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~IO~OAS~Y

OF ES~IO~l7~IN

BOVINE ALL

PLASMAWITSANDWITHOUTCEfROMATOGRAPHY A.J, PETERSON, R.J. FAIRCLOUGH and J.F. SMITH Ministry of Agriculture and Fisheries, Research Division, Ruakura Agricultural Research Centre, Hamilton, New Zealand

Received:

2.1!z i ;lc&

ABSTRACT

of estrad~ol~l7~ (E217fe > in bovine peripheral plasma is . The plasma 1s incubated with an antiserum to E217'-BSA and thex-globulin fraction precipitated with ammonium sulphate. After extraction with diethyl ether E2l7p in the precipitate is estimated by radioimmunoassay using a specific antiserum against E217,#-6-BSA. Plasma concentrations of E217fiduring the normal estrous cycle determined by this method and by a method involving Sephadex LR-20 chromatography range from 4 to 23 pg/ml. descr~e=w

At the start of our investigations on the hormonal effects of various methods of estrous synchronization in dairy and beef cattle, reports on the concentrations of estrogens in bovine peripheral plasma were few end the results inoonsistent (l-4). Consequently it was necessary to develop a reliable assay for plasma steroid estrogen that, because of the large numbess of samples generated in the study, would process rapidly many samples at one time, The present paper describes the development of such an assay capable of measuring the low levels of E2l7p in the peripheral plasma of cows during the estrous cycle. The procedure does not require c~o~to~ap~

and is based on a suggestion of Ismail -et al. (5).

The results obtained by this method are compared with those obtained using radioimmunoassay (RLA) in conjunction with Sephadex X-I-20micro-

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column chromatography. MATERIALS AND METHODS Preparation of antisera: Details of the preparation of the antisera against E217~-6-BSA have been described previously (6). In brief 6-ketoestradiol-17#-6-carboxymethoxime was prepared by the method of Dean et al.(T) and coupled to bovine serum albumin (BSA) by the mixedanhyd%~method of Erlanger et al. (8). It was calculated that 27 E2l7p residues were coupled pz=le of BSA. Antibodies were raised in 2 sheep by injecting the antigen emulsified in Freundts complete adjuvant into 5 subcutaneous sites at 14-day intervals for the first 6 weeks and then at monthly intervals. The antiserum selected for the RIA was obtained from one sheep after 6 months and had a titre of 1:8000. The plasma from this sheep was precipitated with 4C$ ammonium sulphate and the precipitate resuspended with glass-distilled water to give a final dilution of 1:2 of the original plasma and stored in 1 ml aliquots at -2oOc. The antiserum used in the RIA with chromatography was raised against E217p-IT-succinylBSA and was donated by Dr R. Scaremuzzi. Solvents: All solvents were analytical grade and with the exception of the Methyl ether (Fluka, Switzerland) were glass-distilled before use. The diethyl ether was obtained in 11 bottles which were stored in the dark. No bottle lasted more than 5 days and there was no detectable blank during this time. The phospho-buffered saline (BBS) solution and the dextran-coated charcoal suspension were identical to those used previously (9). Radioactive compounds: Estradiol-17~-6,7-5H (40-60 Ci/mmol) and estrone-6,7-5H (40-60 Ci/mmol) were obtained from the Radiochemical Centre, A&&ham, U.K., ' and were ' purified at regular intervals on columns of Sephadex LH-20. Standard steroids: All steroids were obtained from Sigma Chemical Company (St. Louis, U.S.A.) end were prepared as 1 mg/ml stock solutions in ethanol. The working solutions were prepared from these stock solutions at a final dilution of 1 ng/ml. All standard solutions were stored at 4OC. Assav tubes: Disposable polypropylene test tubes (100 mm x 15 mm) with disposable polypropylene stoppers (Laboratory Services Ltd., Auckland, N.Z.) were used as received. Radioactive measurement: The scintillation mixture was prepared by dissolving 8 g PPO in 2 1 toluene and 1 1 Triton X-100 (Rohm Hass). Radioactivities were measured at a preset error of 2$ in a Nuclear Chicago Liquid Scintillation Counter (Isocap 500). The counting efficiency for tritium was 6%. Sample collection: Twelve multiparous dairy cows, with a normal cycle length of 19-23 days, were used. Blood (20 ml) was collected twice aaiiy by means-of indwelling jugular can&as or venepuncture, spun immediately and the plasma frozen at -2O'C pending analysis.

