Radioimmunoassay of Saliva Estrone Sulfate in Pregnant Sows.

Abstract

ABSTRACT. The aim of this study was to establish radioimmunoassay (RIA) for saliva estrone sulfate (E 1 S), and to elucidate changes in saliva E 1 S during pregnancy in the sow. Saliva E 1 S was extracted using a commercially available solid phase column, and the E 1 S 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 E 1 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 E 1 S concentrations. During gestation, the changing patterns of saliva and plasma E 1 S concentrations were essentially the same, and two peaks of E 1 S concentrations were observed, one around day 30 and another just before parturition, although E 1 S concentrations in saliva remained at only 2.4-38.1% (mean 11.4%) of those in plasma E 1 S. 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 E 1 S concentrations. These results suggest that diagnosis of early pregnancy and of normal or abnormal fetal development could be made by measurements of E 1 S in saliva. -KEY WORDS: estrone sulfate, pregnancy, radioimmunoassay, saliva, sow.J. Vet. Med. Sci. 59(9): 759-763, 1997 saliva from the cotton. Saliva samples obtained were mixed with sodium azide (5 mg/ml) and kept frozen at 20°C until assayed for E 1 S. Immediately after saliva collection, blood samples were also collected from the median tail vein into heparinized test tubes. Plasma was separated by immediate centrifugation (1,700 × g, 15 min) and kept frozen at 20°C until assayed for E 1 S.Extraction of E 1 S: Saliva and plasma E 1 S was extracted by a slight modification of the method described by Nakamura [10]. Briefly, saliva and plasma samples (1-4 ml) were diluted with 2 ml borate buffer (0.2 M, pH 8.0) containing 0.05% bovine serum albumin and 0.06% bovine γ-globulin (BSA buffer), and then poured into a 5 ml disposable syringe attached to solid phase columns (SepPak ® PLUS C18 Cartridge, Millpore Co., Ltd., U.S.A.). The sample solutions were allowed to flow into the columns at a rate of 0.2-1 ml per min. The columns were then washed with 4 ml distilled water and with 3 ml diethylether, successively and the effluents were discarded. Finally, five ml acetone was poured into the columns, and the resultant effluent (E 1 S fraction) was recovered into test tubes. The effluents were evaporated to dryness at 50°C under nitrogen gas, and the residues were dissolved in 200 µl of BSA buffer.RIA for E 1 S: Saliva and plasma E 1 S were measured by a modification of RIA described by Nakamura [10], as shown in Fig.

Fecal Estrone Sulfate Profile in Sows during Gestation.

“…In a previous study [15], the authors measured the E1S concentration in the saliva of sows, which is easier to collect than blood. It was noted that the E1S concentration in saliva was much lower than in plasma [15].…”

“…It was noted that the E1S concentration in saliva was much lower than in plasma [15]. A few reports indicated that steroid concentrations in feces were higher than those in saliva [1,2,9,11,25].…”

“…Collection of fecal and blood samples: Five hybrid sows, 5 castrated boars and a total of 147 pregnant sows raised in (standard or test fecal solutions) and plasma E1S by a slight modification of the method described in the previous report [15]. Briefly, fecal solutions and plasma samples (0.5-2 ml) were diluted with 2 ml of 0.02 M phosphate buffer, and then poured into a 10 ml disposable syringe attached to solid phase column (Sep-Pak ® PLUS C18 Cartridge, Waters Co., Milford, Massachusetts, U.S.A.).…”

“…RIA for E1S: E1S in fecal solution and plasma was quantified by a modification of the RIA described in the previous report [15], with estrone-3-sodium sulfate as a standard, estrone sulfate ammonium salt [6.7-3 …”

“…In a previous study [15], the authors measured the E1S concentration in the saliva of sows, which is easier to collect than blood. In recent years, the measurement of the steroid hormone concentration in feces has been reported to be a useful diagnostic tool for wild animals [8,10,12,19,21,26] and pigs [1,2,7,13,20,25], blood collection from which is especially difficult.…”