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Radioimmunoassay for urinary metabolites of prostaglandin F2α
PROSTAGLANDINS
RADIOIMMUNOASSAY FOR URINARY METABOLITES OF PROSTAGLANDIN FZc Granstrijm, E. and Kindahl, Department
H.
of Chemistry, Karolinska Institutet, Stockholm 60, Sweden.
S-104 01
ABSTRACT Antibodies against the main urinary metabolite of PGF2, in the human, 5c,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the w position to bovine serum albumin prior to injection. The resulting antibodies did not distinguish between tetranor compounds varying only in structure at the w carbon, and thus the assay could be used also for other metabolites of PGF2 e.g. the main urinary metabolite in the guinea pig, 5a,7a-%ihydroxy-ll-ketotetranorprostanoic acid. Labeled ligands for the assays were prepared either in vivo by injection of 117,18-3HI-PGF?, into humans after several days' treatment with indomethacin, or in vitro by incubation of 117,18-3HI-lS-keto-13,14-dihydro-PGF3c with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively. The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF2,; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment. The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.
ACKNOWLEDGMENT This investigation was supported by the World Health Organization. The authors are indebted to Miss Ulla Nordin and Mrs. Siv Andell for excellent technical assistance.
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INTRODUCTION Recently, interest has been focussed on levels of prostaglandin metabolites instead of primary prostaglandins as indicators of the endogenous biosynthesis and release of prostaglandins (for a discussion,see Ref. 1). In the circulation, prostaglandins occur mainly as their corresponding 15-keto-13,14-dihydro metabolites, which have considerably longer half-lives than the parent compounds. Furthermore, the nonspecific formation of primary prostaglandins during the sample collection leads to serious problems in attempts to assay these compounds, which is reflected by the large discrepancies in the reported and the calculated levels of e.g. PGF2c in peripheral plasma (2). In recent years, therefore, several radioimmunoassays for 15-keto-13,14-dihydroPGF2u have been developed (3-7). However, since it is known that prostaglandin release often occurs intermittently (cf. 8,9), even the half-life of the 15-keto-13,14-dihydro compounds in thecirculation may be too short, if the blood sampling is not frequent enough. Thus, in some studies measurement of the urinary metabolites of prostaglandins may be more reliable, since a major part of prostaglandins that reach the blood stream is eventually excreted into urine as shorter compounds (1). Mass spectrometric quantitative methods for several urinary prostaglandins have been developed (l), and in recent years a few radioimmunoassays for the main urinary metabolite of PGF2, in the human, Set, 7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid, have been published (7,10-12). The present report describes the development of methods for two major urinary metabolites of PGF2, and PGFlu,e. 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid (human (1)) and 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid (guinea pig (l)), and the application of these methods to various studies, where normal excretion of prostaglandin metabolites as well as excretion during enhanced or diminished production of prostaglandins are measured.
EXPERIMENTAL Preparation
of Conjugate
PROCEDURE
and Immunization
of Rabbits
Methyl Sa,7a-dihydroxy-ll-ketotetranorprosta-l,l6-dioate, 12 mg, was prepared from patients that had received PGF2, for termination of pregnancy (13). The compound was dissolved in 1 ml methanol and 1 ml 2N NaOH was added. After 12 hr in a stoppered test tube at room temperature, the solution was diluted with 20 ml water, and acidified to pH 3.5 by the dropwise addition of 0.2 N HCl. The acidified solution was subsequently percolated through a 10 g column of Amberlite XAD-2 (13), and after washing of the column with 100 ml water, the produ'ct of the hydrolysis, 5a,7a-dihydroxy-ll-ketotetranor-
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-prosta-1,16-dioic acid, was eluted with 100 ml methanol and taken to dryness by evaporation of the solvent. The compound was subsequently kept in 1 ml glacial acetic acid for 1 hour at room temperature to induce the formation of a 6-lactone (13). The acetic acid .was evaporated under a stream of nitrogen and the dry residue, 9.7 mg, vas dissolved in 2 ml dry dimethylformamide. The &-lactone of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid was subsequently coupled selectively at the w carboxyl group to 40 mg bovine serum albumin using 5 mq N,N'-carbonvldiimidazole as coupling reagent (14). The preparation of this conjugate is summarized in Fig. 1.
Fig. 1. Preparation of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid - BSA conjugate for immunization.
After 7 hr at room temperature, the products of the reaction were dialyzed extensively, first against dimethylformamide/ /water for 12 hr and then against water for 2 days. After lyophilization, the dry weight of the conjugate was 42 mg. The molar ratio (prostaglandin metabolite/bovine serum albumin) was not measured. The lyophilized conjugate was divided into eight equal portions and was used for two rabbits. Each portion was dissolved in 1 ml water and emulsified with 1 ml of Freund's complete adjuvant. The rabbits were given this emulsion by subcutaneous injection at multiple sites in the flank area. The injections were given three times with one week's interval. The fourth dose was injected after a rest period of two months. The antibody titer rose rapidly after this booster dose and reached a peak after 10 days, when both the animals were bled.
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Preparation
of Radio-Labeled
Ligand
1. 112L14~1Hlr5a,Za-Dihydroxy-ll-ketotetranor~rosta-l 16__-__ _-__ _____---________ _-,---_L___ -dioic Acid (Homoloqous Tracer) __-__-______-_-____ ___________ This corn ound was prepared in vivo, by injection of 80 UCi 117,18- 3 HI-PGF2, (specific activity 50 Ci/mmole) into a human female, who had taken indomethacin, 50 mg x 4, for four days prior to the injection (13, cf. 30). Before the prostaglandin was given, a 24 hr portion of urine was collected to allow estimation of the endogenous prostaglandin production. Urine was collected for 2 hrs after the injection, and methyl 113,14-3HI-5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioate was purified as described earlier (13). The compound was hydrolyzed and extracted as described above and stored frozen as a stock solution in 5 ml 0.05 M Trisbuffer until use. Yield: 8 uCi. The specific activity of this preparation was calculated as 18 Ci/mmole (see Results).
2. 112L14~'H1~5~LI~~Dlhydroxy-ll-ketotetranor~rostanoic ___- __________-_____ __---___Acid (Heteroloqous Tracer) --_---_-____-_ ----_-_-__113,14-3HI-5a,7a-Dihydroxy-ll-ketotetranorprostanoic acid, the main urinary metabolite of PGF2c in the uinea si9_(31), was prepared by B-oxidation in vitro of 9 17,18HI 15-keto-13,14-dihydro-PGF2,. This latter compound was prepared from 117,18-3HI-PGF2, (80 uCi, specific activity 50 Ci/mmole) by incubation with the high speed supernatant from swine kidney in the presence of EDTA and 5,8,11,14-eicosatetraynoic acid (15). The product obtained 117,18-3HI-15-keto-13,14-dihydro-PGF2c (53 uCi, specific activity 45 Ci/mmole), was incubated with a preparation of washed mitochondria from 7 g of rat liver. The incubation was carried out as described in ref. (16); however, carnitine was added to a final concentration of 0.24 mM as an additional cofactor (17). The products of the incubation were extracted with ether at pH 3 and were purified by reversed phase partition chromatography, system C-38 (16,31). Yield: 18 uCi. The specific activity was not determined.
Preparation
of Standards
1. ___ Preparation of Human Urinary _-__-_____-_ Metabolites _______--__-__-_____-__ The urinary metabolites of PGF2@ were prepared in vivo with known specific activities from urine of a female subject, who had received 5 mg of /98-3HI-PGF2, (specific activity 0.3 uCi/umole) by intravenous infusion during 1 hr, after having taken indomethacin for four days, 50 mg x 4
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(cf. ref. 30). The metabolites were: 5u,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid (the main metabolite, Ib-1); 7ar9cc-dihydroxy-l3-keto(dinor,clrdinor)prost-3-en-1,16-dioic acid (Ib-3); 7a,9a,l3-trihydroxy(dinor,w-dinor)prost-3-en-l, 16-dioic acid (If); 7a,9a,l8-trihydroxy-l3-ketodinorprost-3-enoic acid (IIIa); 7a,9a-dihydroxy-13-ketodinorprost-3-en-1,18-dioic acid (IIIb); 5a,7a,l6-trihydroxy-ll-ketotetranorprostanoic acid (Ic-2); 5a,7a,ll-trihydroxytetranorprosta-1,16-dioic acid (Ie); 5a,7a,ll-trihydroxy(tetranor, w-dinor)prosta-1,14-dioic acid (IVb); 5a,7a-dihydroxy-ll-keto(tetranor,w-dinor)prosta-1,14-dioic acid (IVd); and 7a,9~-dihydroxy(dinor,w-tetranor)prost-3-en-l,l4-dioic acid (II) (see ref. 1). The metabolites were purified as methyl esters according to the procedures described in refs. (13) and (18). All compounds were subsequently subjected to mild alkaline hydrolysis, and extracted after the hydrolysis as described under "Preparation of Conjugate and Immunization of Rabbits" above. As the endogenous contribution could be neglected under these circumstances (28), the specific activities could be regarded as unchanged. Serial dilution of all the ten metabolites were prepared in 0.05 M Tris-buffer.