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Estrus was determined using a vasectomized bull fitted with a chinball harness. Blood (3 1) was withdrawn from 2 steers, the plasma separated and stored at -2OOC until use. RIA usip microcolumns of Senhadex LH-20: Details of this method have been reported previously (9, 10). Plasma (2 ml) was extracted in duplicate with 7 ml diethyl ether. Two controls, in duplicate, were run with each assay, one consisting of bulk steer plasma, the other of the same bulk plasma to which E217p had been added to a final concentration of 50 pg/ml. Microcolumns of 0.5 g Sephadex LH-20 slurried in 2 ml glass insulin syringes were made afresh each dsy and an aliquot of the final column wash was added to each of the standard tubes. Preliminary results, using the method to assay steroid estrogens in bovine peripheral plasma have been reported elsewhere (11). RIA without chromatography: It was hoped that the development of the specific E2l7# antisera would enable E2l7p to be measured on unchromatographed extracts of cow plasma. Unfortunately it was found that there were interfering substance(s) in both steer and cow plasma which flattened the standard curve. Chromatography on the microcolumns of LH-20 overcame this interference (Figure 1). Preliminary experiments fractionating ethereal extracts of steer plasma on 30 cm LH-20 columns have failed to identify the substance but indicate that it is less polar than estrone and is not pigment. To one ml of the duplicate plasma samples was added 0.5 ml of a I:500 dilution of the anti-serum to E2l7p -17-hemisuccinate. This resulted in a final antiserum dilution of I:1500 which bound 9% of E2l7,#in the system. The tubes were shaken on a mechanical sQsker for 10 min then incubated at room temperature for 20 min and at 4 C -for 30 min. During vortex mixing, 1 ml of saturated smmonium sulphate was added to precipitate they-globulin fraction. The resultant concentration of salt was 40$, which precipitated 9% of the antibody-bound steroid (Figure 2). It was important to ensure that the ammonium sulphate was added while the plasma was vortexed so as to avoid locally high concentrations of salt (12) which impair reproducibility. The tubes were spun at 15 000 x g for 20 min, the supernatant aspirated and 1 ml of 0.28 N HCl added to each tube and the tubes vortexed until the precipitate dissolved. Six ml of diethyl ether were added, the tubes capped and then shaken for 10 min. After centrifugation the tubes were placed in a tray containing a mixture of dry ice and acetone and when the aqueous phase had solidified the organic phase was decanted into fresh tubes and evaporated to dryness under nitrogen. A 0.1 ml aliquot of the diluted antiserum to E217#-6-BSA was added to each tube and the tubes shaken for 2 min. E217p-6,7-3H (0.1 uCi in PBS) was then added and the tubes shaken for a further 2 min. All tubes were then covered and incubated at room temperature for 1 hr and then at 4'C for 1.5 hr. One ml of freshly prepared dextran-coated charcoal solution was added and the mixture shaken for 2 min and allowed to stand for 5 min before centrifugation. The tubes were spun at 15 000 ~3 for 10 min and the resultant supernatant decanted carefully into counting vials containing 5 ml of the scintillation mixture. The appropriate series of standards were prepared in duplicate in a similar way with each batch of assays.

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Figure 1: Standard curves obtained during this study r,n=8). t1) Standards added to nonchromatographed extracts of 1 ml steer plasma, 2 standards in ethanol, u7 standards added to chromatographed extracts of 1 ml steer plasma, (4) standards added to 1 ml steer plasma processed by new method.

IO20 30 w so u0u2)I?#picogmms

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30

35

% ammonium

40 sulphate

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Figure 2: Precipitationzof antibody-bound E217p-'H with increasing concentration of ammonium sulphate.

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RESULTS

A.

RIA with column chromatography: The antisera used was raised

against E217k-17-BSA. used at a dilution of I:40 000 aa

cross-reacted

56s ana 42$ with E, (estrone) and E217a (estradiol-17s)respectively. Although E, was separated adequately from the estradiols by the microcolumns, the two estradiol epimers were not.

Recent work (13) has

shown that the contribution of the 17s epimer to total estradiol bind_ing of cow plasma is negligible and the results here reported for estradiol most likely reflect the '7p epimer. Amounts of E217$ of 10, 20 and 50 pg were added

in duplicate to separate 1 ml portions of bulk

steer plasma which were then assayed together with the corresponding duplicate bulk steer plasma samples. The mean recovery, 2 SD, was 74+7$,n=40.

