PROSTAGLANDINS
RADIOIMMUNOASSAY OF 13,14-DIHYDRO-15-KETO PROSTAGLANDIN F IN BOVINEPERIPHERALPLASMA R.J.
Fairclough
New Zealand Ministry
and E. Payne
of Agriculture
Research Ruakura Agricultural Private
and Fisheries,
Division, Research
Centre,
Bag, Hamilton,
New Zealand.
ABSTRACT A radioimmunoassay has been developed for 13,14-dihydro-15keto-prostaglandin F in bovine peripheral plasma. Acidified plasma samples were extracted with diethyl ether and the dried extracts assayed for 13,14-dihydro-15-keto-prostaglandin F using antiserum raised against a 13,14-dihydro-15-keto-prostaglandin F2o-albumin complex. The tracer used for the assay was prepared Polyethylene enzymatically from tritiated prostaglandin Flo. glycol was employed to separate free and bound 13,14-dihydro-15keto-prostaglandin F. The inter-assay coefficient of variation based on 9 determinations of control plasma was 13.8%. The detection limit of the assay was 25 pg 13,14-dihydro-15-ketoprostaglandin F/ml plasma. In 3 cows around estrus there was a complex series of peaks of 13,14-dihydro-15-keto-prostaglandin F concentrations coincident with luteolysis and declining progesterone concentrations. Changes in peripheral plasma concentrations of 13,14-dihydro-15-ketoprostaglandin F in the pregnant cow near term showed a close correlation with prostaglandin F levels in utero-ovarian venous plasma. The concentration of 13,14-dihydro-15-keto-prostaglandin F in 12 men was 114?20 pg/ml plasma. It is concluded that the measurement of peripheral 13,14-dihydro-15-keto-prostaglandin F concentrations may offer a simple and convenient method for monitoring uterine prostaglandin F production in the cow.
ACKNOWLEDGMENTS The authors wish to thank Mrs P. Lane for excellent technical assistance, Dr J.F. Smith and Mr J.T. Hunter for the bovine samples. We are also indebted to Dr K. Kirton of the Upjohn Company for the supply of crystalline 13,14-dihydro-15-keto-PGF2o and for 13,14dihydro-15-keto-PGF2o antiserum (Reference 11, 560-JCC-1461.
266
AUGUST
1975
VOL. 10 NO. 2
PROSTAGLANDINS
INTRODUCTION Previous studies have shown that the onset of luteolysis in the ewe (1,2), COW(~) and sow (4) is accompanied by elevated levels of PGF in utero-ovarian venous plasma. The corresponding levels of PGF in arterial venous plasma are however, very low (5) owing to local tissue metabolism and a very rapid clearance of PGF from the peripheral circulation (6). Experiments in which PGF2, was infused intravenously have shown that PGF2o is rapidly converted into 13,14dihydro- 15-keto- PGF2, (6) . In addition it was shown that the levels of this metabolite were lo-75 times the concentration of the infused PGF2o. (7). On the basis of these findings Granstrijm and Samuelsson (8) proposed that the determination of concentration of 13,14-dihydro15-keto-prostaglandin F (hereinafter referred to as PGFM) in plasma However the may be the best means of monitoring PGF production. radioimmunoassay they developed required a second antibody and the standard curve had a sensitivity of only 50-100 pg. This paper describes a simpler but more sensitive radioimmunoassay for PGFMand provides evidence that the assay may be used as an index of PGF release from the uterus around estrus and parturition in the cow. MATERIALS Prostaglandin Fl,-5,6-3H (62 Ci/m.mol) was purchased from New England Nuclear Corporation, Chicago and checked for radiochemical purity by silicic acid chromatography. Bovine gamma globulin was obtained from the Sigma Chemical Company. Diethyl ether (Fluka Switzerland) was used without prior distillation. Phosphosaline buffer (pH 7.4, 0.01 M) was prepared by dissolving 0..175 g KH2PO4, 0.425 g Na2HP04, 9 g NaCl, 0.1 g merthiolate and 0.372 g EDTA in Scintillation fluid was prepared 1000 ml glass redistilled water. by adding two parts of Toluene (Shell) containing 0.4% PPO and 0.01% POPOP to one part of Triton X-100 (Rohm Hass). A stock solution of 13,-14-dihydro-15-keto-PGF2, was made up in ethanol to a concentration of 20 pg/ml. Working solutions of less than 50 ng/ml were diluted daily from a secondary stock standard of 200 ng/ml. METHODS Preparation
of
labelled
PGFM
The tritiated PGF metabolite was prepared by a modification of the method of Granstrijm (9). Approximately 300 mg aspirin, suspended in 15% gelatin was injected into one male rat to suppress endogenous prostaglandin F (10). The animal was sacrificed six h later and a portion of the lung (2 g) was minced and homogenized in 8 ml Bucher medium and centrifuged at 10 000 g for 15 min. The supernatant was removed and centrifuged at 105 OOb g for 60 min and the supernatant fraction thus obtained was incubated for 3 h at 37OC with 100 uCi
