Susan E. Barrow

Cigarette smoking: profiles of throm☐ane- and prostacyclin-derived products in human urine

Thromboxane (TX) B2, 2,3-dinor-TXB2, 11-dehydro-TXB2, 6-oxoprostaglandin (PG)F1 alpha and 2,3-dinor-6-oxo-PGF1 alpha were measured in 24 h urine samples obtained from 30 apparently healthy chronic cigarette smokers and 37 closely matched non-smoking control subjects. Samples were analysed using a newly developed assay based on immunoaffinity chromatography and capillary column gas chromatography/electron capture negative ion chemical ionisation mass spectrometry. There were significant and comparable increases in the excretion rates of both 2,3-dinor-TXB2 and 11-dehydro-TXB2 in the smoking compared with the non-smoking group (2P less than 0.001). Excretion rates of 2,3-dinor-TXB2 were 418 +/- 35 and 265 +/- 26 pg/mg creatinine in the two groups, respectively. 11-Dehydro-TXB2 excretion rates were 440 +/- 54 and 221 +/- 18 pg/mg creatinine, respectively (mean +/- S.E.). There were significant (2P less than 0.05) positive correlations between average reported cigarette consumption and excretion of both thromboxane metabolites. There were small but significant (2P less than 0.02) increases in the excretion rates of both 6-oxo-PGF1 alpha and 2,3-dinor-6-oxo-PGF1 alpha in the smoking compared with the non-smoking group. There was no significant difference in the rates of excretion of TXB2 in the two groups. The effects of acute cigarette smoke exposure (five cigarettes in 2 h) was also studied in four normally non-smoking healthy volunteers. There was no significant change in the excretion rate of any of the eicosanoids measured during control and smoking periods (at least 2 weeks apart), indicating that increased TXA2 biosynthesis in chronic smokers is unlikely to be a consequence of acute platelet activation.

Effect of vasoactive peptides on prostacyclin synthesis in man

1 Bradykinin, angiotensin II, arginine vasopressin (AVP) or des‐amino‐D‐arginine vasopressin (DDAVP) were administered by intravenous infusion to 10 healthy men. 2 The concentration of 6‐oxo‐prostaglandin F1α (6‐oxo‐PGF1α), the stable hydrolysis product of prostacyclin (PGI2), was measured in plasma using gas chromatography/negative ion chemical ionisation mass spectrometry. 3 Dose‐related increases in plasma concentrations of 6‐oxo‐PGF1α occurred during administration of bradykinin (100–3200 ng kg−1 min−1). The concentrations of 6‐oxo‐PGF1α rose from baseline values in the range < 1.0–4.9 pg ml−1 to 24.9–47.6 pg ml−1 at maximum tolerated infusion rates. 4 There were no changes in the concentrations of 6‐oxo‐PGF1α during administration of angiotensin II, AVP or DDAVP at infusion rates which caused haemodynamic changes.

Elevated plasma 6-keto-prostaglandin F1α in patients in septic shock

Central venous plasma concentrations of 6-keto-prostaglandin F1α (PGF1α), the stable hydrolysis product of prostacyclin (PGI2, a vasodilator and antiaggregatory metabolite of arachidonic acid), were determined in patients with septic shock. In eight nonsurvivors, the median plasma 6-keto-PGF1α level was 229 pg/ml (range 31 to 21,998), compared to 30 pg/ml (range 22 to 194) in six survivors. In three control patients who were not septic or in shock, the levels were less than 4 pg/ml. This study demonstrates that human septic shock is associated with elevated plasma levels of 6-keto-PGF1α, and raises the possibility that increased PGI2 formation may play a role in human septic shock.

Prostaglandin D2 and histamine release in cold urticaria

Prostaglandin (PG) D2 and histamine concentrations have been measured in blood draining cold-challenged forearm skin in patients with cold urticaria. Local venous concentrations of both histamine and PGD2 rose in four patients who developed a whealing response. Plasma histamine concentration increased from a mean resting value of 0.24 +/- 0.09 (SD) ng/ml to peak values of 16.9 to 96.6 ng/ml. Resting concentrations of PGD2 were below the limit of detection (5 pg/ml) in three patients and 62 and 27 pg/ml in the fourth. Peak plasma PGD2 concentration after challenge ranged from 166 to 279 pg/ml. Time course of histamine and PGD2 release was similar with peak concentrations at 6 and 10 minutes, respectively. The maximum clinical response occurred between 10 and 20 minutes after challenge. Our findings demonstrate that PGD2 is produced in association with mast cell degranulation in man, but the amount, relative to histamine, is low. Despite its high potency in production of inflammatory effects, PGD2 probably has only minor direct effects in cold urticaria, although it may act to potentiate other mediators.