The within-assay CV varied from 10 to 1674over the

range of 2 to 50 pg.

The between-assay CV, as measured on both the

bulk steer plasma and the 50 pg control was 2C$.

The least amount

distinguishable from zero amount (P = 0.1 and calculated for duplicate determination) was 3 pg.

The least amount that could be measured in

2 mls of plasma was 5 pg. B.

RIA without chromatogra&y:

There is a constant difference be-

tween the standard curves constructed from E217p alone and those constructed from adding known amounts of E217P to 1 ml samples of steer plasma. This difference represents the basal levels of E217P present in steer plasma and indicates that the method removes effectively the interference present in bovine plasma. 1.

Specificity of ammonium sulphate precipitation: Steer plasma

aliquots (1 ml) to which approximately 100 pg of various tritiated steroids had been added were incubated with the E217$-17-BSA antiserum

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and ther-globulin fraction precipitated and extracted. The percentage of radioactive steroids recovered are shown in table 1.

There was a

substantial percentage of El recovered as would be expected from the cross-reactivity of the antiserum but other steroids tested were recovered in much smaller amounts. 2.

Recovery of E&'&:

Approximately 5 000 cpm of 3H-E2 17f?were added

to duplicate 1 ml samples of steer plasma to which had been added either 0, 10 or 20 pg of unlabelled E217p.

The mean recovery (2 SD)

was 70 + 4$, n = 20. TABLE 1.

Percentage of selected radioactive steroids recovered following ammonium sulphate precipitation and extraction from steer plasma Percentage recovered (n = 4)

Steroid q7/3 El

4-androstene-3,I7-Cone testosterone cortisol ~,17--dihydroxy-5-pregnen-2O-one estrone-j-sulphate

3.

92 54 21 21 11 11 2

Specificity of the E-178-6-BSA antiserum: The cross-reactivity of L

various steroids against the two antisera used was determined by Abrshem*s method (14) and presented in table 2.

The antisera against

E217$-6-BSA is very specific for E217fi.

TABLR 2.

Percentage cross-reactivity of selected steroids against the two antisera used in this paper

Steroid E217P E217@‘

~~17~-6-BSA 100.00 0.82

?&trio1 p-hydrozqr-5-pregnen-20-one testosterone 4-androstene-5,17-dione progesterone

‘Co.1 3.9 ~0.1 d-O.1 < 0.1 c 0.1

E217&l 7-BSA 100.0 42.0 56.0 5.0 4. 0.1 c 0.1 i 0.1 %- 0.1

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Accuracy: Amounts of E217p of 5, 10 and 20 pg were added in duplicate to separate 1 ml portions of the same bulk steer plasma which were then asssyed together with the corresponding duplicate steer plasma samples. The recoveries (2 SD, n = 15) were 80 2 I%,

75 2 7% and 77 2

6% respectively. Precision: Included with each assay were duplicate 1 ml samples of bulk steer plasma with and without 50 pg of added E217@. The betweenassay CV of these controls was 15% (n = IO). The within-asssy CV on 50 duplicate analyses for E217/ chosen at random from 6 consecutive assays and covering the concentration range of 5-15 pg/ml was #. Sensitivity: The sensitivity of the standard curve, derived from the variability of the response at the zero dose level was 2.6 pg. The sensitivity of the method as defined by Borth (15) end calculated from the variability of the reagent blanks was 4.0 pg. c.

Peripheral plasma levels of E21J& during the estrous cycle: The

changes in E217f3concentrations during the normal estrous cycle of 7 COWS

determined by the new method are shown in figure (3). For compar-

ison the changes in estradiol levels during the estrous cycle of 5 other cows measured by the chromatographic method are also included. Values for both methods have been corrected for procedural losses. The observed concentrations of E217@, determined by the chromatographic method range from 4 to 18 pg/ml, in agreement with other reports using similar methods (1, 2, 13). The levels determined by the new method are predominantly within this range and the higher amounts measured around estrus are most likely a consequence of more frequent sampling.

