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Radioimmunoassay of Saliva Estrone Sulfate in Pregnant Sows by maclaren1


									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 (E 1S), and to elucidate changes in
saliva E1S during pregnancy in the sow. Saliva E1 S 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 E1 S 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 E1 S concentrations. During gestation, the changing patterns of
saliva and plasma E1 S 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 E 1 S 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 E1 S in saliva. — KEY WORDS: estrone sulfate, pregnancy, radioimmunoassay,
saliva, sow.
                                                                                                  J. Vet. Med. Sci. 59(9): 759–763, 1997

   In pregnant sows, estrone sulfate (E 1S) 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 E1 S.
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 E1 S: 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            (E1 S) as a standard, estrone sulfate ammonium salt [6.7–
                                                                       3H(N)] (3H-E S; specific activity, 1.48–2.22 TBq/mM) as a
2–10 days during pregnancy, as described previously [9].                             1
Briefly, a wooden chopstick (approximately 20 cm long)                 tracer and anti-estrone-3-sulfate rabbit serum (anti-E 1S
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.,
amounts of saliva was removed from the stick and                       Japan) and 3H-E1S was prepared by Daiichi Pure Chemicals
compressed in a 10–20 ml disposable syringe to squeeze the             Co.. Ltd., Japan. The anti-E1 S serum was generated by
760                                                        T. OHTAKI, ET AL.

                                                                           Fig. 2. The standard curve for radioimmunoassay of
                                                                              estrone sulfate (E1 S) .

                                                                      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
the anti-E1S serum with E1S, estrone glucuronide, estrone,            RESULTS
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
Prior to use, standard E1S and 3H-E1 S were diluted with              obtained by the assay of standard E 1S in quantities ranging
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
250,000 cpm/ml, respectively. Anti-E1S serum was diluted              was estimated as 29.7 pg/tube. Recovery of E1S in swine
1:36,000 with BSA buffer for use.                                     saliva samples was determined by addition of 500, 1,000 or
   Standard E 1S (3.9–1,000 pg) and saliva or plasma extracts         3,000 pg E1 S to 2 ml saliva samples, as shown in Table 1.
dissolved in 200 µl BSA buffer were each mixed with 100               Mean recovery ranged from 92.3 to 108.4%, and for all the
µl anti-E 1S serum solution and allowed to react at 4°C for           9 measurements the value was 99.9%.
12–24 hr. The mixtures were then incubated with 100 µl                   The intra- and inter-assay coefficients of variation were
3H-E S solution at 4°C for 12–24 hr.         The incubated            each calculated from quintuplicate results on 2 pooled saliva

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

                                Added E1 S
                                               No. of assays   Assay value (pg/ml)   Recovery rate (%)

                                     0              3                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 E1 S 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 E 1S concentrations during
                                                                      pregnancy: As shown in Fig. 4, mean E1S concentrations in
                                                                      saliva and plasma fluctuated exhibiting two peaks. The
                                                                      first peak of E 1S 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
                                                                      E 1S concentrations resembled closely the plasma pattern,
                                                                      although E 1S 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 E1 S 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            E 1S 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 E1 S               comparable with the values reported for plasma E 1S assay
concentrations (2,598 pg/ml).                                         in cows [10] and humans [1]. The sensitivity of the assay is
   Correlation between saliva and plasma E 1S: Correlation            29.7 pg/tube and high enough to measure E1S concentrations
between saliva and plasma E1 S 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 (E 1S) concentrations during pregnancy in sows.
762                                                    T. OHTAKI, ET AL.

the present study, the first peak around day 30 and the           REFERENCES
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