2. AcId_~Main_G;inea Preparation of 5a,7a-Dihydgpgy-ll-ketotetranorprostanoic Pis_Metabolifei_____________ -------__________-___--_---_ _____--_____ acid 113,14-3H1-5 a,7a-Dihydroxy-ll-ketotetranorprostanoic was prepared by B-oxidation in vitro of 117,18-3HI-15-keto-13,14-dihydro-PGF2, (100 pg, specific activity 0.3 pCi/ /pmole). The B-oxidation and the purification of the resulting metabolite were carried out as described for the preparation of radiolabeled ligand (heterologous tracer) (cf. also ref. 16). Yield: 18 ug. A serial dilution of the compound was prepared in 0.05 M Tris-buffer. Radioimmunoassay All preparations and dilutions were made using 0.05 M Trisbuffer, pH 7.4, containing 10-3 M EDTA, unless otherwise stated. The antisera obtained from the two rabbits were of similar titers, and one of them was further used in this study. To a series of glass tubes was added 0.1 ml of a serial dilution of the appropriate standard compound (for guinea pig experiacid, and ments, 5a,7a-dihydroxy-ll-ketotetranorprostanoic for human experiments, 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid). The amounts added generally ranged from 1 pg to 300 pg per tube. Both these standards were labelled with tritium (see Preparation, above), however, in these amounts the amounts of tritium added were too low to interfere with the radioimmunoassay. To another series of tubes
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were added 50 or 100 ~1 of a suitable urine dilution (see below) in duplicates. To both series of tubes was added 0.2 ml of a 0.5% solution of bovine y-globulin in buffer and the volume in all tubes was adjusted to 0.5 ml with Tris-buffer. One tenth ml of a suitable antiserum dilution (human experiments, 1:400, final dilution in the assay, 1:2800; guinea pig experiments, 1:1900, final dilution in the assay , 1: 13 300) was added, and after 30 min in room temperature 0.1 ml of a solution of the appropriate radiolabeled ligand was added (for guinea pig experiments (13,14-3HI-5a,7a-dihydroxy-ll-ketotetranorprostanoic acid, and for human experiments 113,14-3HI-5a,7a-dihydroxy-ll-ketotetranorprosta-l,l6-dioic acid). About 4000 dpm were added to each tube, and the whole series was left overnight at +4oC. The tubes were then cooled on ice for 15 min, and 0.7 ml of an ice-cold polyethylene glycol solution (25% PEG, MW 4000, in H20) was added (19). The tubes were vigorously agitated on a Vortex mixer and centrifuged at 1400 x g at +4oC for 1 hr. The supernatant from each tube was decanted into scintillation vials containing 6 ml of Ready-Solv (Beckman Instruments, Inc.). The vials were counted in a Packard Tri-Carb Scintillation Spectrometer, model 3330, equipped with automatic external standardization and a Tele-type writer with a punch. All calculations were performed off-line as a function of logit (32) versus added amounts of prostaglandin metabolite on a Digital equipment PDP 81 computer.
Guinea Pig Experiments 1. Estrous ___-----_Cycles _--Eleven female guinea pigs (nos. l-11) were housed under constant conditions of light and temperature from the age of two months. The animals were given water and commercial diet ad libitum. The experiment was started when the animals were six months old. Estrous behaviour (lordosis) was checked by stroking the posterior of the animals with the fingers in a posterior-anterior direction four to five times daily. This examination in combination with daily inspection of the vaginal membrane was undertaken to establish the day of heat, i.e. day 1 of the estrous cycles. The animals were kept in individual cages during at least two consecutive cycles, and during this period urine was collected at room temperature in one or two portions every day. Each urine portion was diluted to 50, 100 or 200 ml with water, centrifuged, and a small aliquot was stored at -200C until analyzed. The urine was diluted 26-fold with 0.05 M Tris-buffer, pH 7.4, prior to analysis, using an LKB Diluter 2075 (Ultrolab System). 2. Pregnancies and Hysterectomy --___________ _____-__Three of these animals (nos. l-3) were after collection of urine during two estrous cycles housed together with a NOVEMBER
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PROSTAGLANDINS male. The females were checked for symptoms of estrus to establish the heat day, and were after mating again placed in the individual cages for urine collection. In two of the animals (no. 1 and 2) urine was collected in the same way as described above from the second day after mating until day 96 and 97, respectively. The third pregnant animal (no. 3) was hysterectomized on day 33 after mating. The animal was housed as described above for a further period of 11 days, and after killing, autopsy was performed. Body weights were essentially constant over the period of estrous cycle experiments. The animals were not weighed during the pregnancies. 3. Estrogen Treatment ----- -----------178-Estradiol-3-benzoate, 10 ug in 0.1 ml propylene glycol, was injected subcutaneously in four of the guinea pigs (nos. 5-8). These injections were given for 2-9 days.
Experiments 1. Ba&_
in the Human
Excretion
Urine was collected in 24 hr portions for three days from eight apparently healthy human subjects of both sexes to estimate basal excretion of the prostaglandin metabolite. No drugs were allowed from one week before the collection was started. Each 24 hr urine portion was diluted to 2400 ml with distilled water, and 1 ml of this dilution was stored at -200C until analyzed. Prior to analysis the urinary sample was diluted lo-fold with 0.05 M Tris-buffer, pH 7.4. 2. Effect of Anti-Inflammatory -_-Drugs_ ____________________~~~~~~ In three volunteers the effects of indomethacin or acetylsalicylic acid on prostaglandin production were studied. The drugs were given for four days. The dosages were: indomethacin, 50 mg x 4; or acetylsalicylic acid, 650 mg x 4. Urine was collected daily during nine days, starting two days before the administration was commenced, and was treated as described above. Blood samples were in one case taken daily at 9 a.m. in heparinized tubes during the same period. Plasma was isolated, stored frozen, and later analyzed for content of 15-keto-13,14-dihydro-PGF2c (7). 3. Wjection of PGF2e _-__-_--__--_ 198-3HI-PGF2,, 45 ug, specific acitvity 8.0 pCi/pmole, was injected intravenously into one human volunteer. Urine was collected in hourly portions for 12 hours, starting three hours before the injection. The amount of tritium was too low to interfere with the subsequent radioimmunoassay but high enough to allow calculation of the excreted NOVEMBER
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amount of metabolites. Each urinary portion was diluted to 100 ml and a 1 ml aliquot of the dilution stored frozen until analysis. Each aliquot was diluted lo-fold prior to assay, and the aliquots obtained from the three first hrs after the injection were also assayed in a SO-fold dilution. 4. Human Pregnancy ----------_Urine was collected in portions in one human female during the last month of pregnancy. For details, see legend to Fig. 11. The urine was treated as described under 1: Basal Excretion. RESULTS AND DISCUSSION 1. Properties
of the Antiserum
The coupling of the dioic acid metabolite to bovine serum albumin was carried out after the compound had been kept in glacial acetic acid to induce the formation of a g-lactone. The compound should thus be coupled selectively with its wcarboxyl group to amino groups on the protein. However, after the conjugation has taken place, the equilibrium between the open form and the 6-lactone form is no doubt reestablished, particularly at the physiological pH in the rabbit. Thus, the antiserum produced is likely to contain antibodies to both forms. However, since the radioimmunoassay was carried out at pH 1.4, the same equilibrium should be established both for the unlabeled molecules in the standards or samples and for the labeled molecules added as tracer. The heterogenous population of antibodies is thus not likely to be a drawback for the assay. The specificity of the antiserum is shown in Fig. 2. Cross reactivity with the tested C20 prostaglandin compounds was very low (
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1%katodihydroPGF2‘4
I!,-k.todlhydro-POE2
Fig. 2. Specificity of an antiserum against 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid. For the designation of different metabolites tested, see EXPERIMENTAL PROCEDURE or Ref. (1).
did obviously not discriminate well between these compounds; the cross reactivity was greatest when a keto group was present at C-11 as in the main human urinary metabolite, viz. with IVd, Ic-2, and 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid. In these two last-mentioned metabolites the only difference from the main human metabolite is found at the The essentially complete cross reactions with wcarbon. these compounds indicate that the metabolite used for immunization really was coupled entirely at the w position to the bovine serum albumin, since this part of the molecule would then not be recognized by the antibodies. The high cross reactivity with certain tetranor metabolites may influence determination of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid somewhat; however, in the human this major urinary metabolite comprises 22% of the total amount of PGF2c metabolites, whereas the sum of IVd, Ic-2 and 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid11 is only 8% of the total (cf. 18). That the contribution from these three latter metabolites was of minor importance is shown below. On the other hand, the relative non-specificity of the antiserum with respect to ll-ketotetranor compounds renders it useful for studies in other species than the human, e.g. the guinea pig. It also made the use of a heterologous ligand possible, and since 113;14-3HI-5a,7a-dihydroxy-ll-ketotetranorprostanoic acid was possible to prepare by $-oxida1) Granstrijm, E.: Unpublished
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tion in vitro, this heterologous tracer could be obtained with a considerably higher specific activity than the homologous one, which had to be prepared in vivo. This difference was reflected in the dilutions of the antiserum used to obtain the same degree of initial binding in the assay, viz. 1:2800 for the human metabolite, and 1:13300 for the guinea pig metabolite. However, due to the differences in tendencies for these two compounds to form their respective rS-lactones (cf. 13 and 311, the proper labeled ligand was alwa s used in the experiments described below, i.e. 113,14-3H f -5a,7a_dihydroxy -ll-keto-tetranor rosta-1,16-dioic acid for human experiments and [13,14- 5 HI-5e,7a-dihydroxy-ll-ketotetranorprostanoic acid for guinea pig experiments.
2. Properties
of the Assays
Assay of urine: For the assay of urine, either from human -----7--or from guinea pig, various dilutions were tested. Human urine was always diluted to a final volume of 2400 ml/24 hr, and an aliquot of this preparation diluted a further 10 times prior to storage and analysis (see above). In a preliminary study, it was found that analysis of 20 to 200 ~1 of this dilution (1:lO) gave a linear relationship between the volumes of sample added and the resulting readings of metabolite amounts. For all later studies volumes of 50 and 100 ul of this dilution were normally analysed. For guinea pig urine similar tests for parallelism were carried out, and assay of 50 or 100 ul of urine diluted 1:26 was analogously found to give the optimal conditions. Assay-parameters: The lower limit of detection in the assays --_-_-_-_ was 10 pg for the human metabolite assay and 4 pg for the guinea pig metabolite assay. These figures were calculated as the amount of added metabolite which displaced 10% of the bound labeled ligand, and this displacement was always significantly different from the variation around the maximum binding tube. The accuracy of the human assay was determined from two experiments. Fig. 3 shows the results from an experiment in which known amounts of 5a,7a-dihydroxy-ll-ketotetranorprosta1,16-dioic acid had been added to a sample of diluted human urine. The second experiment is shown in Fig. 4. In this experiment, 45 ug of 19$-3HI-PGF2e was given as a bolus injection i.v. to a human subject (see EXPERIMENTAL PROCEDURE). The amounts of 156-3HI-5a,7a-dihydroxy-ll-ketotetranorprosta1,16-dioic acid excreted every hour after this injection could be calculated from the tritium content of the urinary portions and knowledge of the percentage of this metabolite
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among total urinary PGF2c metabolites (average 22%; 20% 1 hr after injection, 25% 6 hrs after injection 1)). The amount of tritium injected was too low to interfere with the radioimmunoassay. The amounts determined by the assay have been corrected for the basal excretion of the metabolite, determined during 3 hrs prior to the injection ( about 0.5 pg/hr in this subject). As can be seen from Fig. 4 a very good correspondence was obtained between the two curves. The slightly higher values found by radioimmunoassay probably reflect the presence of other tetranor metabolites in the urine (see above). The precision of this assay was calculated from a number of duplicate determinations (Table I). The accuracy and precision for the guinea pig assay were determined from corresponding experiments and were found to be of the same order of magnitude.
