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Biomedical subjects

M J Stuart

Publications and source records attributed to M J Stuart.

At least 73 records · Page 4Linked to original sources
PubMed1988

Changes in oxygen tension and effects on cyclooxygenase metabolites: III. Decrease of retinal prostacyclin in kittens exposed to hyperoxia.

The acute phase of oxygen-induced retinopathy is associated with vasoconstriction and occlusion of the retinal vessels. Because this acute vasoobliterative phase could be due to the inhibition in retinal vessels of the production of the potent vasodilator and antithrombotic metabolite prostacyclin, animal experiments were performed to assess this possibility. Eight litters of 27 kittens (four to six days of age) were used. Control kittens were left in room air; hyperoxic kittens were placed in 80% oxygen for 48 hours; recovery kittens were returned to room air for 24 hours following hyperoxic exposure. Following treatments, the animals were killed, retinas isolated, and prostaglandin formation assessed. Retinal tissues produced 6-keto-prostaglandin F1 alpha, prostaglandin F2 alpha, prostaglandin E2, and thromboxane B2 from exogenous arachidonate. A significant (approximately 33%) reduction in retinal 6-keto-prostaglandin F1 alpha (the end product of prostacyclin) was observed both in the hyperoxic and recovery litter mates when compared with controls. Both of the experimental groups also demonstrated a reduction in total retinal prostanoids that paralleled the changes observed in prostacyclin, suggesting that the biochemical effect of hyperoxia on retinal vascular arachidonic acid metabolism occurred at the level of cyclooxygenase. A decrease in the local production of prostacyclin during hyperoxia is consistent with the histologic retinal changes observed during the acute phase of oxygen-induced retinopathy.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Total knee arthroplasty in young adults who have rheumatoid arthritis.

At the Mayo Clinic, between 1978 and 1982, forty-four total knee arthroplasties were performed with cement in twenty-six adults who had rheumatoid arthritis. The age at operation ranged from nineteen to thirty-nine years old. The twenty-six patients were followed for two to eight years (average, five years). At the time of final follow-up, thirty-nine knees were free of pain and five were mildly painful during weight-bearing. The Hospital for Special Surgery knee score improved from a mean of 52 points preoperatively to 84 points postoperatively. There were twenty-four excellent, fourteen good, six fair, and no poor results. Radiographic analysis revealed an incomplete radiolucent line that was more than one millimeter in width adjacent to five tibial and four femoral components in eight knees (18 per cent). The positions of the components had not changed. Postoperatively, there was one transient peroneal palsy and two knees required patellar resurfacing. No revisions were performed for loosening of a prosthesis. We concluded that use of a cemented total knee prosthesis in a patient who has rheumatoid arthritis and is less than forty years old provides a satisfactory result and that the component will not loosen after an average of five years.

Adult↗
PubMed1987

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition.

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

13-Hydroxyoctadeca-9,11-dienoic acid (13-HODE) inhibits thromboxane A2 synthesis, and stimulates 12-HETE production in human platelets.

The effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE), a major lipoxygenase product of endothelial cell linoleic acid metabolism on thrombin-induced platelet thromboxane B2 (TxB2), and 12-hydroxyeico-satetraenoic acid (12-HETE) production was evaluated. 13-HODE inhibited thrombin-induced TxB2 production in human platelets in a concentration-dependent manner. At concentrations of 10 and 30 microM, 13-HODE inhibited TxB2 production by 28 +/- 8% (1SE, n = 5; P less than 0.05) and 48 +/- 6% (P less than 0.01) respectively. 13-HODE (30 microM) also inhibited the production of platelet hydroxyheptadecatrienoic acid (38 +/- 5%, P less than 0.01). A concomitant stimulation of 12-HETE production by 13-HODE was observed (25 +/- 5% and 49 +/- 22% over control values at 10 and 30 microM respectively, P less than 0.01). Our results demonstrate a differential effect of 13-HODE on thrombin stimulated platelet cyclooxygenase and lipoxygenase metabolites.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

13-Hydroxyoctadecadienoic acid (13-HODE) stimulates prostacyclin production by endothelial cells.