AUGUST
1975
VOL. 10 NO. 2
267
PROSTAGLANDINS
(5,6-3H)-PGFlcr and 0.1 mMNAD+. At the completion of the incubation the reaction mixture was acidified to pH 3 with N HCl and the radioactivity was extracted twice with 4 vol diethyl ether. The ether extract was washed twice with distilled water and evaporated to dryness under a stream of air. The labelled PGFMwas purified by thin layer chromatography on silica gel (Eastman Kodak 6061) using the solvent system dioxane:benzene:acetic acid (50:50:2). The area corresponding to the PGF metabolite was scraped off the plate and eluted in 6 ml ethanol. Conversion of 3H-PGF to 3H-PGFM ranged from ZO-50%. Radioimmunoassay
of PGFM
Plasma samples (1.0 ml) in duplicate were acidified to pH 3 with 0.2 ml N HCl and extracted with 4 ml diethyl ether, in disposable polypropylene test tubes (100 x 15 mm). The aqueous phase was frozen in a dry ice/acetone mixture and the extract decacanted into a clean tube. PGFMstandards 0, 0.05, 0.1, 0.2, 0.5, 1.0 and 2.0 ng were pipetted in duplicate into test tubes in which 4 ml diethyl ether had previously been evaporated to allow for any solvent blanks. The standards and samples were then taken to dryness under air. Thr residues were dissolved in 0.1 ml PGFMantiserum (diluted 1:2000 with phosphosaline buffer), 0.1 ml 3H-PGFM (about 4000 cpm in phosphosaline buffer) and 0.1 ml of a 3% w/v bovine gamma globulin solution. After mixing, the tubes were incubated at 23OC for 1 h, and then left overnight at 4OC. Free and bound PGFM were separated by adding 1 ml polyethylene glycol (16.2% w/v) to each tube while vortexing. Following centrifugation at 15 000 g the supernatant was removed by aspiration and the precipitate dissolved in 1 ml distilled water. The contents of each tube was then decanted into a counting vial containing 5 ml Triton scintillation fluid. The vials were counted in a Nuclear Chicago Scintillation counter (Isocap 300) to a preset error of +2%. The PGFMlevels were estimated from the standard curve and the results corrected for a mean recovery of 77%. This figure was based on the amount of labelled metabolite recovered from 6 samples in duplicate. Biological
Studies
Cannulas were inserted into a jugular vein of 3 cows on day Estrus was detected by a vasectomized bull 10 of the estrus cycle. Blood samples were withdrawn every fitted with a chinball harness. 2 h for 10 days into cold heparinized syringes and centrifuged withPlasma was stored at -15’C prior to analysis. In addin 10 min. ition, using the method previously described (ll), catheters were inserted into the jugular and utero-ovarian veins of a fourth cow at about day 240 of gestation. Samples were collected once a day simultaneously from the jugular and utero-ovarian veins until near calving when sampling was more frequent. Samples were also 1600 h.
268
taken from 12 healthy
AUGUST
men between
1975
0800 and
VOL. 10 NO. 2
PROSTAGLANDINS
RESULTS Accuracy and Precision The accuracy of the method was assessed by adding 0, 0.2, 0.5, 1.0 and 2.0 ng PGFM to 0.5 ml bovine plasma. The samples were extractedand assayed by radioimmunoassayas describedpreviously. The results are shown in Fig 1. The intra-assaycoefficientof variation (CV) in 6 replicate determinationsfrom a bovine plasma pool was 13.6%. The corresponding inter-assayCV was 13.8% (n = 9).
I
I
I
I
0.5 I.5 I.0 13,14-dihydro - 15. keto-prostaglandin added ( ng)
Fig. 1
AUGUST
I
2.0 Fea
Accuracy - the PGFM recoveredis plotted as a function of PGFM added. The verticalbars represent the S.D. for 6 determinationswhile the regression was obtained by least squares analysis.
1975
VOL. 10 NO. 2
269
PROSTAGLANDINS
Standard
Curve
The percent binding for the control tubes was 32%. At the 2.0 ng PGFMlevel the percent binding was 8%. Non specific binding of the curve was around 4%. The limit of sensitivity of the standard curve, defined by Ekins and Newman (12) as the variance of the control point at the 95% confidence limit, was 20 pg. For practical purposes, taking into account procedural losses the limit of sensitivity of the assay was 25 pg/ml plasma. Specificity Cross reactivity of the PGFMantiserum for PGFM, 13,14-dihydro 15-keto PGF2o, PGF and Cl6 urinary metabolites of PGF were ;;#a 0.5%, 20%,
RADIOIMMUNOASSAY OF 13,14-DIHYDRO-15-KETO PROSTAGLANDIN F IN BOVINEPERIPHERALPLASMA R.J.