TABLE
I
The precision of measurements of 5u,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid by radioimmunoassay.
Range (Pg)
Mean (Pg)
Coefficient of variation (%I
Number of duplicate determinations
2- 30
18.9
12.3
74
31- 60
43.6
10.3
65
61- 90
73.7
6.8
34
91-120
105.0
7.1
38
121-150
135.8
9.3
31
1) Granstrom,E.:
Unpublished
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io so
b
2.0 sb loo li0 li0 pg Addition of known amounts of Sd, 7d-dihydroxy-ll-ketotctranorprosta-1,16-dioic acid to urine.
Fig. 3. Accuracy of radioimmunoassay for 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid.
12Measured
by RIA
loe-
6-
,
1
1
2
I
3
1
L
1
5
0
6
7
8
1
g
Fig. 4. Excretion of 5a,7a-dihydroxy-llketotetranorprosta-1,16-dioic acid afte 5 intravenous injection of 45 l.lg 198HI-PGFz, to a female subject.
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3. Studies
in the Guinea Pig
A total of 18 estrous cycles have been studied in the guinea pig with respect to the excretion of 5c.,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine. In all the cycles a marked increase of the excretion was seen around days 11-13 compared to the level earlier in the cycle. Figs. 5 and 6 show two cycles each from four guinea pigs (nos. l-4). The elevation of the urinary metabolite level was in agreement with the findings by Blatchley et al. (20), who found an increase of the PGF2c level in the utero-ovarian venous blood of guinea pigs shortly before estrus. In this study, the highest values of the urinary metabolite were generally seen during the two last days of the cycle and the first day of the next cycle. No progesterone determinations were carried out in this study, but the luteolysis should occur during the last few days of the estrous cycle (20), thus, concomitant with the high prostaglandin production. Determination of the first day of the estrous cycle was mainly based on observations of estrous behaviour and the rupture of the vaginal membrane. The correlation was good enough for a reliable determination of the heat day. No vaginal smears were taken in order to avoid the possible influence of mechanical stimulation on prostaglandin production (21). Whether the production and release of prostaglandins are pulsative or not (cf. 20,22) can not be established by analysis of the urinary metabolite level, even with frequent sampling, since it has been shown that even after a bolus intravenous injection of PGF2c, the excretion of the metabolite in the urine is not completed until after 5-6 hrs (Kindahl, H., unpublished observations, cf. also Fig. 4). Blatchley et al. 1972 (20) demonstrated that daily injections of estradiol-benzoate from days 4 to 6 of the cycle give rise to increased levels of PGF20 on day 7 in the utero-ovarian vein. As can be seen from Fig. 7, treatment with estradiol-benzoate on days 3-7 gave a significant increase of the urinary prostaglandin metabolite level after 48 hrs. The level increased further after repeated injections later in the cycle. This animal showed heat after an estrous cycle of 11 days. The same pattern was obtained in three other animals with the same injection scheThe increase after the second dule of estradiol-benzoate. set of injections could not be distinguished from a normal release prior to estrus.
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5 ; ii :: w
50-
O-
16 I
2
3
4
5 6
7
6
9 10 1, 1 DAY
DAY
OF
THE
OF
i’ I3 14 THE
ESTRO
2 3 4 5 6 ESTROUS CYCLE
17 16 2 S CYCLE
3
4
7
6 6
6 9 10 1, 12 13 14 15 16 1 2
7 8
I’
9 10 11 12 13 14 15 I
:
b
2 3 4
Fig. 5. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine of guinea pigs no. 1 (upper panel) and no. 2 (lower panel).
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t
L
, , , ,, !.TyiJyi-, 5
6
7
8
9 10 II 12 1314 15 16 DAY
0’
-7
1
OF THE ESTROUS
1L’ 1151 161
CYCLE
2
3
16 17
DAY
OF
THE
ESTROUS
CYCLE
Fig. 6. Excretion of 5a,7vdihydroxy-ll-ketotetranorprostanoic acid in the urine of guinea pigs no. 3 (upper panel) and no. 4 (lower panel).
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A
loo-
L E P g
I=
-
50-
ii
-
:: w
_
I
16 17 1 2 3 L 5 6 7 6 9 10 11 12 13 14
DAY
OF
THE
ESTROUS
CYCLE
Fig. 7. Urinary metabolite levels in guinea pig no. 5. Arrows denote time of injection of 10 ug 176estradiol-3-benzoate.
b. Pregnancies -__ _-_____ In none of the pregnant animals could any increase of the prostaglandin urinary metabolite level be seen during days 11-13 as in the case with the nonpregnant animals (Figs. 8,9 and 10). This is in accordance with the findings by other authors (23,24). It seems likely that the conceptus prevents the prostaglandin production during this period. Maule Walker and Poyser (23) have suggested that the conceptus will give rise to an anti-luteolytic hormone. Wlodawer et al. (25) found an inhibitor of the prostaglandin synthetase system present in the microsomal fraction of the endometrium in the nonpregnant guinea pig. The level of this inhibitor might vary during the estrous cycle leading to variable synthesis of prostaglandins. Whether the anti-luteolytic hormone of pregnancy in the guinea pig is identical with this endometrial inhibitor needs further evaluation. In all three animals a slight increase of the metabolite level started around day 25 after mating, and high levels comparable to those normally occurring during luteolysis (cf. Figs 5 and 6) were obtained around day 35 in the two that were allowed to fulfill the period of gestaanimals, tion. This increase is concomitant with the known decrease
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in the corpus luteum activity in the guinea pig at mid-term (26). During the latter part of the pregnancy the placenta contributes to the progesterone production. In the two animals that were allowed to fulfill the gestation period (nos. 1 and Z),the levels of the urinary metabolite were different after this increase between days 2545. The urinary metabolite level of female no. 1 decreased to values around twice the level found before day 25 after mating. It could not be clearly demonstrated if the obtained level of the prostaglandin metabolite was caused by increased synthesis from the uterus or by contribution from the rapidly growing fetuses. The prostaglandin metabolite levels of the second female increased to extremely high levels after day 45 with shortlasting peaks of a few days duration until the time of parturition. Parturition took place in the cages, and thus, urine from the day of delivery was contaminated with some blood and amniotic fluid. Female no. 1 delivered 2 fetuses and female no. 2 delivered 4 after a pregnancy period of 71 days each. The litter was in both cases removed and the lactation of the females ceased without any complications. With respect to the prostaglandin metabolite, both animals showed a similar pattern
Fig. 8. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine during pregnancy in guinea pig no. 1. The vaginal closure membrane showed a minimal opening during days 27-35 after mating.
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PROSTAGLANDINS
around parturition with the highest value found the day after the delivery. The level remained high for 3 and 5 days, respectively, after the parturition. Through the same days the vagina was completely open. The animals showed heat 20 and 19 days, respectively, after the delivery. The prostaglandin metabolite levels during these post-partal estrous cycles were in accordance with the levels found during the estrous cycles prior to the mating. Changes in the vaginal membrane during pregnancy were observed in all the three pregnant females (see Figs. 8, 9 and 10). The reason for these minimal openings in the vaginal membrane is not completely known (27), but in two of the animals (nos. 1 and 3) these changes coincided with the increase of the prostaglandin metabolite. In animal no. 2 the minimal opening was seen after a period of fairly high prostaglandin production.
Fig. 9. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine during pregnancy in guinea pig no. 2. The vaginal closure membrane showed a minimal opening during days 37-40 after mating. 776
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_I
;
r @
HYSTERECTOMY
1
DAY
AFTER
MATING
Fig. 10. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine in guinea pig no. 3. The vaginal closure membrane showed a minimal opening during days 24-36 after mating. Female no. 3 was hysterectomized on day 33 after mating, and the uterus contained five fetuses. After hysterectomy, the level of the urinary metabolite rapidly decreased to a basal level. The autopsy 11 days after hysterectomy revealed no gross pathological changes in the abdomen. Both ovaries were unaffected with a total of five corpora lutea and several minor follicles. From this experiment it could be concluded that 1) the increased level of the urinary metabolite was caused by the uterus or its contents, 2) the uterus contributed only to a minor extent to the production of prostaglandins under normal, basal conditions. It is obvious that hysterectomy, partial or total, will aid in understanding the role of prostaglandins in the utero-ovarian relationship. Studies of this kind, both in connection with normal cycles, with pregnancy, and with estradiol treatment, are in progress in our laboratory.
4. Studies
in the Human
a. __-___-_--__--_ Basal Excretion Table II shows the basal excretion of 5a,7a-dihydroxy-llketotetranorprosta-1,16-dioic acid, measured by radioimmunoassay, in nine healthy human subjects. Some of these subjects have earlier been studied with the mass spectrometric method
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PROSTAGLANDINS
developed by Hamberg (28). The results obtained in the previous study were: subject JAG 59.0 pg/24 hr; IB 39.7 pg/24 hr; HK 20.8 pg/24 hr; KG 15.9 pg/24 hr; SH 13.6 l.rg/24hr; and UN 13.6 ug/24 hr (28). Thus, the values obtained by radioimmunoassay agree well with these earlier found mass spectrometric data.