The effect of 13-hydroxyoctadecadienoic acid (13-HODE), an endogenous lipoxygenase metabolite of linoleic acid, on prostacyclin production by fetal bovine aortic endothelial cells was evaluated. Time-dependent release of radioimmunoassayable 6KPGF1 alpha in the presence of 13-HODE (10 uM) was stimulated by 39%, 27%, and 34% at 10, 30 and 120 min respectively. 13-HODE (10 uM) had no effect on the conversion of exogenous [14C] arachidonic acid (AA) to prostacyclin. When the effect on AA release was evaluated in [14C] AA prelabeled cells, 13-HODE (10nM) stimulated the release of AA from membrane phospholipids. Analysis of cellular phospholipids revealed a significant decrease in phosphatidylethanolamine. Our results demonstrate that 13-HODE stimulates prostacyclin production by enhancing AA release from phospholipids.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

Endothelial cell proliferation may be mediated via the production of endogenous lipoxygenase metabolites.

Endogenous regulators of endothelial cell proliferation have not been clearly defined. We investigated whether the cyclooxygenase and/or lipoxygenase metabolites are involved in this process, and report that lipoxygenase products can modulate endothelial cell growth. Nordihydroguaiaretic acid--a lipoxygenase inhibitor, inhibited endothelial cell proliferation as well as DNA synthesis. 5,8,11,14-Eicosatetraynoic acid--an inhibitor of both lipoxygenase and cyclooxygenase also inhibited endothelial cell DNA synthesis, while indomethacin--a selective cyclooxygenase inhibitor did not affect cell proliferation or DNA synthesis. While arachidonic acid stimulated DNA synthesis, this effect was completely abolished by nordihydroguaiaretic acid. These results demonstrate that products of the lipoxygenase pathway can affect endothelial cell proliferation.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1987

Analysis of blood HETE levels by selected ion monitoring with ricinoleic acid as the internal standard.

Selected ion monitoring of mass fragments of peaks from capillary gas chromatograms permits a sensitive and selective analysis of positional isomers of hydroxy-eicosanoids (as their methyl ester, trimethylsilyl ether derivatives). Because deuterated analogs of these HETEs are not readily available, stable isotope dilution cannot be easily performed. We have developed a method for the quantitation of HETEs using ricinoleic acid (12-hydroxy-oleic acid) as an internal standard. Ricinoleic acid is monitored at m/z 187, while 5-, 12-, and 15-HETEs (those commonly found in biological systems) are monitored at m/z 255, 295 and 225 respectively. The procedure has proven linear for levels from 0.03 to greater than 3.0 nmol HETE per nmol standard. There is however significant "crossover" among the HETEs, requiring preliminary resolution of the various isomers by reverse phase HPLC in samples containing more than one HETE. Recovery of authentic standards added to various blood samples through the extraction, resolution and analyses averaged over 90%. Using this procedure, mean plasma concentrations were found to be less than 0.1 microM for all HETEs tested, while serum levels were approximately 0.3, 0.8 and 0.1 microM for 5-, 12-, and 15-HETE respectively. Serum formed in the presence of 2mM arachidonic acid plus 25 microM A23187, to maximize HETE production, contained an average of 4, 22 and 1 microM 5-, 12- and 15-HETE respectively. Thus while normal human plasma contains sufficient quantities of the various HETEs to produce some of the biological effects attributed to these metabolites, there is potential for an even greater production when sufficient substrate is available.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1987

Cyclooxygenase inhibition reduces placental transfer: reversal by carbacyclin.

The effect of the cyclooxygenase inhibitors, indomethacin and ibuprofen, on diffusional transfer in the human placenta was assessed with the dual-perfused isolated placental lobe. Antipyrine, a freely diffusible substance, was used as an indicator of placental transfer efficiency. Each inhibitor (100 mumol/L) was perfused for 30 minutes after a baseline period, resulting in a significant reduction in antipyrine clearance. During a subsequent washout period, ibuprofen-inhibited antipyrine clearance returned to baseline values, whereas indomethacin-inhibited clearance remained reduced. An additional 30 minutes perfusion of 500 mumol/L of ibuprofen resulted in a further reduction in antipyrine clearance compared with 100 mumol/L of ibuprofen, suggesting a dosage-related effect. The perfusion of each inhibitor caused a reduced production of 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) in the fetal circulation. The simultaneous perfusion of carbacyclin, a prostacyclin analogue, at 100 nmol/L and 1 mumol/L resulted in a dosage-dependent reversal of the effects of ibuprofen (500 mumol/L) on antipyrine clearance. The results indicate that the inhibition of cyclooxygenase activity reduces placental transfer and that the effects of these inhibitors are reversed by carbacyclin. This study suggests that the use of cyclooxygenase inhibitors during pregnancy could compromise the developing fetus by reducing placental transfer.

