509
FOR FREE AND CONJUGATED TRIENBOLONE AND FOR TRIENBOLONE ACETATE IN BOVINE TISSUE AND PLASMA SAMPLES RADIOIMMUNOASSAYS
B. Hoffmann and G. Oettel Institut fiirPhysiologie der Stidd. Versuchs- und Forschungsanstalt fiirMilchwirtschaft Technische Universitgt Mtinchen 8050 Freising-Weihenstephan Received
l/13/76 ABSTRACT
A specific, sensitive, precise and accurate radioimmunoassay has been developed for the quantitation of the synthetic anabolic steroid trienbolone acetate (TBA) and its major metabolites, free and conjugated trienbolone (TBOH) in bovine tissues and plasma. With the extraction procedure described unspecific interference with the antigen-antibody reaction could be ruled out. The assay can significantly detect amounts of more than 40 pg TBOH and 70 pg TBA. 0.1 2.0 g tissue and 0.1 - 1 ml plasma are sufficient for 1 determination. Residues (range 0.1 - 2.0 rig/g))were stillR present in calves 69 days after implantation of "Revalor" (20 mg estradiol-178 and 140 mg TBA) with the highest concentrations found in liver and TBA could only be quantitated in fat. Trienbolone acetate (TBA; 17B-hydroxyestra-4,9,11-triene3-one-17B-acetate)
is a potent synthetic anabolic agent,
and is administered in several countries as a subcutaneous implant alone or in combination with estradiol-17B
(1, 2).
Based on radio-tracer studies the major metabolite of TBA in tissues, plasma and urine has been identified to be trienbolone
(TBOH, 178-hydroxyestra-4,9,11-triene-3-one),
Significant traces of TBA could only be found in fat (3). In the present paper a radioimmunoassay
(RIA) for the deter-
mination of both, TBA and TBOH, in bovine tissue samples and plasma is described in order to qualitatively and quantitatively
identify possible residues in tissues regu-
Vo'owne 27, Nwnber 4
s
~BILOXDM
April,
1976
larlr used for human consumption. The methods and results have already been discussed briefly elsewhere
(4, 5).
MATERIALS AND METHODS Special glassware: Homogenization and initial extraction were performed in glass-scintillation vials with tin foillined screw-caps. For RIA disposable glasstubes (14 - 75 mm) were siliconized (Siliconlbsung Serva, Heidelberg, Germany) to minimize adsorption. Solvents and reagents: Ether "pro narcosi" (Hoechst, Germany) was supplied in 100 ml bottles. All other solvents and reagents were "analytical grade" and the solvents were distilled before use; demineralized water, B-glucuronidase (20 U/mg) from Escherichia coli (Boehringer, Mannheim, Germany) and Sephadex-LH-20 (Pharmacia, Fine Chemicals, Uppsala, Sweden) were used. Standards: Unlabelled and 3H-labelled TBA (6 7-T-TBA. 58.2 Ci/mmole) as well as unlabe led and 3H-;abelled'TBOH (6,7-T-TBOH; 51.5 Ci/mmole) and sH-labelled TBOH-glucuronide (6,7-T-TBOH-17-glucuronide, 0.93 Ci/mmole) were obtained from Roussel Uclaf (Paris, France). Purity was checked by thin-layer chromatography (silica-gel: chloroform/ethyl acetate = 2/l)and radio-scanning. Stock solutions were prepared in methanol or a mixture of benzene/methanol (95/5). Due to a slight hydrolysis of the 3H-TBA and a slight oxidation of the 3H-TBOH these stock solutions were purified in 3 months intervals by applying the column chromatography described below. Antisera: After synthesis of TBA-3-(o-carboxy-methyl)-oximeBSA (6) and TBOH-17-hemisuccinate-BSA (7) rabbits were immunized and antisera were obtained from various bleedings. The cross-reactivity of the antisera used for this study are shown in figs. 1 and 2. While the antiserum against TBA-3-(o-carboxy-methyl)-oximeBSA only reacted with TBA (fig. 1) the antiserum against TBOH-17-HS-BSA showed significant cross-reactions with all trienic-steroids BBOH, TBA and estra-4,9,11-triene-3,17dione (triendione)], but not with the other compounds tested (fig. 2). For assay purposes both antisera were used in a dilution of 1 : 3500.
