A Radioimmunoassay for Prostaglandin Ax in Human Peripheral Blood* PRISCILLA ZIA, MICHAEL GOLUB, AND RICHARD HORTON Section of Endocrinology, Department of Medicine, University of Southern California, School of Medicine, Los Angeles, California, 90033 possessing very low cross reactivity to other prostaglandins (PGA2, PGE, PGB and PGF). Activated florisil or ammonium sulfate can be used to separate bound from free prostaglandin. This whole blood or plasma method yields blank values of only 2 ± 2 pg per sample with a between assay precision determined by duplicate analysis of 8% and interassay precision of 3%. The mean whole blood PGAi concentration in 27 subjects is 2.5 ± 1.6 (SD) ng per 100 ml. No significant sex difference in PGAi levels was noted and values were similar whether measured in whole blood or in cooled plasma rapidly prepared and extracted. These values of PGA! are much lower than those RIA values reported by others for "PGA" using antibodies with lower specificities. (/ Clin Endocrinol Metab 41: 245, 1975)

ABSTRACT. A specific, sensitive and accurate radioimmunoassay (RIA) method for the measurement of prostaglandin A, (PGAi) in either human whole blood or plasma is described. Whole blood is immediately lysed with distilled water containing tritiated indicator. When plasma is assayed, the blood samples are handled at 4 C and rapidly centrifuged. The lysate or plasma is adjusted to pH 5 with buffer and quickly extracted with 5% methanol in dichloromethane. The whole blood or plasma extract is then purified by Sephadex LH20 chromatography using the system methanol: methylene chloride (5:95) which separates the major groups PGA, PGE and PGF. The RIA is then performed using an antiserum generated in rabbits from PGA! coupled to bovine thyroglobulin. The antibody is highly specific,

T

Solvents and compounds

Received October 16, 1974. Supported by a grant from the National Institutes of Health (HL 13476). * Presented in part to the annual meeting of the Endocrine Society in Atlanta, Georgia, on June 14,1974.

Solvents for extraction and chromatography (Matheson, Coleman and Bell or J. T. Baker Chemical Co., spectro grade) were used without further purification. Standard prostaglandins were kindly provided by Dr. J. Pike of the Upjohn Co., Kalamazoo, Mich. In addition to the chromatographic data obtained by Upjohn indicating a high degree of purity, PGAj was further checked by its optical density in methanol at 218 millimicrons, e2i8 = 10,900 (11), and the batch used as RIA standard was >92% pure. Tritiated prostaglandins (Ax, E t and E2) were purchased from New England Nuclear Corp., Boston, Mass. [3H]PGAj with specific activity of 50/uCi per 0.15 (xg (700 dpm per pg) was used as the labeled indicator. Purity was periodically checked by our Sephadex LH-20 chromatography system. Proof of purity was provided by an experiment in which an aliquot of [3H]PGAx was added to [14C]PGA, and the mixture passed through the Sephadex LH-20 system (12). No significant change in the original [3H/14C] ratio was noted after chromatography. Sephadex LH-20 with particle size 20-100 /xm

HE A and E prostaglandins possess natriuretic and vasodepressor activities suggesting that they may play a role in blood pressure regulation (1-4). A recent report also suggests that PGAx in subpressor dosage in man can selectively stimulate aldosterone secretion without altering other known adrenal control mechanisms (5). However, specific methods for the various prostaglandins must be developed before the many biologic and potentially hormonal actions of prostaglandins can be placed in proper perspective. Several radioimmunoassays for prostaglandin A or E in human blood have been reported (6-9). However, the separation techniques and antibody specificities have not allowed assay of prostaglandin Ax or A2 to be performed (6-10). As part of an ongoing program, we report a RIA for PGAX in peripheral blood.

Materials and Methods

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ZIA, GOLUB AND HORTON

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was obtained from Pharmacia Co., Piscataway, New Jersey. Florisil (60-100 mesh, Fisher Scientific Company, Fairlawn, New Jersey) was prepared by washing a large batch 20 times with distilled water, stirring, allowing the material to partically settle for a few minutes and decanting the fines. The remaining material was dried at 120 C for 2 days and stored in a tight container inside a dessicator.* The conjugating materials were obtained from K and K labs., Los Angeles, California. All glassware was acid washed or disposable. Tritium counting was performed in 10 ml of PPO-POPOP in toluene (Spectrafluor, AmershamSearle). 10% NCS solubilizer (Amersham-Searle) was added when aqueous solutions were counted. The efficiency of counting with a Nuclear Chicago Mark I spectrometer of these slightly quenched samples was 55%. Formation of piostaglandin Ay-bovine thyroglobulin conjugate. This conjugate was prepared according to the method for conjugating PGF 2a described by Caldwell and et al. (13) with some modifications. Bovine thyroglobulin (Tg) from Sigma Chemical Company, St. Louis, Missouri, was used as a protein carrier instead of bovine serum albumin (BSA) (14,15). The molar incorporation ratio of the prostaglandin A, conjugate was determined by comparing the specific radioactivity of the conjugate to the specific radioactivity of the PGAj before coupling and was calculated to be 226. Immunization. Five hundred micrograms of the conjugate was injected into New Zealand white rabbits according to the method of Vaitukaitis et al. (16). The animals received booster doses of 100 fig at one month and subsequently were boosted every two weeks. Antibodies were detected 2 months after the initial immunization. Radioimmunoassay

procedure

Sample collection. Blood was drawn into an iced heparinized syringe and the whole blood extracted immediately (see below). Plasma was * Calcium sulfate (purified) and ammonium sulfate (crystals) were obtained from Matheson, Coleman and Bell, and ammonium sulfate was further purified by recrystallization.

JCE & M • 1975 Vol 41 • No 2

prepared by immediate centrifugation at 2000 rpm at 4 C for 10 min. The plasma was quickly separated and extracted. Whole blood. Five ml of freshly drawn (on ice) whole blood was immediately lysed with equal volumes of deionized water containing 700 dpm of [3H]PGA! (1 pg) in a 100 ml conical tube. The pH of the lysate was adjusted to 5 by addition of 1 ml 0.5M phosphate buffer (pH 5) and the contents immediately extracted by vigorous shaking with 7-10 volumes of 5% methanol in methylene chloride. After centrifugation for 10 min at 2000 rpm, three layers were evident, a semisolid disk between the aqueous and solvent layers. The upper aqueous layer was decanted. The solid was broken and the organic solvent filtered through sodium sulfate and glass wool and dried under vacuum. Plasma. Similarly, before extracting plasma (4 ml), the pH was adjusted to 5 with 1 ml 0.5M phosphate buffer. The mixture was then extracted with 40 ml of 5% methanol in methylene chloride. Chromatographic purification. A Sephadex LH-20 column (80 x lcm), prepared as described previously (12), was used for the separation of prostaglandin A type from E and F types. The dried extract was placed on the column with 1 ml x 3 washes of the mobile solvent, (5% methanol in dichloromethane). The column was then eluted with the same mobile solvent. A 20 ml fraction (65-85 ml), which recovers 90% of the added PGAj, was collected in a counting vial. The sample was dried and redissolved in 1.5 ml methanol and two 0.5 ml aliquots were transferred to 10 x 13 mm glass tubes for assay. The remaining one-third was dried and counted for 40 min to reduce counting error of the recovery aliquot to

A radioimmunoassay for prostaglandin A1 in human peripheral blood.

A specific, sensitive and accurate radioimmunoassay (RIA) method for the measurement of prostaglandin A1 (PGA1) in either human whole blood or plasma ...
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