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M J Stuart

Publications and source records attributed to M J Stuart.

At least 91 records · Page 5Linked to original sources
PubMed1985

Re-examination of the assay for plasma prostanoids by solid-phase extraction, and radioimmunoassay.

A method for the routine assay of plasma thromboxane and prostacyclin metabolites, thromboxane B2 and 6 keto-prostaglandin F1 alpha (employing solid phase extraction to remove the metabolites from interfering substances) and using commercially available antisera for their radioimmunoassay has been investigated. Modifications of previously published procedures have been explored, including the use of non-explosive solvents in the extraction procedure, and inclusion of a non-cross reacting internal standard, prostaglandin D2, to estimate recovery through the extraction procedures. Both the original and modified procedures can produce high blank values, which apparently result from the solid-phase extraction. These high backgrounds are constant for a given lot of solid phase extraction cartridges and can be corrected by subtraction. The modified method is linear with volume of plasma assayed to as little as one ml, and all cross-reacting material in normal human plasma was found to co-chromatograph with authentic standards on reverse-phase high performance liquid chromatography. Values for normal adult plasmas were found to be 0.44 +/- .15 pmol/ml 6 keto PGF1 alpha (mean +/- SD) and .103 +/- .07 for thromboxane B2. The method reported provides a convenient, reproducible way to assay these important plasma prostanoids.

Adolescent↗
PubMed1985

In vitro comparison of the efficacy of cyclooxygenase inhibitors on the adult versus neonatal platelet.

Neonates manifest more hemorrhagic tendencies when exposed to either acetylsalicylic acid (ASA) or indomethacin in comparison to adults. We therefore assessed the susceptibility of neonatal and adult platelets to the effects of these cyclooxygenase inhibitors in vitro. Baseline thromboxane B2 production in response to thrombin was similar in platelets from the adult and neonate. Following exposure to varying concentrations (0.5-100 microM) of either ASA or indomethacin, platelet thromboxane B2 was inhibited to a similar extent in both adult and neonatal platelets. Our study demonstrates that the enhanced tendency to bleeding observed in the neonate following exposure to ASA or indomethacin is not due to an enhanced susceptibility of the neonatal platelet enzyme to the effects of cyclooxygenase inhibition.

Adult↗
PubMed1985

Effect of changes in oxygen tension on vascular and platelet hydroxyacid metabolites. II. Hypoxia increases 15-hydroxyeicosatetraenoic acid, a proangiogenic metabolite.

Current strict O2 management may be precipitating more severe retinopathy of prematurity than would occur with a more lenient approach. Hypoxemia in an animal model has also been found to worsen retinal neovascularization. It has recently been shown that the hydroxyeicosatetraenoic acids can modulate angiogenesis. 15-Hydroxyeicosatetraenoic acid is proangiogenic, whereas 12-hydroxyeicosatetraenoic acid is an antiangiogenic metabolite. In vitro exposure of paired human neonatal vessels (n = 7) to hypoxia enhanced the production of total vascular hydroxyacids (232 +/- 36 pmol/mg of protein [experimental group] nu 168 +/- 31 pmol [control group]; P less than .01). The increase in vascular 15-hydroxyeicosatetraenoic acid under hypoxic conditions was even more significant (P less than .001). However, platelet production of 12-hydroxyeicosatetraenoic acid was not significantly affected by hypoxia. These observations suggest a possible biochemical basis for the abnormal angiogenic process that occurs during the proliferation phase of the retinopathy of prematurity. The production of local hydroxyeicosatetraenoic acids in tissues manifesting abnormal neovascularization needs to be further evaluated.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1985

Inhibition of human malignant neuroblastoma cell DNA synthesis by lipoxygenase metabolites of arachidonic acid.

In vivo studies have shown that inhibitors of cyclooxygenase metabolism of arachidonic acid may diminish growth and metastasis of certain tumors. Because cyclooxygenase inhibition may increase the production of lipoxygenase products of arachidonic acid metabolism, we have investigated the effect of two such products, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on tumor cell proliferation in vitro. When neuroblastoma cells (SK-N-SH) in culture were treated with 12-HETE for 18 hr, incorporation of [3H]thymidine was inhibited up to 64% at concentrations from 20 to 50 microM. Under the same conditions, 15-HETE resulted in inhibition of up to 46%, while arachidonic acid had no apparent effect. When evaluated in the presence of serum, 12-HETE at a concentration of 120 microM produced a 20.6 +/- 2.8% (S.E.) inhibition of the increase in total DNA content over 48 hr, while 15-HETE at this concentration produced a 16.5 +/- 5.3% inhibition. We conclude that 12-HETE, the product of platelet lipoxygenase, and 15-HETE, a product of neutrophil and lymphocyte lipoxygenases, can inhibit human neuroblastoma cell growth in vitro and may play a role in the effect of cyclooxygenase inhibitors on tumor growth in vivo.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1985

Megakaryocyte thromboxane production induced by platelet stimuli during in vitro culture.