Synthesis of leukotriene B and other conjugated triene lipoxygenase products by blood cells of the rainbow trout, Salmo gairdneri

Stimulation of whole blood from rainbow trout with the calcium ionophore, A23187 (20 microM), produced leukotrienes B4 and B5 at concentrations in the range 22-30 ng.ml-1 and 8-24 ng.ml-1, respectively. Their identification and quantification was achieved using reverse-phase high-performance liquid chromatography, combined capillary column gas chromatography-electron capture chemical ionization mass spectrometry and ultraviolet spectroscopy. A number of other lipoxygenase products were also detected, but only partially analysed. The fatty acid composition of the leucocytes, which are presumed to be the site of leukotriene synthesis, was determined by thin-layer and gas-liquid chromatography to enable a comparison of the relative levels of the polyunsaturated fatty acids, which act as substrates for the synthesis of these lipoxygenase products. Arachidonic (20:4(n - 6)), eicosapentaenoic (20:5(n - 3)) and docosahexaenoic (22:6(n - 3)) acids represented approx. 6, 5 and 40%, respectively, of the total fatty acid content.

Analysis of picomolar concentrations of 6-oxo-prostaglandin F1 in biological fluids

Abstract A highly sensitive and specific assay for the quantitation of 6-oxo-prostaglandin F1, the stable hydrolysis product of prostacyclin, is described. The method involves the addition of [3,3′,4,4′-2H4]-6-oxo-prostaglandin F1 as internal standard, extraction from biological fluids using μBondapak C18 reversed-phase Sep-Paks, and preliminary purification by normal-phase chromatography. Following conversion to the methoxime, tris-trimethylsilyl, pentafluorobenzyl derivative, samples were analysed using combined capillary column gas chromatography negative ion chemical ionisation mass spectrometry. Fragment ions at m/z 614 (1H) and 618 (2H) [M  C7H2F5]− were monitored for quantitation. This method was used for the measurement of endogenous levels of 6-oxo-prostaglandin F1 in human urine and for the determination of prostacyclin release from rat peritoneal mast cells and from rat aortic rings incubated in human plasma.

Eicosanoid generation and effects on the aggregation of thrombocytes from the rainbow trout, Oncorhynchus mykiss

Fish blood lacks anucleate platelets but contains a nucleated cell type termed the thrombocyte that is thought to be functionally analogous. Thrombocytes were purified from the peripheral blood of the rainbow trout, Oncorhynchus mykiss, by a two step gradient centrifugation method. Following this procedure, the recovered thrombocytes were 78-86% pure as defined by immunoreactivity to a panel of monoclonal antibodies and were of variable morphology from round to spindle-shaped. Incubation of thrombocyte suspensions with either calcium ionophore, A23187, platelet-activating factor or a thromboxane (TX) mimetic, U-46619, generated a range of eicosanoids derived from arachidonic acid including 12-hydroxyeicosatetraenoic acid (12-HETE), TXB2, prostaglandin (PG) E2, leukotriene (LT) B4 and lipoxin (LX) A4. The equivalent products derived from eicosapentaenoic acid were also formed. Co-incubation of thrombocytes with either erythrocytes or granulocytes/monocytes in the presence of calcium ionophore did not result in the formation of any further new lipoxygenase products. Incubation of isolated thrombocytes in plasma-free conditions with U-46619 (0.03-10 microM) resulted in a rapid, dose-dependent aggregatory response. This effect was markedly augmented in the presence of mammalian fibrinogen (400 micrograms ml-1). Thrombin (0.1-1.3 units ml-1), like U-46619, was also a potent proaggregatory compound for trout thrombocytes. LXA4 and LTB4 had limited aggregatory potential and then only at high concentrations (10 microM), while 12-HETE and PAD had no significant effect at all concentrations tested. These results demonstrate that some of the eicosanoids released during the activation of trout thrombocytes are involved in the aggregatory behaviour of this cell type.

Effects of dietary fatty acids on eicosanoid-generating capacity, fatty acid composition and chemotactic activity of rainbow trout (Oncorhynchus mykiss) leucocytes.