S
Txsmox~rn
511
Figure 1 Cross reactions of various steroids with antibodies against TBA-3-(o-carboxy-methyl)-oxime-BSA
i 1
100
90 80
0
B
;
:
i
e
0
-il
compounds
. .
g 70% .j 6050? 'Z 40z 30X 20trienbotone
tested:
bstradiol-
17 6
bstradiot
-17 (I
0
estrone
.
estriol
.
estradiolbenzoetb
0
diethylstilbestrol
0
DEA
.
testosterone
.
progesterone
.
Corlicos1eronb
.
17a-hydrcmiprogestbronb
0
trienbolone
0
triendlone
.
androstendionb
SCdStb I
3.0
I
I
4.0
5.0 ng
compound
Figure 2 Cross-reactions of various steroids with antibodies against TBOH-17-hemisuccinate-BSA 0 .
: .
compounds
tbStbd:
l
estrediol-
17 t3
6
ettrediol-
17 b
0
estrone
.
estriol
.
estrediolbbnzoetb
o
diethylstilbestrol
m
dibnestrol
.
testosterone
l
progesterone
l
corticosterone
trlenbotmb trbndione lrienbolonb
1
I
1.0
I
2.0
lcb!Ste I
3.0
I
4.0ng
compound
512
S
TDEOIDCI
RIA-procedure, counting and assay evaluation: RIA was performed as described elesewhere (8). Brieflv the wrocedure involves the simultaneous addition of iabelled and c8ld steroid to the antibody, an in tial incubation at 37 C & followed by an incubation at 4 C. Bound and free steroids are separated by charcoal adsorption. After centrifugation the supernatant was decanted into disposable polyethylene counting vials already containing 10 ml scintillation fluid [7 g PPO, 0.3 g PPO, 100.g naphtaline in 1 1 dioxane (techn. grade) (94. All samples were counted to a pre-set error of 3 %. With each series of samples (n = 32) 2 calibration curves (range 0 - 2 ng) were developed. To correct for the additional counts in the biological samples resulting from the internal standard, the following approximation was applied.
cPmo CPmTK
=
x
cJ?mo +
‘PmT
cpmw
CpmTK: corrected cpm-value for biological sample cpmo : cpm in reference sample of calibration curve with no charcoal added cpmT : cpm determined in biological sample cp"w : calculated amount of cpm in biological sample resulting from internal standard The readings then obtained from the calibration curve were corrected for the aliquot, the general recovery and size of the tissue- and plasma sample extracted. Extraction procedures for free TBA and TBOH: 0.1 - 2.0 g of minced muscular-, liver- and kidney-tissue are homogenized for 15 sec. on an Ultra Turrax (Janke & Kunckel, Staufen, Germany) after the addition of 2000 dpm 3H-TBOH and 2 to 4 ml of water. The homogenate is extracted with ether (IO 15 m 1. To minced fatty tissue 2000 dpm of each 3H-TBOH and 3H-TBA and 2 ml of H 0 are added and the sample is then directly extracted with 35 ml ether. For extraction the samples are vigorously shaken on a Vortex-mixer, briefly centrifuged and the ether is decanted after freezing the aqueous phase. The extraction is repeated in the cases of liver- and kidney-tissue. After evaporation of the ether the extracts are taken up in 5 ml of 70 % methanol and a solvent parti&ion against 2 ml of petroleum ether (boiling range 50 - 70 C) is performed. The petroleum ether is discarded and this step is repeated 2 more times. By adding 0.1 ml 1 n HCl emulsions could be broken which tended to
513 occur in cases of liver and kidney extracts. For these 2 tissues it was also necessary to perform a second partition between 5 ml CC1 /CHCL (5/l) and 2 times 1 ml 0.5 n NaOH and 1 ml H 0. After e?aporation of the 70 % methanol (muscle, fat? and the CC1 /CHCl -mixture (liver, kidney) 1 ml of H 0 was added to afl samales which were then extracted with 2 &mes 2 ml of ether. The pooled ether extracts were evaporated and the samples subjected to column chromatography on Sephadex LH 20 (length 90 mm, diameter 8 mm), using benzene/methanol (95/5) for elution. TBOH could be collected from ml 5.0 to ml 10.0 and in the case of fat TBA from ml 1.5 to ml 4.0. The fractions were dried down, dissolved in 1.0 ml methanol and 0.2 ml were removed for counting and recovery determination and 0.6 ml for RIA. In case of plasma (0.05 - 0.5 ml) H 0 (2.0 ml) was added and a simple extraction with ether (5 rnz)was performed. The ether was evaporated and the extract directly submitted to RIA. Extraction procedure for conjugated TBOH: After removal of the free steroids with the initial zextraction (see above) 500 mU R-glucuronidase are added to the remaining aqueous phase and the samples are tgen incubated for 1 hour at 37OC. With the addition of H-TBOH there-after the procedure is from there on identical with the one described for free TBOH. Reliability criteria: The precision, accuracy, reproducibility and sensitivity of the methods were evaluated by determining the recovery of labelled and unlabelled hormone added to tissue samples prior to the extraction, by repetitive duplicate and triplicate assays of unknown samples and by testing various aliquots of individual samples. In addition to the binding characteristics of the antisera the specificity of the method was further investigated by determining the effect of tissue extracts from untreated animals on the course of the calibration curves (IO). Animal experiments: As examples the results obtained in tissues of 3 cxz slaughtered 69 days after the treatment (implantat&on)with the normal, lo-fold and 25-fold dose of Revalor (20 mg estradiol-17B + 140 mg TEA) are given. From another experiment plasma samples were obtained covering a period of 67 days following the implantation of RevalorR.
