Radioimmunoassay of Saliva Estrone Sulfate in Pregnant Sows

Tadatoshi OHTAKI, Masaharu MORIYOSHI, Ken NAKADA, Toshihiko NAKAO, and Keiichiro KAWATA Department of Veterinary Obstetrics and Gynecology, Faculty of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069, Japan

(Received 28 November 1996/Accepted 12 May 1997)

ABSTRACT. The aim of this study was to establish radioimmunoassay (RIA) for saliva estrone sulfate (E1S), and to elucidate changes in saliva E1S during pregnancy in the sow. Saliva E1S was extracted using a commercially available solid phase column, and the E1S fraction obtained was subjected to RIA. The sensitivity of the RIA was 29.7 pg/tube. The intra- and inter-assay coefficients of variation were 5.5–

8.4% and 13.1–19.5%, respectively. Mean recovery for E1S added to saliva samples was as high as 99.9%. A significant positive correlation (r=0.54, n=69, p<0.01) existed between saliva and plasma E1S concentrations. During gestation, the changing patterns of saliva and plasma E1S concentrations were essentially the same, and two peaks of E1S concentrations were observed, one around day 30 and another just before parturition, although E1S concentrations in saliva remained at only 2.4–38.1% (mean 11.4%) of those in plasma E1S. Thus, the present study has made it possible to measure saliva concentrations of E1S and demonstrated a high degree of positive correlation between saliva and plasma E1S concentrations. These results suggest that diagnosis of early pregnancy and of normal or abnormal fetal development could be made by measurements of E1S in saliva. — KEY WORDS: estrone sulfate, pregnancy, radioimmunoassay, saliva, sow. J. Vet. Med. Sci. 59(9): 759–763, 1997

In pregnant sows, estrone sulfate (E1S) in peripheral blood saliva from the cotton. Saliva samples obtained were mixed is first detectable around day 16 of pregnancy and fluctuates with sodium azide (5 mg/ml) and kept frozen at －20°C showing 2 peaks, one on days 23–30 and another just before until assayed for E1S. Immediately after saliva collection, parturition [14]. Since Robertson and King [14] suggested blood samples were also collected from the median tail vein that E1S concentration in the maternal blood reflects the into heparinized test tubes. Plasma was separated by amount of estrogens synthesized and secreted by the immediate centrifugation (1,700 × g, 15 min) and kept blastocyst or fetus [14], early pregnancy diagnosis [2, 19, frozen at －20°C until assayed for E1S. 20] and the diagnosis of normal or abnormal fetal Extraction of E1S: Saliva and plasma E1S was extracted development [3] could be made in terms of maternal blood by a slight modification of the method described by

E1S concentrations. Nakamura [10]. Briefly, saliva and plasma samples (1–4 Because saliva samples can be more easily and safely ml) were diluted with 2 ml borate buffer (0.2 M, pH 8.0) collected compared to blood samples, various steroid containing 0.05% bovine serum albumin and 0.06% bovine hormones in saliva have been measured in humans, cows, γ-globulin (BSA buffer), and then poured into a 5 ml horses and pigs [4, 5, 8, 9, 11–13, 15]. In pigs, saliva disposable syringe attached to solid phase columns (Sep- progesterone and cortisol have been determined for early Pak®PLUS C18 Cartridge, Millpore Co., Ltd., U.S.A.). The pregnancy diagnosis [9] and for an adrenocortical function sample solutions were allowed to flow into the columns at a test [11], respectively. An attempt has been made to rate of 0.2–1 ml per min. The columns were then washed determine saliva E1S in horse [15] but not in the pig. with 4 ml distilled water and with 3 ml diethylether, Therefore, the present study was undertaken to measure successively and the effluents were discarded. Finally, five saliva concentrations of E1S by radioimmunoassay (RIA) ml acetone was poured into the columns, and the resultant and to correlate the changing pattern of saliva E1S with that effluent (E1S fraction) was recovered into test tubes. The of plasma E1S in pregnant sows. effluents were evaporated to dryness at 50°C under nitrogen gas, and the residues were dissolved in 200 µl of BSA MATERIALS AND METHODS buffer. RIA for E1S: Saliva and plasma E1S were measured by a Saliva and blood collections: Five hybrid parous sows modification of RIA described by Nakamura [10], as shown raised in this laboratory were used for the present study. in Fig. 1, using 1, 3, 5 [10]-Estratrien-3-ol-17-one sulfate Saliva samples were collected from each sow at intervals of (E1S) as a standard, estrone sulfate ammonium salt [6.7– 3 3 2–10 days during pregnancy, as described previously [9]. H(N)] ( H-E1S; specific activity, 1.48–2.22 TBq/mM) as a Briefly, a wooden chopstick (approximately 20 cm long) tracer and anti-estrone-3-sulfate rabbit serum (anti-E1S tipped with 2 g of absorbent cotton was inserted into the serum). The standard E1S preparation was provided by Dr. mouth. Subsequently, the cotton soaked with sufficient A. Kanbegawa (Teikoku Hormone Manufacturing Co.. Ltd., 3 amounts of saliva was removed from the stick and Japan) and H-E1S was prepared by Daiichi Pure Chemicals compressed in a 10–20 ml disposable syringe to squeeze the Co.. Ltd., Japan. The anti-E1S serum was generated by 760 T. OHTAKI, ET AL.