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Figure 3: Changes in peripheral plasma estradiol concentration throughout the normal bovipe estrous cycle determined by zh$ chromatographic (@---0, x - S.E., n = 7) and new (O---O, x - S.E., n = 5) methods. DISCUSSION Chromatography is the major factor which limits the number of samples that can be assayed at one time by saturation analysis. The advent of antisera highly specific to certain steroids has enabled, in some asssys, the chromatographic step to be omitted without a significant loss of specificity. One must be judicious, however, when omitting

chromatography for there may be present in the asssy

system other compounds, not necessarily steroids, which msy interfere with the binding of the antibody.

Although there have been previous reports on the RIA of steroid estrogens in bovine peripheral plasma without chromatography, the present paper is the first to report the presence of factors in bovine plasma which interfere with this assw

system. These factors may be

related to those which cause a similar inhibition in the competitive protein binding (CPB) assa;yfor bovine plasma estrogen (16) and their presence may account for the discrepancies in reported concentrations af plasma steroid estrogens in cows (l-4,

13).

The nature

of the

interference remains to be elucidated and its presence renders it es@ential that before the measuxement of bovine plasma estrogens by either CPB or RIA there must be some degree of purification of the plasma extract. The assay

developed

in the present paper circumvents the time-

consuming chromatographic step and removes the interfesence from the sample. It is applicable to the measurement of E217@ in plasma where the concentration of E, is negligible, as is found in peripheral plasma of cows during the estrous cycle. The use of a more specific antiserum to E217p in the initial incubation would make the assay more widely applicable to those plasmas with significant amounts of E,. There is good agreement between the values obtained by this technique and those obtained by RIA after Sephadex IX-20 chxomatography. The assey enables one technician to process at least twice as many samples in duplicate per week as the chromatographic method and is eminently suitable for use in our study on the hormonal effects of estrous s~c~oniaation

in cattle. ACKNOWLEDGMENl'S

The authors thank DC E.Payne for constructive criticism,

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J.G.Ackerley and G.M.Haeard for mnagsment and sampling of cattle and Christine Lane for skilled technical assistance. A.J.P. is a recipient of a New Zealand National Research Advisory Council PostDoctoral Fellowship. RE3XRENCES 1.

2.

5. 6. 7. 8.

9. 10. 11.

12. 17. 14. 15. 16.

Echternkamp, S.E. and Hansel, W.: J. DAIRY SCI., 54, 800 (1971). Wettemann, R.P., Hafs, H.D., Edgerton, L.A. and Swanson, L.V., J. ANIM. SCI. 34, 1020 (1972). Shemesh, M., Ayalon, N. and Lindner, H.R., J. OCR. 55, 73 (1972) Mason, B.D., &ddmamurti, C.R. and Kitts, W.D., J. ENDOCR. 55, 141 (1972). A.R. J. CLIN. ENDOCR. Imail, A.A.A., Niswender, G.D. and IV.idgl.ey, 34, 177 (1972). Fairclouch, R.J., Peterson, A.J. and Payne, E. PROC. N.Z. SCX!. ~~RINOL., Oct. 1973. N-2. Mf!D.J, In Press. Dean, P.D.G., Exley, D. and Johnson, M.W., STEROIDS, 18, 593 Erl,fa~~'l!*F . .) Borek, F., Beiser, S.M. and Leibeman, S. J. BIOL. CHEM., 228, 713 (1958). Peterson, A.J. and Common, R.H. CAN. J. ZOOL., 50, 395 (1972). Wu, C.H. and LunqY, L.E., ST~OIDS, 18, 91 (1971). Peterson, A.J. and Smith, J.F. PROC. N.Z. SOC. ENDOCRINOL., act. 1973. N.Z. MED. J. In Press. Rosner, W. J. CLIN. ENDWR., 34, 983 (1972). Glencross, R.G., Mizmo, I.B., Senior, B.E. and Pope, G.S., ACTA EKOOCRINOL., 73, 374 (1973). Abraham, G.E., J. CLIN. ENDOCR., 29, 866 (1969). Borth, Ii., ACTA ENDOCRINOL., suppl. 147, 33 (1970). Drinan, J.P. and Cox, R.I. PROC. BUST. SOC. =OD. BIOL. 5, 60 (1973).

Radioimmunoassay of estradiol-17beta in bovine peripheral plasma with and without chromatography.

An assay of estradiol-17beta (E217beta) in bovine peripheral plasma is described. The plasma is incubated with an antiserum to E217beta-BSA and the ga...
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