S
514
TDROIDB
RESULTS In order to evaluate unspecific interference with the antigen-antibody-reaction
specificity of the methods was
investigated by comparing standard curves
(0; 0.05; 0.1;
0.2; 0.5; 1.0 ng) with and without tissue- and plasma extracts. All curves were calculated on a computerized program
(Wang 700) as polynomial regression
(3rd degree)
by plotting the ng of the calibration curve (y-axis) against the time (x-axis) necessary to accumulate counts to a pre-set instrument-error
of 3 %. In order to estimate
the variation within curves they were first compared (comparison I) on the basis of the residuals calculated (i.e. difference
(+ or -) between y-value given and y-value
estimated according to the regression equation). There were no differences between the average residuals calculated for individual standard curves with tissue- or plasma extracts;
(TE) and without
(R)
(range of residuals = 3.3 2
3.0 pg - 21.0 2 14.3 pg; table 1.) In a second step (comparison II) the x-values
(time) obtained for calibra-
tion curves with tissue extracts
(TE) were treated as un-
knowns and quantitated on the regression-equation computerized for the regular standard curves (R). In case of TBOH over- and underestimation were equally distributed and within or slightly above the variation of each standard curve. With a constant positive difference
(mean: 50.8 2
19.6 pg) there was a slight tendency of overestimation
in
case of the determination of TBA (table 1). For illustration the curve-comparisons
(computer print-out) for
muscle and liver are shown in figure 3. Recovery of
3 H-TBOH
(z/s) was rather constant and not re-
lated to the amount of tissue extracted case of muscle
(0.1 - 2.0 g) in
(58.2/6.7 %, n = 215) and fat (57.9/4.6 %,
n = 209). In liver and kidney it decreased from 58.8/7.1 %
TBA
11.3( 6.9)
liver
12.6( 7.2)
8.5( 7.9)
10.5( 6.5)
3.6( 1.6)
8.3( 5.9)
8.1( 5.9)
3.3( 3.0)
ll.O( 6.7)
18.2(10.7)
7.8( 4.6)
50.8 (19.6)
18.6 (13.7)
11.6 ( 9.8)
23.2 ( 7.7)
30.1 (23.4)
26.6 (22.2)
36.1 (14.9)
comparison II: x-values for TE evaluated on R. mean difference x (s) in pg between y-value given and estimated
TE = standard curve developed in presence of tissue extracts
R = regular standard curve
fat
fat
kidney (conjugated)
5.6( 6.1)
8.2( 5.9)
muscle
kidney
21.0(14.3)
comparison I: residuals x (s) in pg R TE
plasma
hormone sample
TBOH
Table 1
Evaluation of assay specificity by comparing standard curves (0 - 1 ng) developed regularly (R) and in the presence of tissue extract (TB). Comparison of average residuals as estimated on a polynomial regression 3rd degree (comparison I) and by treating y-values of (TE) as unknowns on (R) (comparison II).