Megakaryocytes isolated from guinea pigs produced thromboxane (assayed by radioimmunoassay as thromboxane B2) in response to the platelet aggregatory stimuli arachidonic acid, thrombin, and the calcium ionophore A23187. The relative responses to these stimuli were similar in megakaryocytes and platelets from the same animals. When the megakaryocytes were maintained in short-term in vitro culture, all three stimuli still elicited thromboxane production. Following overnight in vitro culture, thromboxane production in response to thrombin decreased, overall, to under 60% of control values, increased approximately threefold in response to A23187, but did not show any alteration in response to arachidonic acid. Requirements for calcium were virtually unchanged. These results demonstrate that megakaryocytes contain all of the pathways needed for arachidonic acid mobilization from phospholipids in response to thrombin or A23187 and conversion of that arachidonate to thromboxane. These pathways are retained by the cells in short-term in vitro culture.

Animals↗
PubMed1984

Kinetic analyses of the effects of hyperoxia and hypoxia on vascular cyclooxygenase activity in vitro.

Kinetic analyses were performed to understand the mechanism of hyperoxic induced inhibition of prostacyclin synthesis by human umbilical arteries. Brief exposure of arterial segments to oxygen resulted in over 30% decrease in Vmax of cyclooxygenase in treated vessels. In contrast, cyclooxygenase from hypoxic arterial segments showed approximately a 49% increase in Vmax. There were no significant differences in apparent Km values. These studies suggest that the decreased production of prostacyclin by hyperoxic tissue is due to cyclooxygenase inactivation.

Arachidonic Acid↗
PubMed1984

Recovery of megakaryocyte thromboxane production in vitro after aspirin inhibition.

Megakaryocytes isolated in high purity from guinea pigs produced thromboxane B2 in response to exogenously provided arachidonic acid. This production was inhibited by in vitro treatment with acetylsalicylic acid with a concentration response relationship similar to that seen in platelets. During in vitro culture, the aspirin-treated megakaryocytes recovered thromboxane synthetic ability. Following a lag of 12 hours, recovery of megakaryocyte thromboxane production resumed at a rate of 16% of control per day. This recovery was inhibited by the addition of cycloheximide to the culture medium.

Animals↗
PubMed1984

Effects of hydrogen peroxide on vascular arachidonic acid metabolism.

Hydrogen peroxide (H2O2) released by granulocytes during phagocytosis has previously been demonstrated to affect the function of other cellular elements including red cells and platelets. We have evaluated the effect of H2O2 on vascular arachidonic acid (AA) metabolism. Exposure of human vascular segments to H2O2 (25 to 200 microM) results in a concentration dependent inhibition in the ability of these vessels to produce PGI2 either from endogenous stores of AA, or from exogenously provided substrate. The inhibition of PGI2 production was present at 5 minutes post addition of H2O2, with maximal inhibitory effect occurring by 15 minutes. Production of 6 Keto PGF1 alpha from exogenously provided 14C AA was similarly inhibited in isolated microsomes from these vessels, as was the production of the other vascular cyclo-oxygenase metabolites PGE2 and PGF2 alpha. These results demonstrate that the major effect of H2O2 on vascular AA metabolism appears to occur at the cyclo-oxygenase level. Vascular inhibition of PGI2 formation caused by the local release of H2O2 from phagocytizing cellular elements may play a role in the pathophysiology of the inflammatory process.

6-Ketoprostaglandin F1 alpha↗
PubMed1984

Differences in thromboxane production between neonatal and adult platelets in response to arachidonic acid and epinephrine.

In this study, we have investigated the possible role of the pro-aggregatory arachidonic acid (AA) metabolite thromboxane, in the impaired function of neonatal platelets. In platelet-rich plasma thromboxane production (measured by radioimmunoassay of thromboxane B2) was not different between neonates and adults when stimulated by thrombin (at 0.1 or 1.0 U/ml) or collagen (70 micrograms/ml) although neonatal platelets produced decreased thromboxane (TBX2) postepinephrine stimulation. In response to 1 U/ml thrombin, adult and neonatal platelet-rich plasmas produced mean values of 3.41 +/- 0.35 (SEM) and 3.11 +/- 0.49 pmol of TXB2/10(6) platelets, respectively. Production of TXB2 in response to 0.1 U/ml thrombin was not dissimilar between neonates (1.01 +/- 0.46 pmol) and adults (1.04 +/- 0.38 pmol). When collagen was used as the aggregating agent, TXB2 production was also not significantly different with values of 2.44 +/- 0.48 and 1.90 +/- 0.46 pmol/10(6) platelets produced by adult and neonatal platelet-rich plasma, respectively. In response to 200 microM epinephrine, adult platelets produced 1.03 +/- 0.39 pmol TXB2/10(6) platelets while neonatal platelet TXB2 production was significantly decreased (0.15 +/- 0.04; P less than 0.05). Thromboxane production in response to AA, however, was markedly elevated in neonatal platelet-rich plasma. When 200 and 400 microM concentrations of AA were used as the aggregating stimuli, neonatal platelet rich plasma produced 3.17 +/- 0.77 and 8.0 +/- 1.47 pmol TXB2/10(6) platelets, respectively. These values were significantly elevated P less than 0.02 and less than 0.005) when compared to mean values of 0.41 +/- 0.10 and 3.32 +/- 0.15 pmol in adult platelet-rich plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1984