TABLE II Daily excretion of 5a,7e-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid in human subjects as measured by radioimmunoassay.
Amount
Subject
(pg/24 hr) Day 3
Day 1
Day 2
48.0
58.1
Male IB
34.2
28.9
Male HK
15.7
16.3
14.8
Male KG
21.1
17.9
18.2
Male SH
17.2
16.2
18.6
Male GH
16.8
20.2
17.1
Female UN
9.2
11.6
10.8
Female EG
10.9
9.8
13.7
4.8
7.6
8.8
Male JAG
Female
SA
b. Human Pregnancy -----------The results from the human pregnancy are shown in Fig. .l. Normal basal excretion in this human female was around 4.8 vg/24 hr (0.2 pg/hr). During the last month the urinary prostaglandin metabolite levels were elevated about threefold compared to normal excretion, with very high levels during parturition. These figures agree well with those earlier obtained by Hamberg with the mass spectrometric method (29).
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2.5
0
NON20 PREGNANT WEEK
37
38
39
OF PREGNANCY
LO
t’
PARTUS
2 WEEK
J-
IO
OF POST-PARTAL PERIOD
Fig. 11. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid in one pregnant human female. From week 36 urine was collected in 24 hr portions every second day. From 8 days before delivery all urine was collected; during the day of parturition in portions. c. Effect of Anti-Inflammatory ____ Drugs_ _-_-___-__--______-_~~~~-~ The previous experiments all describe measurements of increased excretion of the main urinary metabolite of PGF2c under conditions known to enhance biosynthesis and release of this compound. In order to assess the applicability of this radioimmunoassay also for measurements of decreased levels of the metabolite, a number of analyses were carried out in human volunteers before, during and after administration of certain anti-inflammatory drugs. Figs. 12 and 13 show the effect of orally administered indomethacin and acetylsalicylic acid, respectively. In the first case parallel measurement of 15-keto-13,14-dihydroPGF2c, the main metabolite of PGF2u in plasma, showed a decrease from about 90 pg/ml to 50 pg/ml. At the same time, the levels of the urinary metabolite decreased from 15 to 6 ug/24 hr. Thus, both parameters indicated approximately 50% inhibition of prostaglandin production. The inhibitory effect of acetylsalicylic acid, 650 mg x 4, was in the same order of magnitude, as can be seen from the experiment with volunteer no. 2, shown in Fig. 13.
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779
PROSTAGLANLHNS
120
i!'Oo : '0 60
Plum8
z\60
Od,lld-dihydroxy-
no
-1%ketoprost-5-moic
acid.
40 20
15
Urinrry Sd,7d-dihydroxy-
*
-11-ketotrtrmor-
f 10
prosta-1,16-dloic
C
acid.
I5
*
345678910 Dayx
Fig.
12.
Prostaglandin F2u production before, during and after administration of indomethacin, measured as major metabolites in plasma (upper panel) and urine (lower panel). Acetylsalicylic acid, 650 mg x 4
? DAYS Fig.
780
13.
Urinary excretion of 5a,7u-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid before, during, and after administration of acetylsalicylic acid.
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In the third volunteer indomethacin was administered during four days. A decrease of the excretion of the urinary metabolite from 4 pg/24 hr to 1.2 pg/24 hr was obtained. During the forth day 80 DCi 117,18-3HI-PGF2, was injected (see Preparation of Radio-Labeled Ligand). With the knowledge of the endogenous contribution immediately prior to the injection, it was possibly to estimate the specific activity of the obtained 113,14- HI-5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid (calculated as 18 Ci/ /mmole).
REFERENCES 1. Samuelsson,B., E. Granstrom, Hammarstrom. Prostaglandins, 1975.
K.GrBen, M.Hamberg, and S. Ann. Rev. Biochem. 44:669,
2. Samuelsson,B. Quantitative aspects on prostaglandin thesis in man, Adv. Biosci. 9:7, 1973.
syn-
3. GranstrSm,E. and B.Samuelsson. Development and mass spectrometric evaluation of a radioimmunoassay for 9a, lla-dihydroxy-15-ketoprost-5-enoic acid, FEBS Lett. 26:211, 1972. 4. Cornette,J.C., K.L.Harrison, and K.T.Kirton. Measurement of prostaglandin F2c metabolites by radioimmunoassay, Prostaglandins 5:155, 1974. 5. Levine,L. and R.M.Gutierrez-Cernosek. Levels of 13,14dihydro-15-keto-PGF2U in biological fluids as measured by radioimmunoassay, Prostaglandins 3:785, 1973. 6. Stylos,W.A., S.Burstein, J.Rosenfeld, E.M.Ritzi, and D. J.Watson. A radioimmunoassay for the initial metabolites of the F prostaglandins, Prostaglandins 4:553, 1973. 7. GranstrBm,E. and H.Kindahl. Radioimmunoassays for prostaglandin metabolites, In: Adv. in Prostaglandin and Thromboxane Research, Vol.1 (Eds. B.Samuelsson and R.Paoletti) Raven Press, New York, p. 81, 1976. 8. Barcikowski,B., J.C.Carlson, L.Wilson, and J.A.McCracken. The effect of endogenous and exogenous estradiol-178 on the release of prostaglandin F2c from the ovine uterus, Endocrinology 95:1340, 1974. 9. Kindahl,H., L.-E.Edqvist, A.Bane, and E.Granstrom. Blood levels of progesterone and 15-keto-13,14-dihydroprostaglandin F2a during the normal oestrous cycle and early pregnancy in heifers, Acta endocr. (Kbh.) 82:134, 1976. 10. Ohki,S., T.Hanyu, K.Imaki, N.Nakazawa, and F.Hirata. Radioimmunoassays of prostaglandin F2c and prosUx+";l:,"_ F2,-main urinary metabolite with prostaglandinsine methylester amide, Prostaglandins 6:137, 1974.
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PROSTAGLANDINS
11.
Cornette,J.C., K.T.Kirton, W.P.Schneider, F.F.Sun, R.A. Johnson, and E.G.Nidy. Preparation and quantitation of urinary metabolites of prostaglandin F2c by radioimmunoassay, Prostaglandins 9:323, 1975.
12. Ohki,S., K.Imaki, F.Hirata, T.Hanyu, and N.Nakazawa. Radioimmunoassay of main urinary metabolite of prostaglandin F~cx, Prostaglandins 10:549, 1975. 13. Granstrdm,E. and B.Samuelsson. On the metabolism of prostaglandin F2c in female subjects, J. Biol. Chem. 246:5254, 1971. as coupling reagent 14. Axen,U. N,N'-Carbonyldiimidazole the preparation of bovine serum albumin conjugates, Prostaglandins 5:45, 1974.
for
15. Grgen,K., E.Granstrdm, B.Samuelsson, and U.Axen. Methods for quantitative analysis of PGF2 , PGE2, 9c,lla-dihydroxy-15-keto-prost-5-enoic aci,% and 9a,lla,l5-trihydroxy-prost-5-enoic acid from body fluids using deuterated carriers and gas chromatography-mass spectrometry, Anal. Biochem. 54~434, 1973. 16. Hamberg,H. Metabolism of prostaglandins in rat liver mitochondria, Eur. J. Biochem. 6:135, 1968. 17. Fritz,I.B. The metabolic consequences of the effects of carnitine on long-chain fatty acid oxidation, In: Cellular Compartmentalization and Control of Fatty Acid Metabolism. (Ed. F.C.Gran), UniversitetsfBrlaget, Oslo, p. 39, 1968. 18. Granstrom,E. and B.Samuelsson. On the metabolism of prostaglandin F2c in female subjects. II. Structures six metabolites, J. Biol. Chem. 246:7470, 1971.
of
19. Van Orden,D.E. and D.B.Farley. Prostaglandin F2c radioimmunoassay utilizing polyethylene glycol separation technique, Prostaglandins 4:215, 1973. 20. Blatchley,F.R., B.T.Donovan, E.W.Horton, and N.L.Poyser. The release of prostaglandins and progestin into the utero-ovarian venous blood of guinea-pigs during the oestrous cycle and following oestrogen treatment, J. Physiol. 223:69, 1972. 21. Roberts,J.S., B.Barcikowski, L.Wilson, R.C.Skarnes, and J.A.McCracken. Hormonal and related factors affecting the release of prostaglandin F2c from the uterus, J. Ster. Biochem. 6:1091, 1975. 22. Earthy,M., C.Bishop, and J.D.Flack. Progesterone and prostaglandin F concentration in utero-ovarian venous plasma of cyclic guinea-pigs, J. Endocrinol. 64:llp, 1975. 23. Maule Walker,F.M. and N.L.Poyser. Production of prostaglandins by the early pregnant guinea-pig uterus in vitro, J. Endocrinol. 61:265, 1974. 782
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24.
Blatchley,F.R., F.M.Maule Walker, and N.L.Poyser. Progesterone, prostaglandin F2c, and oestradiol in the utero-ovarian venous plasma of non-pregnant and early, unilaterally pregnant guinea-pigs, J. Endocrinol. 67: 225, 1975.
25. Wlodawer,P., H.Kindahl, and M.Hamberg. Biosynthesis of prostaglandin F2c from arachidonic acid and prostaglandin endoperoxides in the uterus, Biochim. Biophys. Acta 431:603, 1976. 26. Kovacic,N.M.I. Endocrinology of reproduction, In: Reproduction and Breeding Techniques for Laboratory Animals. (Ed. E.S.E.Hafez), Lea & Febiger, Philadelphia, p. 5, 1970. 27. Ford,D.H., R.C.Webster, and W.C.Young. Rupture of the. vaginal closure membrane during pregnancy in the guinea pig, Anat. Rec. 109:707, 1951. 28. Hamberg,M. Quantitative studies on prostaglandin synthesis in man. II. Determination of the major urinary metabolite of prostaglandins Flc and F2c, Anal. Biochem. 55:368, 1973. 29.