Antipyrine↗
PubMed1987

Decreased plasma arachidonic acid binding capacity in neonates.

Arachidonic acid (AA) metabolites have been implicated in neonatal pathologic states such as respiratory distress syndrome (RDS). Since free (nonprotein bound) AA is the substrate for synthesis of these compounds, a decreased capacity to bind AA in neonatal plasma could contribute to these disorders. AA binding was assayed by equilibrium dialysis in plasma samples from healthy adults and various infant groups. Plasma from these infant groups bound significantly less AA than adult plasma. Premature infants with RDS and premature infants receiving intralipid had the lowest capacity to bind AA. The increased availability of free AA may be important in neonatal pathophysiologic states involving arachidonate metabolites.

Adult↗
PubMed1987

Enhancement of ovulation rate in gilts by increasing dietary energy and administering insulin during follicular growth.

Two experiments were conducted to examine influences of dietary energy and insulin on ovulation rate and patterns of luteinizing hormone (LH), follicle stimulating hormone (FSH), glucose, insulin and estradiol in gilts during 6 d before estrus. In Exp. 1, 36 gilts were given altrenogest for 14 d to synchronize estrus. In a factorial arrangement, gilts were fed one of two levels of dietary energy (5,771 or 9,960 kcal metabolizable energy (ME)/d), and given one of two levels of porcine insulin (0 or .1 IU/kg body weight iv every 6 h). Dietary treatments began 4 d before and insulin treatments began 1 d after the last day of altrenogest, respectively, and lasted until 24 h after estrus. Main effect means for number of corpora lutea were 14.0 +/- 1.3 and 17.6 +/- .9 for 5,771 and 9,960 kcal ME (P less than .05), and 14.6 +/- 1.0 and 17.0 +/- .9 for 0 and .1 IU insulin (P less than .05). Number of LH peaks on d 3 was greater for gilts that received 9,960 kcal than 5,771 kcal (3.3 +/- .2 vs 2.7 +/- .2; P less than .05), and for .1 than 0 IU insulin (3.2 +/- .2 vs 2.7 +/- .2; P less than .05). During the first 24 h of sampling, concentrations of LH and FSH were greater (P less than .05) in gilts receiving 9,960 kcal ME plus insulin than for other treatment combinations. Concentrations of estradiol were not affected by treatments. In Exp. 2, two formulations of insulin were evaluated for influence on ovulation rate. All gilts received altrenogest and 9,960 kcal ME/d as in Exp. 1. Then on the first day after altrenogest, seven gilts each received short-acting insulin (as in Exp. 1), long-acting insulin (zinc suspension, 1.0 IU/kg body weight every 18 to 24 h), or served as controls. Ovulation rates were increased (P less than .05) by both insulin preparations (15.6, control; 19.1, short-acting; 18.5, long-acting; SE = 1.2). Concentrations of LH tended to be greater after short-acting insulin, but differences were not significant (P = .13). We conclude that increases in ovulation rate produced by dietary energy and insulin are not necessarily accompanied by changes in gonadotropins or estradiol.

Animals↗
PubMed1986

Time-dependent inhibition of platelet cyclooxygenase by indomethacin is slowly reversible.

Indomethacin has been characterized in vitro as a time-dependent, irreversible inhibitor of cyclo-oxygenase, yet its effects on human platelets have been found to be reversible in vivo. To understand this apparent contradiction, we have investigated the kinetics of recovery of platelet thromboxane production after a single dose of indomethacin. The inhibition of platelet thromboxane production was greater than would be expected from the levels of indomethacin found in the plasma suggesting that the time-dependent inhibition occurs in vivo. Yet recovery of platelet thromboxane production was faster than expected for an irreversible inhibitor, with 50% of control values being regained within 24 hours after ingestion of the drug. When platelets were isolated and resuspended in homologous drug-free plasma, slow recovery of thromboxane production was seen to occur with 50% of control activity regained in 100 minutes. This recovery was much slower than that seen from a competitive inhibitor of cyclo-oxygenase, ibuprofen. Ibuprofen-treated platelets recovered nearly completely immediately on being resuspended in drug-free plasma. When microsomes were isolated from platelets, then treated with indomethacin, no time-dependent recovery of activity was seen. The recovery of cyclo-oxygenase after indomethacin inhibition appears to be limited to the unperturbed enzyme in its natural milieu.