Fairclough
New Zealand Ministry
and E. Payne
of Agriculture
Research Ruakura Agricultural Private
and Fisheries,
Division, Research
Centre,
Bag, Hamilton,
New Zealand.
ABSTRACT A radioimmunoassay has been developed for 13,14-dihydro-15keto-prostaglandin F in bovine peripheral plasma. Acidified plasma samples were extracted with diethyl ether and the dried extracts assayed for 13,14-dihydro-15-keto-prostaglandin F using antiserum raised against a 13,14-dihydro-15-keto-prostaglandin F2o-albumin complex. The tracer used for the assay was prepared Polyethylene enzymatically from tritiated prostaglandin Flo. glycol was employed to separate free and bound 13,14-dihydro-15keto-prostaglandin F. The inter-assay coefficient of variation based on 9 determinations of control plasma was 13.8%. The detection limit of the assay was 25 pg 13,14-dihydro-15-ketoprostaglandin F/ml plasma. In 3 cows around estrus there was a complex series of peaks of 13,14-dihydro-15-keto-prostaglandin F concentrations coincident with luteolysis and declining progesterone concentrations. Changes in peripheral plasma concentrations of 13,14-dihydro-15-ketoprostaglandin F in the pregnant cow near term showed a close correlation with prostaglandin F levels in utero-ovarian venous plasma. The concentration of 13,14-dihydro-15-keto-prostaglandin F in 12 men was 114?20 pg/ml plasma. It is concluded that the measurement of peripheral 13,14-dihydro-15-keto-prostaglandin F concentrations may offer a simple and convenient method for monitoring uterine prostaglandin F production in the cow.
ACKNOWLEDGMENTS The authors wish to thank Mrs P. Lane for excellent technical assistance, Dr J.F. Smith and Mr J.T. Hunter for the bovine samples. We are also indebted to Dr K. Kirton of the Upjohn Company for the supply of crystalline 13,14-dihydro-15-keto-PGF2o and for 13,14dihydro-15-keto-PGF2o antiserum (Reference 11, 560-JCC-1461.
266
AUGUST
1975
VOL. 10 NO. 2
PROSTAGLANDINS
INTRODUCTION Previous studies have shown that the onset of luteolysis in the ewe (1,2), COW(~) and sow (4) is accompanied by elevated levels of PGF in utero-ovarian venous plasma. The corresponding levels of PGF in arterial venous plasma are however, very low (5) owing to local tissue metabolism and a very rapid clearance of PGF from the peripheral circulation (6). Experiments in which PGF2, was infused intravenously have shown that PGF2o is rapidly converted into 13,14dihydro- 15-keto- PGF2, (6) . In addition it was shown that the levels of this metabolite were lo-75 times the concentration of the infused PGF2o. (7). On the basis of these findings Granstrijm and Samuelsson (8) proposed that the determination of concentration of 13,14-dihydro15-keto-prostaglandin F (hereinafter referred to as PGFM) in plasma However the may be the best means of monitoring PGF production. radioimmunoassay they developed required a second antibody and the standard curve had a sensitivity of only 50-100 pg. This paper describes a simpler but more sensitive radioimmunoassay for PGFMand provides evidence that the assay may be used as an index of PGF release from the uterus around estrus and parturition in the cow. MATERIALS Prostaglandin Fl,-5,6-3H (62 Ci/m.mol) was purchased from New England Nuclear Corporation, Chicago and checked for radiochemical purity by silicic acid chromatography. Bovine gamma globulin was obtained from the Sigma Chemical Company. Diethyl ether (Fluka Switzerland) was used without prior distillation. Phosphosaline buffer (pH 7.4, 0.01 M) was prepared by dissolving 0..175 g KH2PO4, 0.425 g Na2HP04, 9 g NaCl, 0.1 g merthiolate and 0.372 g EDTA in Scintillation fluid was prepared 1000 ml glass redistilled water. by adding two parts of Toluene (Shell) containing 0.4% PPO and 0.01% POPOP to one part of Triton X-100 (Rohm Hass). A stock solution of 13,-14-dihydro-15-keto-PGF2, was made up in ethanol to a concentration of 20 pg/ml. Working solutions of less than 50 ng/ml were diluted daily from a secondary stock standard of 200 ng/ml. METHODS Preparation
of
labelled
PGFM
The tritiated PGF metabolite was prepared by a modification of the method of Granstrijm (9). Approximately 300 mg aspirin, suspended in 15% gelatin was injected into one male rat to suppress endogenous prostaglandin F (10). The animal was sacrificed six h later and a portion of the lung (2 g) was minced and homogenized in 8 ml Bucher medium and centrifuged at 10 000 g for 15 min. The supernatant was removed and centrifuged at 105 OOb g for 60 min and the supernatant fraction thus obtained was incubated for 3 h at 37OC with 100 uCi