Hamberg,M. Quantitative studies on prostaglandin synthesis in man. III. Excretion of the major urinary metabolite of prostaglandins Flc and F2c during pregnancy, Life Sciences 14:247, 1974.
30. Hamberg, M. Inhibition man, Biochem. Biophys.
of prostaglandin synthesis in Res. Commun. 49:720, 1972.
31. Granstrbm, E. and B. Samuelsson. The structure of the main urinary metabolite of prostaglandin F2c in the guinea pig, Eur. J. Biochem. 10:411, 1969. 32. Rodbard, D.., W. Bridson, tion of radioimmunoassay 770, 1969.
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1976
and P.L. Rayford. Rapid calcularesults, J. Lab. Clin. Med. 74:
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RADIOIMMUNOASSAY FOR URINARY METABOLITES OF PROSTAGLANDIN FZc Granstrijm, E. and Kindahl, Department
H.
of Chemistry, Karolinska Institutet, Stockholm 60, Sweden.
S-104 01
ABSTRACT Antibodies against the main urinary metabolite of PGF2, in the human, 5c,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the w position to bovine serum albumin prior to injection. The resulting antibodies did not distinguish between tetranor compounds varying only in structure at the w carbon, and thus the assay could be used also for other metabolites of PGF2 e.g. the main urinary metabolite in the guinea pig, 5a,7a-%ihydroxy-ll-ketotetranorprostanoic acid. Labeled ligands for the assays were prepared either in vivo by injection of 117,18-3HI-PGF?, into humans after several days' treatment with indomethacin, or in vitro by incubation of 117,18-3HI-lS-keto-13,14-dihydro-PGF3c with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively. The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF2,; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment. The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.
ACKNOWLEDGMENT This investigation was supported by the World Health Organization. The authors are indebted to Miss Ulla Nordin and Mrs. Siv Andell for excellent technical assistance.
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PROSTAGLANDINS
INTRODUCTION Recently, interest has been focussed on levels of prostaglandin metabolites instead of primary prostaglandins as indicators of the endogenous biosynthesis and release of prostaglandins (for a discussion,see Ref. 1). In the circulation, prostaglandins occur mainly as their corresponding 15-keto-13,14-dihydro metabolites, which have considerably longer half-lives than the parent compounds. Furthermore, the nonspecific formation of primary prostaglandins during the sample collection leads to serious problems in attempts to assay these compounds, which is reflected by the large discrepancies in the reported and the calculated levels of e.g. PGF2c in peripheral plasma (2). In recent years, therefore, several radioimmunoassays for 15-keto-13,14-dihydroPGF2u have been developed (3-7). However, since it is known that prostaglandin release often occurs intermittently (cf. 8,9), even the half-life of the 15-keto-13,14-dihydro compounds in thecirculation may be too short, if the blood sampling is not frequent enough. Thus, in some studies measurement of the urinary metabolites of prostaglandins may be more reliable, since a major part of prostaglandins that reach the blood stream is eventually excreted into urine as shorter compounds (1). Mass spectrometric quantitative methods for several urinary prostaglandins have been developed (l), and in recent years a few radioimmunoassays for the main urinary metabolite of PGF2, in the human, Set, 7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid, have been published (7,10-12). The present report describes the development of methods for two major urinary metabolites of PGF2, and PGFlu,e. 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid (human (1)) and 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid (guinea pig (l)), and the application of these methods to various studies, where normal excretion of prostaglandin metabolites as well as excretion during enhanced or diminished production of prostaglandins are measured.
EXPERIMENTAL Preparation
of Conjugate
PROCEDURE
and Immunization
of Rabbits
Methyl Sa,7a-dihydroxy-ll-ketotetranorprosta-l,l6-dioate, 12 mg, was prepared from patients that had received PGF2, for termination of pregnancy (13). The compound was dissolved in 1 ml methanol and 1 ml 2N NaOH was added. After 12 hr in a stoppered test tube at room temperature, the solution was diluted with 20 ml water, and acidified to pH 3.5 by the dropwise addition of 0.2 N HCl. The acidified solution was subsequently percolated through a 10 g column of Amberlite XAD-2 (13), and after washing of the column with 100 ml water, the produ'ct of the hydrolysis, 5a,7a-dihydroxy-ll-ketotetranor-
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-prosta-1,16-dioic acid, was eluted with 100 ml methanol and taken to dryness by evaporation of the solvent. The compound was subsequently kept in 1 ml glacial acetic acid for 1 hour at room temperature to induce the formation of a 6-lactone (13). The acetic acid .was evaporated under a stream of nitrogen and the dry residue, 9.7 mg, vas dissolved in 2 ml dry dimethylformamide. The &-lactone of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid was subsequently coupled selectively at the w carboxyl group to 40 mg bovine serum albumin using 5 mq N,N'-carbonvldiimidazole as coupling reagent (14). The preparation of this conjugate is summarized in Fig. 1.
Fig. 1. Preparation of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid - BSA conjugate for immunization.
After 7 hr at room temperature, the products of the reaction were dialyzed extensively, first against dimethylformamide/ /water for 12 hr and then against water for 2 days. After lyophilization, the dry weight of the conjugate was 42 mg. The molar ratio (prostaglandin metabolite/bovine serum albumin) was not measured. The lyophilized conjugate was divided into eight equal portions and was used for two rabbits. Each portion was dissolved in 1 ml water and emulsified with 1 ml of Freund's complete adjuvant. The rabbits were given this emulsion by subcutaneous injection at multiple sites in the flank area. The injections were given three times with one week's interval. The fourth dose was injected after a rest period of two months. The antibody titer rose rapidly after this booster dose and reached a peak after 10 days, when both the animals were bled.
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Preparation
of Radio-Labeled
Ligand
1. 112L14~1Hlr5a,Za-Dihydroxy-ll-ketotetranor~rosta-l 16__-__ _-__ _____---________ _-,---_L___ -dioic Acid (Homoloqous Tracer) __-__-______-_-____ ___________ This corn ound was prepared in vivo, by injection of 80 UCi 117,18- 3 HI-PGF2, (specific activity 50 Ci/mmole) into a human female, who had taken indomethacin, 50 mg x 4, for four days prior to the injection (13, cf. 30). Before the prostaglandin was given, a 24 hr portion of urine was collected to allow estimation of the endogenous prostaglandin production. Urine was collected for 2 hrs after the injection, and methyl 113,14-3HI-5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioate was purified as described earlier (13). The compound was hydrolyzed and extracted as described above and stored frozen as a stock solution in 5 ml 0.05 M Trisbuffer until use. Yield: 8 uCi. The specific activity of this preparation was calculated as 18 Ci/mmole (see Results).
2. 112L14~'H1~5~LI~~Dlhydroxy-ll-ketotetranor~rostanoic ___- __________-_____ __---___Acid (Heteroloqous Tracer) --_---_-____-_ ----_-_-__113,14-3HI-5a,7a-Dihydroxy-ll-ketotetranorprostanoic acid, the main urinary metabolite of PGF2c in the uinea si9_(31), was prepared by B-oxidation in vitro of 9 17,18HI 15-keto-13,14-dihydro-PGF2,. This latter compound was prepared from 117,18-3HI-PGF2, (80 uCi, specific activity 50 Ci/mmole) by incubation with the high speed supernatant from swine kidney in the presence of EDTA and 5,8,11,14-eicosatetraynoic acid (15). The product obtained 117,18-3HI-15-keto-13,14-dihydro-PGF2c (53 uCi, specific activity 45 Ci/mmole), was incubated with a preparation of washed mitochondria from 7 g of rat liver. The incubation was carried out as described in ref. (16); however, carnitine was added to a final concentration of 0.24 mM as an additional cofactor (17). The products of the incubation were extracted with ether at pH 3 and were purified by reversed phase partition chromatography, system C-38 (16,31). Yield: 18 uCi. The specific activity was not determined.
Preparation
of Standards
1. ___ Preparation of Human Urinary _-__-_____-_ Metabolites _______--__-__-_____-__ The urinary metabolites of PGF2@ were prepared in vivo with known specific activities from urine of a female subject, who had received 5 mg of /98-3HI-PGF2, (specific activity 0.3 uCi/umole) by intravenous infusion during 1 hr, after having taken indomethacin for four days, 50 mg x 4
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(cf. ref. 30). The metabolites were: 5u,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid (the main metabolite, Ib-1); 7ar9cc-dihydroxy-l3-keto(dinor,clrdinor)prost-3-en-1,16-dioic acid (Ib-3); 7a,9a,l3-trihydroxy(dinor,w-dinor)prost-3-en-l, 16-dioic acid (If); 7a,9a,l8-trihydroxy-l3-ketodinorprost-3-enoic acid (IIIa); 7a,9a-dihydroxy-13-ketodinorprost-3-en-1,18-dioic acid (IIIb); 5a,7a,l6-trihydroxy-ll-ketotetranorprostanoic acid (Ic-2); 5a,7a,ll-trihydroxytetranorprosta-1,16-dioic acid (Ie); 5a,7a,ll-trihydroxy(tetranor, w-dinor)prosta-1,14-dioic acid (IVb); 5a,7a-dihydroxy-ll-keto(tetranor,w-dinor)prosta-1,14-dioic acid (IVd); and 7a,9~-dihydroxy(dinor,w-tetranor)prost-3-en-l,l4-dioic acid (II) (see ref. 1). The metabolites were purified as methyl esters according to the procedures described in refs. (13) and (18). All compounds were subsequently subjected to mild alkaline hydrolysis, and extracted after the hydrolysis as described under "Preparation of Conjugate and Immunization of Rabbits" above. As the endogenous contribution could be neglected under these circumstances (28), the specific activities could be regarded as unchanged. Serial dilution of all the ten metabolites were prepared in 0.05 M Tris-buffer.