Adult↗
PubMed1986

Bleeding time in hemophilia A: potential mechanisms for prolongation.

Prolongation of bleeding time has been previously observed in hemophilia, although no cause has been elucidated. We measured bleeding time, platelet aggregation, nucleotide release, and thromboxane B2 (TXB2), plasma 6-keto-PGF 1 alpha, platelet-associated IgG (PAIgG), and circulating immune complexes in 31 unselected patients with severe hemophilia A and in 17 controls. In 85% of patients with hemophilia A, the bleeding time was greater than 2 SD above the control level (greater than 8 minutes). Sixty-six percent of patients with hemophilia A had circulating immune complexes, and there was a striking relationship between the presence of these complexes and prolonged bleeding time. Plasma 6-keto-PGF 1 alpha levels were significantly elevated in the patient group, and correlated with bleeding time changes. Platelet aggregation and nucleotide release were normal in the patients with hemophilia, although reduced platelet TXB2 biosynthesis was noted in 26%. No correlation was demonstrated between bleeding time and impairment of platelet TXB2 formation. Seventy-two percent of the patients with hemophilia A had elevated levels of PAIgG, and an inverse relationship between PAIgG and platelet count was observed. No relationship was noted between platelet count and bleeding time. This study indicates that the majority of patients with hemophilia A have prolonged bleeding times. The close correlation between bleeding time, plasma 6-keto-PGF 1 alpha levels, and the presence of circulating immune complexes suggests a role for immune complex-mediated defects in vascular function as the basis for bleeding time prolongation.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

15-Hydroxy-5,8,11,13-eicosatetraenoic acid inhibits human vascular cyclooxygenase. Potential role in diabetic vascular disease.

Human umbilical arteries converted arachidonic acid to three hydroxyeicosatetraenoic acids (HETEs) as well as prostaglandins. The mono-HETEs have been identified by reverse-phase high pressure liquid chromatography and gas chromatography-mass spectroscopy as 15-HETE and 11-HETE. 15-HETE in arterial segments appears to be derived mainly via the 15-lipoxygenase pathway, whereas 11-HETE, and the presumed di-HETE(s) were products of cyclooxygenase. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, stimulated prostanoid production with a concomitant inhibition of 15-HETE formation. These results suggested that 15-HETE may function as an endogenous regulator of prostacyclin. In human umbilical arterial microsomes, 15-HETE was found to inhibit 6-keto-prostaglandin F1 alpha and total prostanoid production in a concentration-dependent manner (median inhibition constant [IC50] of 52 +/- 3 and 63 +/- 4 microM respectively). The relative distribution of prostaglandins, however, remained unaffected, indicating that the site of action was cyclooxygenase. Kinetic analysis revealed that 15-HETE was a competitive inhibitor of the enzyme. Although no changes in maximum velocity occurred, the apparent Km was significantly different (9.3 +/- 6.9 microM [1 SD] for control vs. 37.6 +/- 17.7 microM for the 15-HETE-treated enzyme). Furthermore, the inhibitory effect of 15-HETE on prostacyclin production was confirmed using cultured bovine endothelial cells. In this cell system, not only did 15-HETE inhibit endogenous prostacyclin production, but also the conversion of exogenous [1-14C]arachidonic acid to prostacyclin (IC50 of 40 +/- 17 microM). No effect on arachidonic acid release was noted. To investigate whether our in vitro finding that 15-HETE inhibited prostacyclin production could be relevant to the in vivo situation, our final studies were performed on vasculature obtained from the diabetic milieu. We found that the production of 15-HETE was significantly increased in vasculature obtained from the infant of the diabetic mother (1.14 +/- 0.26 pmol/mg) when compared to control neonates (0.77 +/- 0.22; P less than 0.01). A concomitant decrease in prostacyclin production was seen (51.6 +/- 12.6 pmol/mg in infants of diabetic mothers vs. 71 +/- 22.3 in controls). Moreover, an inverse correlation between these two eicosanoids was also noted. Our results suggest a potential in vivo regulatory role for 15-HETE on prostacyclin production.

Animals↗
PubMed1985

Abnormalities in vascular arachidonic acid metabolism in the infant of the diabetic mother.