Fig. 2. The standard curve for radioimmunoassay of

estrone sulfate (E1S) .

solutions were mixed 250 µl of dextran-coated charcoal solution [0.2 M borate buffer solution containing 0.5% charcoal (Norit A; Kishida Chemical Industries, Ltd., Japan) and 0.05% dextran (DextranT70; Pharmacia Fine Chemicals AB, Uppsala, Sweden), pH 8.0], left at 4°C for 10 min and subsequently centrifuged at 4°C for 15 min (1,700 × g). Two-hundred µl of the supernatant was emulsified with 3 ml scintillator (Scintisol® EX-H, Wako Pure Chemical Industries, Ltd., Japan), and radioactivity was assayed by a Fig. 1. Flowchart of the procedure in RIA method. scintillation counter (Liquid scintillation system LSC-703, Aloka Co., Ltd., Japan). The quantities of E1S in saliva and Cosmo Bio Co., Ltd. Japan using anti 6-oxo-estrone-3- plasma samples were estimated by interpolation from a sulfate-6-CMO-BSA as the antigen. According to the standard curve. information from the manufacturer, the cross-reactivities of RESULTS the anti-E1S serum with E1S, estrone glucuronide, estrone, estradiol-3-sulfate, estradiol-3-glucuronide and estriol sulfate were 100, 5.4, 4.0, 1.5, 0.8 and 0.8%, respectively. Parameter for reliability of E1S RIA: The standard curve 3 obtained by the assay of standard E1S in quantities ranging Prior to use, standard E1S and H-E1S were diluted with BSA buffer at concentration of 3.9–1,000 pg/200 µl and from 3.9 to 1,000 pg is shown in Fig. 2. The sensitivity was estimated as 29.7 pg/tube. Recovery of E1S in swine 250,000 cpm/ml, respectively. Anti-E1S serum was diluted 1:36,000 with BSA buffer for use. saliva samples was determined by addition of 500, 1,000 or 3,000 pg E1S to 2 ml saliva samples, as shown in Table 1. Standard E1S (3.9–1,000 pg) and saliva or plasma extracts dissolved in 200 µl BSA buffer were each mixed with 100 Mean recovery ranged from 92.3 to 108.4%, and for all the 9 measurements the value was 99.9%. µl anti-E1S serum solution and allowed to react at 4°C for 12–24 hr. The mixtures were then incubated with 100 µl The intra- and inter-assay coefficients of variation were 3 each calculated from quintuplicate results on 2 pooled saliva H-E1S solution at 4°C for 12–24 hr. The incubated

Table 1. Recovery of varying amounts of estrone sulfate (E1S) added to swine saliva samples

Added E S 1 No. of assays Assay value (pg/ml) Recovery rate (%) (pg/ml)

03 11 ± 0.6* – 250 3 241 ± 21.6 92.3 500 3 506 ± 57.4 99.0 1,500 3 1,638 ± 162.4 108.4 Total mean 99.9

* Mean ± S.E. RIA OF SALIVA E1S IN PREGNANT SOWS 761

from a plot of the two parameters is given in Fig. 3. A significant positive correlation existed between saliva and

plasma E1S concentrations. The correlation coefficient was 0.547 (p<0.01).

Changes in saliva and plasma E1S concentrations during pregnancy: As shown in Fig. 4, mean E1S concentrations in saliva and plasma fluctuated exhibiting two peaks. The

first peak of E1S concentrations was evident on days 26–32 in saliva and on days 24–30 in plasma. The steroid in both saliva and plasma remained at baseline levels during mid-

pregnancy. Saliva and plasma E1S, however, began to rise gradually around days 70–80 and reached a peak concentration (the second peak) on days 110–114 (just before parturition). Thus, the changing patterns of saliva

E1S concentrations resembled closely the plasma pattern, although E1S concentrations in saliva remained at only 2.4– 38.1% (mean 11.4%) of those in plasma.

Fig. 3. Correlation between saliva and plasma estrone sulfate DISCUSSION (E1S) concentrations in sows . In the present study, first the reliability of the RIA method samples with different mean E1S concentrations. The intra- as a procedure for measuring saliva E1S in sows has been assay coefficient of variation was 5.5% for the sample with demonstrated. Variations in individual recovery rates for the lower E1S concentrations (302 pg/ml) and for the other E1S added to saliva samples are in small range, and mean sample with the higher E1S concentrations (2,233 pg/ml) it recovery for steroid is as high as 99.9%. The intra- and was 8.4%, while the inter-assay coefficient of variation for inter-assay coefficients of variation obtained for saliva E1S the sample with the lower E1S concentrations (324 pg/ml) RIA in the present study are rather small and closely was 19.5% and 13.1% for the sample with the higher E1S comparable with the values reported for plasma E1S assay concentrations (2,598 pg/ml). in cows [10] and humans [1]. The sensitivity of the assay is

Correlation between saliva and plasma E1S: Correlation 29.7 pg/tube and high enough to measure E1S concentrations between saliva and plasma E1S concentrations were not only in the plasma but also in the saliva. examined in 69 pregnant sows. The regression line obtained Two peaks of plasma E1S concentrations are evident in

Fig. 4. Changes in saliva and plasma estrone sulfate (E1S) concentrations during pregnancy in sows. 762 T. OHTAKI, ET AL. the present study, the first peak around day 30 and the REFERENCES second peak just before parturition. This agrees quite well with the finding of Robertson and King [14], although they 1. Ciotti, P. M., Franceschetti, F., Bulletti, C., Jasonni, V. M., and Bolelli,G. F. 1989. Rapid and specific RIA of serum carried out serial estimations of plasma E1S in only a single gilt at implantation, throughout pregnancy, and at estrone sulfate with selective solid phase extration. J. Steroid parturition. Biochem. 32: 473–474. 2. Cunningham, N. F., Hattersley. J. J. P., and Wrathall, A. E. The present study has first described changes in saliva 1983. Pregnancy diagnosis in sows based on serum oestrone E1S concentrations during pregnancy in the sow. Moriyoshi sulphate concentration. Vet. Rec. 113: 229–233. et al. [9] have reported that saliva and plasma progesterone 3. 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