2. AcId_~Main_G;inea Preparation of 5a,7a-Dihydgpgy-ll-ketotetranorprostanoic Pis_Metabolifei_____________ -------__________-___--_---_ _____--_____ acid 113,14-3H1-5 a,7a-Dihydroxy-ll-ketotetranorprostanoic was prepared by B-oxidation in vitro of 117,18-3HI-15-keto-13,14-dihydro-PGF2, (100 pg, specific activity 0.3 pCi/ /pmole). The B-oxidation and the purification of the resulting metabolite were carried out as described for the preparation of radiolabeled ligand (heterologous tracer) (cf. also ref. 16). Yield: 18 ug. A serial dilution of the compound was prepared in 0.05 M Tris-buffer. Radioimmunoassay All preparations and dilutions were made using 0.05 M Trisbuffer, pH 7.4, containing 10-3 M EDTA, unless otherwise stated. The antisera obtained from the two rabbits were of similar titers, and one of them was further used in this study. To a series of glass tubes was added 0.1 ml of a serial dilution of the appropriate standard compound (for guinea pig experiacid, and ments, 5a,7a-dihydroxy-ll-ketotetranorprostanoic for human experiments, 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid). The amounts added generally ranged from 1 pg to 300 pg per tube. Both these standards were labelled with tritium (see Preparation, above), however, in these amounts the amounts of tritium added were too low to interfere with the radioimmunoassay. To another series of tubes
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PROSTAGLANDINS
were added 50 or 100 ~1 of a suitable urine dilution (see below) in duplicates. To both series of tubes was added 0.2 ml of a 0.5% solution of bovine y-globulin in buffer and the volume in all tubes was adjusted to 0.5 ml with Tris-buffer. One tenth ml of a suitable antiserum dilution (human experiments, 1:400, final dilution in the assay, 1:2800; guinea pig experiments, 1:1900, final dilution in the assay , 1: 13 300) was added, and after 30 min in room temperature 0.1 ml of a solution of the appropriate radiolabeled ligand was added (for guinea pig experiments (13,14-3HI-5a,7a-dihydroxy-ll-ketotetranorprostanoic acid, and for human experiments 113,14-3HI-5a,7a-dihydroxy-ll-ketotetranorprosta-l,l6-dioic acid). About 4000 dpm were added to each tube, and the whole series was left overnight at +4oC. The tubes were then cooled on ice for 15 min, and 0.7 ml of an ice-cold polyethylene glycol solution (25% PEG, MW 4000, in H20) was added (19). The tubes were vigorously agitated on a Vortex mixer and centrifuged at 1400 x g at +4oC for 1 hr. The supernatant from each tube was decanted into scintillation vials containing 6 ml of Ready-Solv (Beckman Instruments, Inc.). The vials were counted in a Packard Tri-Carb Scintillation Spectrometer, model 3330, equipped with automatic external standardization and a Tele-type writer with a punch. All calculations were performed off-line as a function of logit (32) versus added amounts of prostaglandin metabolite on a Digital equipment PDP 81 computer.
Guinea Pig Experiments 1. Estrous ___-----_Cycles _--Eleven female guinea pigs (nos. l-11) were housed under constant conditions of light and temperature from the age of two months. The animals were given water and commercial diet ad libitum. The experiment was started when the animals were six months old. Estrous behaviour (lordosis) was checked by stroking the posterior of the animals with the fingers in a posterior-anterior direction four to five times daily. This examination in combination with daily inspection of the vaginal membrane was undertaken to establish the day of heat, i.e. day 1 of the estrous cycles. The animals were kept in individual cages during at least two consecutive cycles, and during this period urine was collected at room temperature in one or two portions every day. Each urine portion was diluted to 50, 100 or 200 ml with water, centrifuged, and a small aliquot was stored at -200C until analyzed. The urine was diluted 26-fold with 0.05 M Tris-buffer, pH 7.4, prior to analysis, using an LKB Diluter 2075 (Ultrolab System). 2. Pregnancies and Hysterectomy --___________ _____-__Three of these animals (nos. l-3) were after collection of urine during two estrous cycles housed together with a NOVEMBER
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PROSTAGLANDINS male. The females were checked for symptoms of estrus to establish the heat day, and were after mating again placed in the individual cages for urine collection. In two of the animals (no. 1 and 2) urine was collected in the same way as described above from the second day after mating until day 96 and 97, respectively. The third pregnant animal (no. 3) was hysterectomized on day 33 after mating. The animal was housed as described above for a further period of 11 days, and after killing, autopsy was performed. Body weights were essentially constant over the period of estrous cycle experiments. The animals were not weighed during the pregnancies. 3. Estrogen Treatment ----- -----------178-Estradiol-3-benzoate, 10 ug in 0.1 ml propylene glycol, was injected subcutaneously in four of the guinea pigs (nos. 5-8). These injections were given for 2-9 days.
Experiments 1. Ba&_
in the Human
Excretion
Urine was collected in 24 hr portions for three days from eight apparently healthy human subjects of both sexes to estimate basal excretion of the prostaglandin metabolite. No drugs were allowed from one week before the collection was started. Each 24 hr urine portion was diluted to 2400 ml with distilled water, and 1 ml of this dilution was stored at -200C until analyzed. Prior to analysis the urinary sample was diluted lo-fold with 0.05 M Tris-buffer, pH 7.4. 2. Effect of Anti-Inflammatory -_-Drugs_ ____________________~~~~~~ In three volunteers the effects of indomethacin or acetylsalicylic acid on prostaglandin production were studied. The drugs were given for four days. The dosages were: indomethacin, 50 mg x 4; or acetylsalicylic acid, 650 mg x 4. Urine was collected daily during nine days, starting two days before the administration was commenced, and was treated as described above. Blood samples were in one case taken daily at 9 a.m. in heparinized tubes during the same period. Plasma was isolated, stored frozen, and later analyzed for content of 15-keto-13,14-dihydro-PGF2c (7). 3. Wjection of PGF2e _-__-_--__--_ 198-3HI-PGF2,, 45 ug, specific acitvity 8.0 pCi/pmole, was injected intravenously into one human volunteer. Urine was collected in hourly portions for 12 hours, starting three hours before the injection. The amount of tritium was too low to interfere with the subsequent radioimmunoassay but high enough to allow calculation of the excreted NOVEMBER
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amount of metabolites. Each urinary portion was diluted to 100 ml and a 1 ml aliquot of the dilution stored frozen until analysis. Each aliquot was diluted lo-fold prior to assay, and the aliquots obtained from the three first hrs after the injection were also assayed in a SO-fold dilution. 4. Human Pregnancy ----------_Urine was collected in portions in one human female during the last month of pregnancy. For details, see legend to Fig. 11. The urine was treated as described under 1: Basal Excretion. RESULTS AND DISCUSSION 1. Properties
of the Antiserum
The coupling of the dioic acid metabolite to bovine serum albumin was carried out after the compound had been kept in glacial acetic acid to induce the formation of a g-lactone. The compound should thus be coupled selectively with its wcarboxyl group to amino groups on the protein. However, after the conjugation has taken place, the equilibrium between the open form and the 6-lactone form is no doubt reestablished, particularly at the physiological pH in the rabbit. Thus, the antiserum produced is likely to contain antibodies to both forms. However, since the radioimmunoassay was carried out at pH 1.4, the same equilibrium should be established both for the unlabeled molecules in the standards or samples and for the labeled molecules added as tracer. The heterogenous population of antibodies is thus not likely to be a drawback for the assay. The specificity of the antiserum is shown in Fig. 2. Cross reactivity with the tested C20 prostaglandin compounds was very low (
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1%katodihydroPGF2‘4
I!,-k.todlhydro-POE2
Fig. 2. Specificity of an antiserum against 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid. For the designation of different metabolites tested, see EXPERIMENTAL PROCEDURE or Ref. (1).
did obviously not discriminate well between these compounds; the cross reactivity was greatest when a keto group was present at C-11 as in the main human urinary metabolite, viz. with IVd, Ic-2, and 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid. In these two last-mentioned metabolites the only difference from the main human metabolite is found at the The essentially complete cross reactions with wcarbon. these compounds indicate that the metabolite used for immunization really was coupled entirely at the w position to the bovine serum albumin, since this part of the molecule would then not be recognized by the antibodies. The high cross reactivity with certain tetranor metabolites may influence determination of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid somewhat; however, in the human this major urinary metabolite comprises 22% of the total amount of PGF2c metabolites, whereas the sum of IVd, Ic-2 and 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid11 is only 8% of the total (cf. 18). That the contribution from these three latter metabolites was of minor importance is shown below. On the other hand, the relative non-specificity of the antiserum with respect to ll-ketotetranor compounds renders it useful for studies in other species than the human, e.g. the guinea pig. It also made the use of a heterologous ligand possible, and since 113;14-3HI-5a,7a-dihydroxy-ll-ketotetranorprostanoic acid was possible to prepare by $-oxida1) Granstrijm, E.: Unpublished
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tion in vitro, this heterologous tracer could be obtained with a considerably higher specific activity than the homologous one, which had to be prepared in vivo. This difference was reflected in the dilutions of the antiserum used to obtain the same degree of initial binding in the assay, viz. 1:2800 for the human metabolite, and 1:13300 for the guinea pig metabolite. However, due to the differences in tendencies for these two compounds to form their respective rS-lactones (cf. 13 and 311, the proper labeled ligand was alwa s used in the experiments described below, i.e. 113,14-3H f -5a,7a_dihydroxy -ll-keto-tetranor rosta-1,16-dioic acid for human experiments and [13,14- 5 HI-5e,7a-dihydroxy-ll-ketotetranorprostanoic acid for guinea pig experiments.