The infant of the diabetic mother has an increased incidence of thromboses in utero and in the neonatal period. In the adult with diabetes a decrease in prostacyclin formation has been suggested as a cause for the atherothrombotic tendency. We therefore evaluated arachidonic acid metabolism in infants of diabetic mothers. Endogenous radioimmunoassayable 6-keto prostaglandin F1 alpha (PGF1 alpha) was normal in umbilical vessels obtained from the infants of diabetic mothers whose glucose homoeostasis was maintained when compared with control values. Nevertheless, a significant inhibition of vascular production of 6-keto PGF1 alpha was observed in infants born to mothers with raised HbA1C concentrations. A decrease in the concentration of plasma 6-keto PGF1 alpha was also seen in the infants of diabetic mothers when compared with control neonates. The correlation observed between plasma 6-keto PGF1 alpha concentrations and endogenous vascular prostacyclin formation in the infants of diabetic mothers indicates that the in vitro deficiency of prostacyclin formation reflects a concomitant in vivo abnormality.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Formation of 11-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid in human umbilical arteries is catalyzed by cyclooxygenase.

Human umbilical arteries convert arachidonic acid into three hydroxy-eicosatetraenoic acids as well as 6-ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and D2 and thromboxane B2. Two of these hydroxy derivatives of arachidonic acid were purified by reverse-phase HPLC and identified by GC-MS as 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) while a third, presumed dihydroxy derivative has not yet been identified. Both the cyclooxygenase and HETE synthesizing activities were found to be localized mainly in the microsomal fraction (100 000 X g pellet) (51 and 61% of total, respectively), and approx. 25% of both activities was found in the 10 000 X g pellet. The formation of these HETEs was inhibited by the cyclooxygenase inhibitors indomethacin and aspirin but not by the lipoxygenase inhibitor nordihydroguaiaretic acid. Production of immunoreactive 15-HETE as well as 6-ketoprostaglandin F1 alpha were also decreased significantly when arterial segments were incubated in the presence of either indomethacin or aspirin. Indomethacin inhibited the formation of both prostanoids and HETEs by microsomes in a concentration-dependent and time-dependent manner. The ID50 values for indomethacin against HETE synthesizing activity and against cyclooxygenase were 4.5 and 3.8 microM, respectively. The inactivation constants were found to be 0.09 and 0.08 min-1 for HETE synthesizing activity and cyclooxygenase, respectively. These two microsomal activities were solubilized in parallel with Tween-20. Incubation with three distinct monoclonal antibodies against different epitopes on cyclooxygenase precipitated both cyclooxygenase and HETE synthesizing activity. Each of these activities was recovered in the immune pellets. These studies demonstrate that in human umbilical arteries 11-HETE, 15-HETE and a presumed di-HETE are the products of cyclooxygenase.

Arachidonic Acid↗
PubMed1985

Insemination management for a one-injection prostaglandin F(2)alpha synchronization system. II. One versus two inseminations following detection of estrus.

Following detection of estrus in an estrus synchronization system, 216 dairy heifers were inseminated (A.I.) randomly either soon after detected estrus (1X) or soon after detected estrus and again 10 to 12 h later (2X). Average h from detection of estrus to A.I. was 1.8+/-0 for 1X and 1.1+/-0 and 11.1+/-0.4 for 2X. During the regimen, heifers were checked visually for estrus daily for five consecutive days with 16.0 and 17.3% showing estrus and receiving A.I. in the 1X and 2X groups, respectively. Those not seen in estrus were injected with 25 mg PGF(2)alpha with observations for estrus and A.I. continuing for five more days. Response rates as indicated by estrus following prostaglandin F(2)alpha (PGF(2)alpha) were 74.7 and 75.8% for 1X and 2X, respectively. Percentages of heifers in estrus <24, 25 to 48, 49 to 72, 73 to 96 and >96 h after PGF(2)alpha were 3.7, 22.8, 47.1, 15.4 and 11.0, respectively. Based on rectal palpation for pregnancy between 45 and 60 days after A.I., conception rates of 70.2% for 1X and 68.6% for 2X did not differ significantly (P>0.05). Progesterone concentrations at injection for heifers not responding to PGF(2)alpha were lower than was seen in responding heifers (2.7 vs 5.8 ng/ml) (P<0.01). Data from the present experiment supports the conclusion of an earlier experiment that satisfactory conception can be achieved with a single, established daily insemination period.

Journal Article↗