Rainbow trout, Oncorhynchus mykiss, were maintained on isocalorific diets in which either sunflower, menhaden or Fosol oils were used as the dietary source of fatty acids. At intervals over a period of 6 months, head kidney leucocytes were isolated and used for the analysis of their fatty acid composition and eicosanoid-generating capacity. Major changes in fatty acid composition were apparent within 4 weeks on the diets, with fish fed sunflower oil diets showing a 2.1-fold increase in total n-6 fatty acids and a 2.3-fold decrease in n-3 fatty acids, compared with the original basal levels. By week 8 the fatty acid composition changes were greater in the sunflower-fed fish, but thereafter remained relatively stable to the end of the experiment at week 24. Leucocytes from the fish maintained for > 8 weeks on the sunflower oil containing diet produced significantly lower percentages of 5-series lipoxygenase products derived from eicosapentaenoic acid including 12-hydroxyeicosapentaenoic acid, leukotriene B5 and lipoxin A5 compared with those cells from fish fed either menhaden or Fosol based diets. Unlike the fatty acid composition, differences in lipoxygenase product profiles between the dietary groups increased throughout the experiment and by week 24 the arachidonic acid/eicosapentaenoic acid derived product ratios were approx. 14:1 in the sunflower oil-fed fish compared with approx. 1:1.5 in the menhaden oil-fed fish. A functional consequence of these differing ratios was seen in the ability of supernatants containing these products to cause the in vitro locomotion of trout neutrophils. Supernatants from sunflower oil-fed fish were less chemo-attractive than supernatants from menhaden or Fosol oil-fed fish.

Prostacyclin and thromboxane biosynthesis in mild essential hypertension.

The possibility that prostacyclin or thromboxane biosynthesis is abnormal in patients with established mild essential hypertension was investigated in 46 patients. These eicosanoids have opposing effects both on vascular smooth muscle and on platelets. An imbalance in their biosynthesis could therefore influence both vascular tone and predisposition to thrombosis. We studied the relation between blood pressure and the biosynthesis of prostacyclin and thromboxane A2 by measuring urinary excretion rates of stable breakdown products of prostacyclin (6-oxo-prostaglandin F1 alpha and 2,3-dinor-6-oxo-prostaglandin F1 alpha) and of thromboxane A2 (thromboxane B2 and 2,3-dinor-thromboxane B2) using immunoaffinity chromatography and gas chromatography/electron capture mass spectrometry. Excretion rates of both of the prostacyclin-derived products ranged from less than 5 to more than 100 ng/g creatinine; each was significantly negatively correlated with blood pressure (r = 0.36-0.45). A reduction of 2,3-dinor-6-oxo-prostaglandin F1 alpha excretion of 100 ng/g creatinine was associated with an increase in arterial pressure of 14 mm Hg (systolic) and 8 mm Hg (diastolic) in patients who had been without antihypertensive medication for 2 weeks. The same reduction in 6-oxo-prostaglandin F1 alpha excretion was associated with an increased pressure of 19 mm Hg (systolic) and 12 mm Hg (diastolic) (2p less than 0.05 for diastolic pressure and 2p less than 0.01 for systolic pressure in each case). There were similar correlations between the excretion rates of these products and blood pressure in the same patients while they were receiving antihypertensive therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of prostaglandin D2 and identification of metabolites in human plasma during intravenous infusion

A stable isotope dilution assay has been developed for the quantification of prostaglandin D2 (PGD2) in plasma. Samples are analysed by capillary column gas chromatography/negative ion chemical ionisation mass spectrometry (GC/NICIMS). The method employs an internal standard of [2H6]PGD2, prepared biosynthetically by incubation of rat peritoneal mast cells with deuterated arachidonic acid. No PGD2 was detected in peripheral venous plasma samples obtained from 10 healthy male volunteers (detection limit = 5 pg ml-1). Following intravenous infusion of PGD2 at increasing incremental infusion rates ranging from 16-256 ng kg-1 min-1, a dose related increase in the plasma concentration of PGD2 was observed. Plasma levels at 128 ng kg-1 min-1 ranged from 724-1444 pg ml-1. The major circulating metabolites of PGD2, during infusion, were identified as 13,14-dihydro-15-oxo-PGF2 alpha and 15-oxo-PGF2 alpha.