2. Properties
of the Assays
Assay of urine: For the assay of urine, either from human -----7--or from guinea pig, various dilutions were tested. Human urine was always diluted to a final volume of 2400 ml/24 hr, and an aliquot of this preparation diluted a further 10 times prior to storage and analysis (see above). In a preliminary study, it was found that analysis of 20 to 200 ~1 of this dilution (1:lO) gave a linear relationship between the volumes of sample added and the resulting readings of metabolite amounts. For all later studies volumes of 50 and 100 ul of this dilution were normally analysed. For guinea pig urine similar tests for parallelism were carried out, and assay of 50 or 100 ul of urine diluted 1:26 was analogously found to give the optimal conditions. Assay-parameters: The lower limit of detection in the assays --_-_-_-_ was 10 pg for the human metabolite assay and 4 pg for the guinea pig metabolite assay. These figures were calculated as the amount of added metabolite which displaced 10% of the bound labeled ligand, and this displacement was always significantly different from the variation around the maximum binding tube. The accuracy of the human assay was determined from two experiments. Fig. 3 shows the results from an experiment in which known amounts of 5a,7a-dihydroxy-ll-ketotetranorprosta1,16-dioic acid had been added to a sample of diluted human urine. The second experiment is shown in Fig. 4. In this experiment, 45 ug of 19$-3HI-PGF2e was given as a bolus injection i.v. to a human subject (see EXPERIMENTAL PROCEDURE). The amounts of 156-3HI-5a,7a-dihydroxy-ll-ketotetranorprosta1,16-dioic acid excreted every hour after this injection could be calculated from the tritium content of the urinary portions and knowledge of the percentage of this metabolite
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among total urinary PGF2c metabolites (average 22%; 20% 1 hr after injection, 25% 6 hrs after injection 1)). The amount of tritium injected was too low to interfere with the radioimmunoassay. The amounts determined by the assay have been corrected for the basal excretion of the metabolite, determined during 3 hrs prior to the injection ( about 0.5 pg/hr in this subject). As can be seen from Fig. 4 a very good correspondence was obtained between the two curves. The slightly higher values found by radioimmunoassay probably reflect the presence of other tetranor metabolites in the urine (see above). The precision of this assay was calculated from a number of duplicate determinations (Table I). The accuracy and precision for the guinea pig assay were determined from corresponding experiments and were found to be of the same order of magnitude.
TABLE
I
The precision of measurements of 5u,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid by radioimmunoassay.
Range (Pg)
Mean (Pg)
Coefficient of variation (%I
Number of duplicate determinations
2- 30
18.9
12.3
74
31- 60
43.6
10.3
65
61- 90
73.7
6.8
34
91-120
105.0
7.1
38
121-150
135.8
9.3
31
1) Granstrom,E.:
Unpublished
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1976
observations.
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io so
b
2.0 sb loo li0 li0 pg Addition of known amounts of Sd, 7d-dihydroxy-ll-ketotctranorprosta-1,16-dioic acid to urine.
Fig. 3. Accuracy of radioimmunoassay for 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid.
12Measured
by RIA
loe-
6-
,
1
1
2
I
3
1
L
1
5
0
6
7
8
1
g
Fig. 4. Excretion of 5a,7a-dihydroxy-llketotetranorprosta-1,16-dioic acid afte 5 intravenous injection of 45 l.lg 198HI-PGFz, to a female subject.
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3. Studies
in the Guinea Pig
A total of 18 estrous cycles have been studied in the guinea pig with respect to the excretion of 5c.,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine. In all the cycles a marked increase of the excretion was seen around days 11-13 compared to the level earlier in the cycle. Figs. 5 and 6 show two cycles each from four guinea pigs (nos. l-4). The elevation of the urinary metabolite level was in agreement with the findings by Blatchley et al. (20), who found an increase of the PGF2c level in the utero-ovarian venous blood of guinea pigs shortly before estrus. In this study, the highest values of the urinary metabolite were generally seen during the two last days of the cycle and the first day of the next cycle. No progesterone determinations were carried out in this study, but the luteolysis should occur during the last few days of the estrous cycle (20), thus, concomitant with the high prostaglandin production. Determination of the first day of the estrous cycle was mainly based on observations of estrous behaviour and the rupture of the vaginal membrane. The correlation was good enough for a reliable determination of the heat day. No vaginal smears were taken in order to avoid the possible influence of mechanical stimulation on prostaglandin production (21). Whether the production and release of prostaglandins are pulsative or not (cf. 20,22) can not be established by analysis of the urinary metabolite level, even with frequent sampling, since it has been shown that even after a bolus intravenous injection of PGF2c, the excretion of the metabolite in the urine is not completed until after 5-6 hrs (Kindahl, H., unpublished observations, cf. also Fig. 4). Blatchley et al. 1972 (20) demonstrated that daily injections of estradiol-benzoate from days 4 to 6 of the cycle give rise to increased levels of PGF20 on day 7 in the utero-ovarian vein. As can be seen from Fig. 7, treatment with estradiol-benzoate on days 3-7 gave a significant increase of the urinary prostaglandin metabolite level after 48 hrs. The level increased further after repeated injections later in the cycle. This animal showed heat after an estrous cycle of 11 days. The same pattern was obtained in three other animals with the same injection scheThe increase after the second dule of estradiol-benzoate. set of injections could not be distinguished from a normal release prior to estrus.
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5 ; ii :: w
50-
O-
16 I
2
3
4
5 6
7
6
9 10 1, 1 DAY
DAY
OF
THE
OF
i’ I3 14 THE
ESTRO
2 3 4 5 6 ESTROUS CYCLE
17 16 2 S CYCLE
3
4
7
6 6
6 9 10 1, 12 13 14 15 16 1 2
7 8
I’
9 10 11 12 13 14 15 I
:
b
2 3 4
Fig. 5. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine of guinea pigs no. 1 (upper panel) and no. 2 (lower panel).
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PROSTAGLANDINS
t
L
, , , ,, !.TyiJyi-, 5
6
7
8
9 10 II 12 1314 15 16 DAY
0’
-7
1
OF THE ESTROUS
1L’ 1151 161
CYCLE
2
3
16 17
DAY
OF
THE
ESTROUS
CYCLE
Fig. 6. Excretion of 5a,7vdihydroxy-ll-ketotetranorprostanoic acid in the urine of guinea pigs no. 3 (upper panel) and no. 4 (lower panel).
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PROSTAGLANDINS
A
loo-
L E P g
I=
-
50-
ii
-
:: w
_
I
16 17 1 2 3 L 5 6 7 6 9 10 11 12 13 14
DAY
OF
THE
ESTROUS
CYCLE
Fig. 7. Urinary metabolite levels in guinea pig no. 5. Arrows denote time of injection of 10 ug 176estradiol-3-benzoate.
b. Pregnancies -__ _-_____ In none of the pregnant animals could any increase of the prostaglandin urinary metabolite level be seen during days 11-13 as in the case with the nonpregnant animals (Figs. 8,9 and 10). This is in accordance with the findings by other authors (23,24). It seems likely that the conceptus prevents the prostaglandin production during this period. Maule Walker and Poyser (23) have suggested that the conceptus will give rise to an anti-luteolytic hormone. Wlodawer et al. (25) found an inhibitor of the prostaglandin synthetase system present in the microsomal fraction of the endometrium in the nonpregnant guinea pig. The level of this inhibitor might vary during the estrous cycle leading to variable synthesis of prostaglandins. Whether the anti-luteolytic hormone of pregnancy in the guinea pig is identical with this endometrial inhibitor needs further evaluation. In all three animals a slight increase of the metabolite level started around day 25 after mating, and high levels comparable to those normally occurring during luteolysis (cf. Figs 5 and 6) were obtained around day 35 in the two that were allowed to fulfill the period of gestaanimals, tion. This increase is concomitant with the known decrease
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in the corpus luteum activity in the guinea pig at mid-term (26). During the latter part of the pregnancy the placenta contributes to the progesterone production. In the two animals that were allowed to fulfill the gestation period (nos. 1 and Z),the levels of the urinary metabolite were different after this increase between days 2545. The urinary metabolite level of female no. 1 decreased to values around twice the level found before day 25 after mating. It could not be clearly demonstrated if the obtained level of the prostaglandin metabolite was caused by increased synthesis from the uterus or by contribution from the rapidly growing fetuses. The prostaglandin metabolite levels of the second female increased to extremely high levels after day 45 with shortlasting peaks of a few days duration until the time of parturition. Parturition took place in the cages, and thus, urine from the day of delivery was contaminated with some blood and amniotic fluid. Female no. 1 delivered 2 fetuses and female no. 2 delivered 4 after a pregnancy period of 71 days each. The litter was in both cases removed and the lactation of the females ceased without any complications. With respect to the prostaglandin metabolite, both animals showed a similar pattern
Fig. 8. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine during pregnancy in guinea pig no. 1. The vaginal closure membrane showed a minimal opening during days 27-35 after mating.
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around parturition with the highest value found the day after the delivery. The level remained high for 3 and 5 days, respectively, after the parturition. Through the same days the vagina was completely open. The animals showed heat 20 and 19 days, respectively, after the delivery. The prostaglandin metabolite levels during these post-partal estrous cycles were in accordance with the levels found during the estrous cycles prior to the mating. Changes in the vaginal membrane during pregnancy were observed in all the three pregnant females (see Figs. 8, 9 and 10). The reason for these minimal openings in the vaginal membrane is not completely known (27), but in two of the animals (nos. 1 and 3) these changes coincided with the increase of the prostaglandin metabolite. In animal no. 2 the minimal opening was seen after a period of fairly high prostaglandin production.
Fig. 9. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine during pregnancy in guinea pig no. 2. The vaginal closure membrane showed a minimal opening during days 37-40 after mating. 776
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PROSTAGLANDINS
_I
;
r @
HYSTERECTOMY
1
DAY
AFTER
MATING
Fig. 10. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprostanoic acid in the urine in guinea pig no. 3. The vaginal closure membrane showed a minimal opening during days 24-36 after mating. Female no. 3 was hysterectomized on day 33 after mating, and the uterus contained five fetuses. After hysterectomy, the level of the urinary metabolite rapidly decreased to a basal level. The autopsy 11 days after hysterectomy revealed no gross pathological changes in the abdomen. Both ovaries were unaffected with a total of five corpora lutea and several minor follicles. From this experiment it could be concluded that 1) the increased level of the urinary metabolite was caused by the uterus or its contents, 2) the uterus contributed only to a minor extent to the production of prostaglandins under normal, basal conditions. It is obvious that hysterectomy, partial or total, will aid in understanding the role of prostaglandins in the utero-ovarian relationship. Studies of this kind, both in connection with normal cycles, with pregnancy, and with estradiol treatment, are in progress in our laboratory.
