not hydrolyze the nitrile group of the drug to the corresponding carboxylic acid, but, instead, produced a sulfonated derivative. Analysis of the product using several spectroscopic techniques indicated that the sulfonic acid moiety was introduced into the verapamil molecule at the 6-position of the 3,4-dimethoxyphenyl ring distant to the chiral center.

Chemical Phenomena↗
PubMed1985

Alpha-methyl-para-tyrosine effects in mice selectively bred for differences in sensitivity to ethanol.

The responses of catecholamine systems in long sleep (LS) and short sleep (SS) mice to alpha-methyl-p-tyrosine (AMPT) have been examined. Marked differences were found between LS and SS mice in the dose necessary for maximal brain catecholamine depletion and in the time-course of the catecholamine depletion. Brain catecholamines in the LS mice were depleted by lower doses of AMPT and the levels remained depressed for longer periods of time in this line of mice. These differences may be explained only partially by an increased susceptibility of the LS mice to the hypothermia and toxic effects caused by AMPT administration, as they persist with non-toxic AMPT dosage regimens and under conditions where the degree of hypothermia is comparable in both lines of mice. In addition, there were no differences between the Ki values for the effect of AMPT on the tyrosine hydroxylase from striata of these mouse lines. The primary cause of the heightened response to AMPT in LS mice would appear to be pharmacokinetic in nature, as brain and plasma peak levels of AMPT in LS mice were greater and the levels remained higher for a longer time. The depletion of brain tyrosine by AMPT combined with the lower affinity of the LS striatal tyrosine hydroxylase for the substrate tyrosine may also contribute to the heightened response in LS mice.

Animals↗
PubMed1985

Effect of alcohols on arachidonic acid metabolism in murine mastocytoma cells and human polymorphonuclear leukocytes.

The effects of alcohols on the formation of leukotrienes, 5-HETE and prostaglandin D2 in mastocytoma cells and human neutrophils were studied. In murine mastocytoma cells, alcohols appear to have at least two different effects on the production of these arachidonic acid metabolites. At low levels of cellular arachidonic acid achieved after stimulation with calcium ionophore A23187 or addition of low levels of exogenous arachidonic acid, alcohols appear to have a general inhibitory effect on the production of lipoxygenase metabolites. In the presence of higher concentrations of cellular arachidonic acid, ethanol and methanol stimulated the production of lipoxygenase metabolites, but had no large stimulatory effect on the cyclo-oxygenase metabolite, prostaglandin D2. Under these conditions, n-propanol and t-butanol have inhibitory effects on leukotriene production. Human neutrophils are less sensitive to ethanol than mastocytoma cells, but stimulatory effects were still found at high ethanol concentrations (220-430 mM).

Alcohols↗