Effects of hyperoxia and hypoxia on vascular prostacyclin formation in vitro.

Exposure to high oxygen (O2) concentrations, especially in the neonate, is associated with the development of pathologic syndromes characterized by vascular involvement including the retinopathy of prematurity. Some of the initial vascular changes observed appear consistent with a reduction in prostacyclin formation. Exposure of human umbilical arteries to oxygen resulted in more than 30% inhibition in the ability of the vessels to produce prostacyclin either from endogenous stores of arachidonic acid or from exogenously provided substrate. In contrast, hypoxia (which more closely approximates the fetal environment) resulted in more than 30% stimulation in the production of prostacyclin from either endogenous or exogenous arachidonic acid. When microsomes were prepared from treated arterial segments, these effects persisted. In vitro results suggest that neonates exposed to O2 after delivery may experience a marked decrease in vascular prostacyclin formation. Inhibition of the production of this potent vasodilator and antithrombotic metabolite could play an important role in the acute exudative phase of O2 toxicity.

6-Ketoprostaglandin F1 alpha↗
PubMed1982

Effects of acetylsalicylic-acid ingestion on maternal and neonatal hemostasis.

In a case-control study, we evaluated the effects of maternal ingestion of acetylsalicylic acid (aspirin) within 10 days of delivery on maternal and neonatal hemostasis. Only one of 34 control maternal-neonatal pairs (3 per cent) had hemostatic abnormalities. In 10 pairs, when maternal aspirin ingestion occurred within five days of delivery, 6 of 10 mothers and 9 of the 10 infants had bleeding tendencies. Seven maternal-neonatal pairs in which aspirin was ingested 6 to 10 days before delivery were free of clinical bleeding. Among seven other mothers who ingested aspirin in the immediate post-partum period four of the seven (57 per cent) also had impaired hemostasis. Neonatal hemostatic abnormalities included numerous petechiae over the presenting part, hematuria, a cephalhematoma, subconjunctival hemorrhage, and bleeding from a circumcision. Maternal bleeding was confined to excessive intrapartum or post-partum blood loss. We conclude that aspirin should be avoided during pregnancy. If ingestion has occurred within five days of delivery, the neonate should be evaluated for the presence of bleeding.

Adult↗
PubMed1982

Effect of homocysteine and homocystine on platelet and vascular arachidonic acid metabolism.

Normal hemostasis depends in part on the balance achieved between proaggregatory and prothrombotic platelet thromboxane A2, measured as its stable end-product thromboxane B2 (TXB2), and vascular prostacyclin (PGI2), which inhibits platelet aggregation and is antithrombotic. Cystathionine-beta-synthase deficiency is characterized by a high frequency of thromboembolic disease. We therefore studied, in vitro, the effects of homocysteine and related compounds on platelet TXB2 and vascular PGI2 formation. In paired samples of platelet rich plasma, which had been preincubated with L-homocystine (1 mM), mean production of the two platelet cyclooxygenase products, TXB2 and 12-hydroxy-5, 8,10-heptadecatrienoic acid increased significantly from control levels [13.6% +/- 1.9 to 19.8% +/- 2.1 (P less than 0.02) TXB2 and 29.8% +/- 4.2 to 39.4% +/- 4.1 (P less than 0.01) HHT]. In the presence of D,L-homocysteine (1 mM), mean TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid production was also significantly increased [12.7% +/- 1.5 to 16.9% +/- 1.5 (P less than 0.01) TXB2 and 27% +/- 4 to 31% +/- 4.1 (P less than 0.02) HHT]. Cystine, cysteine, or methionine (1 mM) did not have similar effects in this test system. Homocysteine and homocystine were without effect on the synthesis of vascular PGI2 by umbilical artery segments [control, 0.22 +/- 0.03 to 0.21 +/- 0.03 ng/mg with D,L-homocysteine and 0.20 +/- 0.04 control to 0.19 +/- 0.04 ng/mg with D,L-homocystine]. A homocyst(e)ine-induced increase in platelet thromboxane production in the absence of an increase in vascular prostacyclin, if present in vivo, may contribute to the vascular thromboses characteristic of human homocystinemias (homocystinurias).

Arachidonic Acid↗