4. Studies
in the Human
a. __-___-_--__--_ Basal Excretion Table II shows the basal excretion of 5a,7a-dihydroxy-llketotetranorprosta-1,16-dioic acid, measured by radioimmunoassay, in nine healthy human subjects. Some of these subjects have earlier been studied with the mass spectrometric method
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developed by Hamberg (28). The results obtained in the previous study were: subject JAG 59.0 pg/24 hr; IB 39.7 pg/24 hr; HK 20.8 pg/24 hr; KG 15.9 pg/24 hr; SH 13.6 l.rg/24hr; and UN 13.6 ug/24 hr (28). Thus, the values obtained by radioimmunoassay agree well with these earlier found mass spectrometric data.
TABLE II Daily excretion of 5a,7e-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid in human subjects as measured by radioimmunoassay.
Amount
Subject
(pg/24 hr) Day 3
Day 1
Day 2
48.0
58.1
Male IB
34.2
28.9
Male HK
15.7
16.3
14.8
Male KG
21.1
17.9
18.2
Male SH
17.2
16.2
18.6
Male GH
16.8
20.2
17.1
Female UN
9.2
11.6
10.8
Female EG
10.9
9.8
13.7
4.8
7.6
8.8
Male JAG
Female
SA
b. Human Pregnancy -----------The results from the human pregnancy are shown in Fig. .l. Normal basal excretion in this human female was around 4.8 vg/24 hr (0.2 pg/hr). During the last month the urinary prostaglandin metabolite levels were elevated about threefold compared to normal excretion, with very high levels during parturition. These figures agree well with those earlier obtained by Hamberg with the mass spectrometric method (29).
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PROSTAGLANDINS
2.5
0
NON20 PREGNANT WEEK
37
38
39
OF PREGNANCY
LO
t’
PARTUS
2 WEEK
J-
IO
OF POST-PARTAL PERIOD
Fig. 11. Excretion of 5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid in one pregnant human female. From week 36 urine was collected in 24 hr portions every second day. From 8 days before delivery all urine was collected; during the day of parturition in portions. c. Effect of Anti-Inflammatory ____ Drugs_ _-_-___-__--______-_~~~~-~ The previous experiments all describe measurements of increased excretion of the main urinary metabolite of PGF2c under conditions known to enhance biosynthesis and release of this compound. In order to assess the applicability of this radioimmunoassay also for measurements of decreased levels of the metabolite, a number of analyses were carried out in human volunteers before, during and after administration of certain anti-inflammatory drugs. Figs. 12 and 13 show the effect of orally administered indomethacin and acetylsalicylic acid, respectively. In the first case parallel measurement of 15-keto-13,14-dihydroPGF2c, the main metabolite of PGF2u in plasma, showed a decrease from about 90 pg/ml to 50 pg/ml. At the same time, the levels of the urinary metabolite decreased from 15 to 6 ug/24 hr. Thus, both parameters indicated approximately 50% inhibition of prostaglandin production. The inhibitory effect of acetylsalicylic acid, 650 mg x 4, was in the same order of magnitude, as can be seen from the experiment with volunteer no. 2, shown in Fig. 13.
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1976 VOL. 12 NO. 5
779
PROSTAGLANLHNS
120
i!'Oo : '0 60
Plum8
z\60
Od,lld-dihydroxy-
no
-1%ketoprost-5-moic
acid.
40 20
15
Urinrry Sd,7d-dihydroxy-
*
-11-ketotrtrmor-
f 10
prosta-1,16-dloic
C
acid.
I5
*
345678910 Dayx
Fig.
12.
Prostaglandin F2u production before, during and after administration of indomethacin, measured as major metabolites in plasma (upper panel) and urine (lower panel). Acetylsalicylic acid, 650 mg x 4
? DAYS Fig.
780
13.
Urinary excretion of 5a,7u-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid before, during, and after administration of acetylsalicylic acid.
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PROSTAGLANDINS
In the third volunteer indomethacin was administered during four days. A decrease of the excretion of the urinary metabolite from 4 pg/24 hr to 1.2 pg/24 hr was obtained. During the forth day 80 DCi 117,18-3HI-PGF2, was injected (see Preparation of Radio-Labeled Ligand). With the knowledge of the endogenous contribution immediately prior to the injection, it was possibly to estimate the specific activity of the obtained 113,14- HI-5a,7a-dihydroxy-ll-ketotetranorprosta-1,16-dioic acid (calculated as 18 Ci/ /mmole).
REFERENCES 1. Samuelsson,B., E. Granstrom, Hammarstrom. Prostaglandins, 1975.
K.GrBen, M.Hamberg, and S. Ann. Rev. Biochem. 44:669,
2. Samuelsson,B. Quantitative aspects on prostaglandin thesis in man, Adv. Biosci. 9:7, 1973.
syn-
3. GranstrSm,E. and B.Samuelsson. Development and mass spectrometric evaluation of a radioimmunoassay for 9a, lla-dihydroxy-15-ketoprost-5-enoic acid, FEBS Lett. 26:211, 1972. 4. Cornette,J.C., K.L.Harrison, and K.T.Kirton. Measurement of prostaglandin F2c metabolites by radioimmunoassay, Prostaglandins 5:155, 1974. 5. Levine,L. and R.M.Gutierrez-Cernosek. Levels of 13,14dihydro-15-keto-PGF2U in biological fluids as measured by radioimmunoassay, Prostaglandins 3:785, 1973. 6. Stylos,W.A., S.Burstein, J.Rosenfeld, E.M.Ritzi, and D. J.Watson. A radioimmunoassay for the initial metabolites of the F prostaglandins, Prostaglandins 4:553, 1973. 7. GranstrBm,E. and H.Kindahl. Radioimmunoassays for prostaglandin metabolites, In: Adv. in Prostaglandin and Thromboxane Research, Vol.1 (Eds. B.Samuelsson and R.Paoletti) Raven Press, New York, p. 81, 1976. 8. Barcikowski,B., J.C.Carlson, L.Wilson, and J.A.McCracken. The effect of endogenous and exogenous estradiol-178 on the release of prostaglandin F2c from the ovine uterus, Endocrinology 95:1340, 1974. 9. Kindahl,H., L.-E.Edqvist, A.Bane, and E.Granstrom. Blood levels of progesterone and 15-keto-13,14-dihydroprostaglandin F2a during the normal oestrous cycle and early pregnancy in heifers, Acta endocr. (Kbh.) 82:134, 1976. 10. Ohki,S., T.Hanyu, K.Imaki, N.Nakazawa, and F.Hirata. Radioimmunoassays of prostaglandin F2c and prosUx+";l:,"_ F2,-main urinary metabolite with prostaglandinsine methylester amide, Prostaglandins 6:137, 1974.
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11.
Cornette,J.C., K.T.Kirton, W.P.Schneider, F.F.Sun, R.A. Johnson, and E.G.Nidy. Preparation and quantitation of urinary metabolites of prostaglandin F2c by radioimmunoassay, Prostaglandins 9:323, 1975.
12. Ohki,S., K.Imaki, F.Hirata, T.Hanyu, and N.Nakazawa. Radioimmunoassay of main urinary metabolite of prostaglandin F~cx, Prostaglandins 10:549, 1975. 13. Granstrdm,E. and B.Samuelsson. On the metabolism of prostaglandin F2c in female subjects, J. Biol. Chem. 246:5254, 1971. as coupling reagent 14. Axen,U. N,N'-Carbonyldiimidazole the preparation of bovine serum albumin conjugates, Prostaglandins 5:45, 1974.
for
15. Grgen,K., E.Granstrdm, B.Samuelsson, and U.Axen. Methods for quantitative analysis of PGF2 , PGE2, 9c,lla-dihydroxy-15-keto-prost-5-enoic aci,% and 9a,lla,l5-trihydroxy-prost-5-enoic acid from body fluids using deuterated carriers and gas chromatography-mass spectrometry, Anal. Biochem. 54~434, 1973. 16. Hamberg,H. Metabolism of prostaglandins in rat liver mitochondria, Eur. J. Biochem. 6:135, 1968. 17. Fritz,I.B. The metabolic consequences of the effects of carnitine on long-chain fatty acid oxidation, In: Cellular Compartmentalization and Control of Fatty Acid Metabolism. (Ed. F.C.Gran), UniversitetsfBrlaget, Oslo, p. 39, 1968. 18. Granstrom,E. and B.Samuelsson. On the metabolism of prostaglandin F2c in female subjects. II. Structures six metabolites, J. Biol. Chem. 246:7470, 1971.
of
19. Van Orden,D.E. and D.B.Farley. Prostaglandin F2c radioimmunoassay utilizing polyethylene glycol separation technique, Prostaglandins 4:215, 1973. 20. Blatchley,F.R., B.T.Donovan, E.W.Horton, and N.L.Poyser. The release of prostaglandins and progestin into the utero-ovarian venous blood of guinea-pigs during the oestrous cycle and following oestrogen treatment, J. Physiol. 223:69, 1972. 21. Roberts,J.S., B.Barcikowski, L.Wilson, R.C.Skarnes, and J.A.McCracken. Hormonal and related factors affecting the release of prostaglandin F2c from the uterus, J. Ster. Biochem. 6:1091, 1975. 22. Earthy,M., C.Bishop, and J.D.Flack. Progesterone and prostaglandin F concentration in utero-ovarian venous plasma of cyclic guinea-pigs, J. Endocrinol. 64:llp, 1975. 23. Maule Walker,F.M. and N.L.Poyser. Production of prostaglandins by the early pregnant guinea-pig uterus in vitro, J. Endocrinol. 61:265, 1974. 782
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24.
Blatchley,F.R., F.M.Maule Walker, and N.L.Poyser. Progesterone, prostaglandin F2c, and oestradiol in the utero-ovarian venous plasma of non-pregnant and early, unilaterally pregnant guinea-pigs, J. Endocrinol. 67: 225, 1975.
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