AUGUST
1975
VOL. 10 NO. 2
267
PROSTAGLANDINS
(5,6-3H)-PGFlcr and 0.1 mMNAD+. At the completion of the incubation the reaction mixture was acidified to pH 3 with N HCl and the radioactivity was extracted twice with 4 vol diethyl ether. The ether extract was washed twice with distilled water and evaporated to dryness under a stream of air. The labelled PGFMwas purified by thin layer chromatography on silica gel (Eastman Kodak 6061) using the solvent system dioxane:benzene:acetic acid (50:50:2). The area corresponding to the PGF metabolite was scraped off the plate and eluted in 6 ml ethanol. Conversion of 3H-PGF to 3H-PGFM ranged from ZO-50%. Radioimmunoassay
of PGFM
Plasma samples (1.0 ml) in duplicate were acidified to pH 3 with 0.2 ml N HCl and extracted with 4 ml diethyl ether, in disposable polypropylene test tubes (100 x 15 mm). The aqueous phase was frozen in a dry ice/acetone mixture and the extract decacanted into a clean tube. PGFMstandards 0, 0.05, 0.1, 0.2, 0.5, 1.0 and 2.0 ng were pipetted in duplicate into test tubes in which 4 ml diethyl ether had previously been evaporated to allow for any solvent blanks. The standards and samples were then taken to dryness under air. Thr residues were dissolved in 0.1 ml PGFMantiserum (diluted 1:2000 with phosphosaline buffer), 0.1 ml 3H-PGFM (about 4000 cpm in phosphosaline buffer) and 0.1 ml of a 3% w/v bovine gamma globulin solution. After mixing, the tubes were incubated at 23OC for 1 h, and then left overnight at 4OC. Free and bound PGFM were separated by adding 1 ml polyethylene glycol (16.2% w/v) to each tube while vortexing. Following centrifugation at 15 000 g the supernatant was removed by aspiration and the precipitate dissolved in 1 ml distilled water. The contents of each tube was then decanted into a counting vial containing 5 ml Triton scintillation fluid. The vials were counted in a Nuclear Chicago Scintillation counter (Isocap 300) to a preset error of +2%. The PGFMlevels were estimated from the standard curve and the results corrected for a mean recovery of 77%. This figure was based on the amount of labelled metabolite recovered from 6 samples in duplicate. Biological
Studies
Cannulas were inserted into a jugular vein of 3 cows on day Estrus was detected by a vasectomized bull 10 of the estrus cycle. Blood samples were withdrawn every fitted with a chinball harness. 2 h for 10 days into cold heparinized syringes and centrifuged withPlasma was stored at -15’C prior to analysis. In addin 10 min. ition, using the method previously described (ll), catheters were inserted into the jugular and utero-ovarian veins of a fourth cow at about day 240 of gestation. Samples were collected once a day simultaneously from the jugular and utero-ovarian veins until near calving when sampling was more frequent. Samples were also 1600 h.
268
taken from 12 healthy
AUGUST
men between
1975
0800 and
VOL. 10 NO. 2
PROSTAGLANDINS
RESULTS Accuracy and Precision The accuracy of the method was assessed by adding 0, 0.2, 0.5, 1.0 and 2.0 ng PGFM to 0.5 ml bovine plasma. The samples were extractedand assayed by radioimmunoassayas describedpreviously. The results are shown in Fig 1. The intra-assaycoefficientof variation (CV) in 6 replicate determinationsfrom a bovine plasma pool was 13.6%. The corresponding inter-assayCV was 13.8% (n = 9).
I
I
I
I
0.5 I.5 I.0 13,14-dihydro - 15. keto-prostaglandin added ( ng)
Fig. 1
AUGUST
I
2.0 Fea
Accuracy - the PGFM recoveredis plotted as a function of PGFM added. The verticalbars represent the S.D. for 6 determinationswhile the regression was obtained by least squares analysis.
1975
VOL. 10 NO. 2
269
PROSTAGLANDINS
Standard
Curve
The percent binding for the control tubes was 32%. At the 2.0 ng PGFMlevel the percent binding was 8%. Non specific binding of the curve was around 4%. The limit of sensitivity of the standard curve, defined by Ekins and Newman (12) as the variance of the control point at the 95% confidence limit, was 20 pg. For practical purposes, taking into account procedural losses the limit of sensitivity of the assay was 25 pg/ml plasma. Specificity Cross reactivity of the PGFMantiserum for PGFM, 13,14-dihydro 15-keto PGF2o, PGF and Cl6 urinary metabolites of PGF were ;;#a 0.5%, 20%,
