Lipoxygenase-dependent mechanisms in hypertension.

This study was designed to examine the contribution of lipoxygenase products to mechanisms of vascular contraction and elevated blood pressure in rats with aortic coarctation-induced hypertension. In cytosolic fractions of aortae taken from hypertensive rats, 12-lipoxygenase protein was increased as compared to normotensive controls. Aortic rings from hypertensive, but not from normotensive rats, exhibited a basal tone which was reduced 74+/-12 and 71+/-22%, respectively, by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10(-5) mol/L) and 5,8,11-eicosatriynoic acid (ETI, 10(-5) mol/L). CDC (8 mg/kg s.c.) did not affect the blood pressure of normotensive rats but decreased that of hypertensive rats from 182+/-6 to 151+/-10 mm Hg. The blood pressure lowering effect of CDC was blunted in hypertensive rats pretreated with indomethacin or antibodies against 5,6-dihydro-prostaglandin I2. These data suggest contribution of lipoxygenase-derived products to mechanisms underlying aortic smooth muscle basal tone and elevated blood pressure in rats with aortic coarctation-induced hypertension. The vasodepressor effect of CDC depends on a mechanism involving vasodilatory prostaglandins.

Measurement by radioimmunoassay of prostaglandins as their methyl oximes.

Antisera have been raised to the following prostaglandins as their methyl oximes; PGE2, PGD2, 13-14-dihydro-15-oxo PGE2, 13,14-dihydro-15-oxo PGF2 alpha, 6-oxo PGF1 alpha, 6-oxo PGE1 and thromboxane B2. These antisera have good specificity and sensitivity and their use allows the immediate treatment of biological fluids with oximating solution which prevents sample decomposition during storage. A methyl oximating reagent is described which gives greater than 95% conversion of PGs to their methyl oximes by treating samples at 20 degrees C overnight. The use of this reagent allows easy and reliable sample derivatisation prior to assay with the above antisera. Since the antisera (with the exception of that for thromboxane B2) do not recognise the underivatised PG or the oxime (=NOH) form, oxime formation can be carried out in parallel with methyl oxime formation and the oximated portion of the sample can act as a "reference" for the methyl oximated portion, which will allow non specific interference to be recognised.

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2.

A sensitive and specific radioimmunoassay for prostaglandin D2 has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D2 with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D2-methoxamine coupled to bovine serum albumin. A (125I)-Histamide prostaglandin D2-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D1-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D2 in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D2 destruction. The unstability of this prostaglandin is due to the presence of a beta-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.

Development of antibody-mediated extraction followed by GC/MS (antibody/GC/MS) and its application to iloprost determination in plasma.

A new analytical principle has been developed combining the features of both radioimmunoassay and GC/MS. Its application in eicosanoid analysis was tested with the prostacyclin analogue, iloprost. The iloprost antibody, generally employed in RIA measurements, was coupled to Sepharose 4B and used as stationary phase for extraction of the drug. Variations in recovery were corrected by using deuterated iloprost as an internal standard. The samples were derivatized and quantitated by negative-ion chemical ionization-mass spectrometry. Reproducibility was 2.3 % at the 50 pg/ml level and the limit of detection was 5 pg for 1 ml samples. With plasma volumes of up to 20 ml, 0.25 pg/ml could be determined. Antibody/GC/MS proved superior to radioimmunoassay due to its higher specificity and sensitivity and superior to GC/MS with conventional clean-up procedures because of a higher sample capacity.

Antibodies to 5,6-dihydroprostaglandin I2 trap endogenously produced prostaglandin I2 in the rat circulation.

Rabbit immunoglobulins raised against 5,6-dihydroprostaglandin I2 which crossreact with prostaglandin I2 were infused intravenously into Inactin-anaesthetised male adult rats. Clearance of intravenously administered [3H]prostaglandin I2 from the blood, which is normally rapid (t 1/2 approx. 45 s), was delayed strikingly in the presence of antibody (t 1/2 approx. 60 min). The antibodies also sequestered the endogeneously synthesized prostaglandin I2 and inhibited its metabolism. The rate of appearance of endogenous prostaglandin I2 in the circulation of the rat was measured in the following way: arterial blood samples (0.5 ml) were withdrawn before, during and at various time intervals (up to 180 min) after infusion of antibodies had terminated; the prostaglandins were extracted from the blood with ethanol, and the extracts were assayed by radioimmunoassay (before and after separation by high-pressure liquid chromatography) for the following prostaglandins: 6-keto-F1 alpha, E2, F2 alpha and 13,14-dihydro-15-keto-metabolites of E2 and F2 alpha. Rapid and specific time-related increments of prostaglandin I2 (detected serologically as 6-keto-F1 alpha) were observed. At 180 min these increases ranged from 1500- to 2500-fold over preinfusion levels. No significant increases were observed in the other prostaglandins measured; nor were there increases in 6-keto-F1 alpha when saline or immunoglobulins from non-immune plasma were infused into rats. When measured by these procedures, no appreciable differences in 6-keto-F1 alpha production were found between Japanese normotensive and spontaneously hypertensive rats.