(n =
41)
to
35.5/6.1
%
(n = 10) when 0.2 and 2.0 g of 3 tissue were extracted, respectively. Recovery of H-TBA from fat slightly decreased when 0.1 and 1.0 g were extracted
(34.7/4.4 %, n = 8 and 27.6/6.1 %, n = 26). Re-
covery of 3H-TBOH from plasma yielded 91.2/4.9 % (n = 169) making routine internal standardization unnecessary. Hydrolysis as tested in several control experiments by the addition of 3H-TBOH-glucuronide to various tissue samples yielded a recovery of 85.8/3.1 % as free 3H-TBOH. As can be seen from figure 4 the extraction of various amounts of tissue gave linear regressions
(b between 0.994
and 1.00) with calculated intercepts equal from 0.167 ng (liver) to zero (muscle). The coefficient of variation was between 4.5 % (fat, TBOH) and 12.6 % (fat, TBA) when the results were expressed as rig/g tissue. For plasma a mean value of 2.3 + 0.2 ng TBOH/ml was obtained, when 0.05, 0.1 and 0.2 ml were extracted. Similarly as in these experiments a linear regression was obtained for the recovery of unlabelled TBOH and TBA resp. added prior to the extraction
(table 2). The calculated
intercepts showed a range equal from 2 to 61 pg. As can be seen from the recovery of TBOH from liver and plasma, the intra-assay variation was between 2.0 and 8.0 %, while the inter-assay variation showed a range of 2.3 to 13.4 % (table 2). Reproducibility was examined for all tissues and for plasma by repeatedly assaying the same samples over a period of 4-
6 weeks and the overall coefficient of variation
varied between 5.2 8 (TBOH, kidney) and 18.7 % (TBA, fat) (table 3).
3.0.
3.5.
SCALE
LIVER
: 1
?ROtd
.o 10
00
to
5.0
10
00
00
1
1
I
1.5.
pz.0.
OR
+TE
P
MUSCLE
62.5
3.0.
3.5
4.0
L.5
1.5.
4.0.
5.0
5.07
.:-
:.*
::~
time
$’
.a’1
.
Figure 3 Agreement of calibration curves with (TE) and #thout (R) tissue-extract; curves expressed as polynomial regression of 3 degree (computer print-out)
0.202/0.026 12.9 6.8 0.468/0.032 0.872/'0.065 7.4 5.0 2.0 3.1
0.201/0.011 0.498/0.010 1.024/0.023
0.197/0.026 0.537/0.057 7.031/0.057 0.249/0.010 0.516/0.033 0.951/0.022 0.196/0.022
0.543/0.057 1.042/0.112 0.241,'0.014 0.503/0.040 0.952/0.060
7 16 15
6 5 6
16 16 16
8 16 8
a
::
6
6 6
0.2 0.5 1.0
0.2 liver 0.5 (TEOH) (oneexperiment)I*'
0.2 0.5 1.0
0.2 0.5 1.0
0.2 0.5 1.0
0.2
0.5 1 .o
kidney (TKW
fat (=I
fat CJJW
Pb==
CJ.‘KN
(one experiment)
(TKW
muscle
8.0 6.3
5.9
11.4 10.6 10.8
4.0 6.4 2.3
13.4 10.7 5.6
(%I
(ng)
canpound
W
aIKxlnt added
sample and
recovery parameter VIZ n Z/S
Recoveq of unlabelledTWH and TE!Aresp. from various tissuesand plAsma
Table 2
I
1
5 * 2 z
4 -5 3
3
us .z
6
1
2 amount of tissue extracted
0.5
(9)
TBOH and TEA (fatonly) determinedin variousmrountsof tissues.Mean values of dqAicates (x), triplicates(xx) and quadruplicates(xxx)
Figure 4
S
519
TBEOXDI
Table 3 Repetitive determination of TBOH and TBA resp. in tissues and plasma over a period of 4 - 6 weeks Statistical sample parameter wle TROH n G (ng/g) +s VK (%)
16
and liver TBOH
compound fat kidney TBOH TBOH
16
18
examined fat Plasma TBA
16
10
13
1.402
5.146
5.538
14.091
0.814
4.520
0.168
0.819
0.287
1.328
0.152
0.830
5.2
9.4
11.9
15.9
18.7
18.0
Based on the variation within the standard curves with and without tissue extracts obtained for the present experiments, the lower limit of sensitivity for the determination of TBOH is around 40 pg while it is about 70 pg for the determination of TBA in fat. "In viva" studies: The results obtained in 3 animals, treated with the normal dose of Revalor R (20 mg estradiol1713+ 140 mg TBA), the lo-fold and 25-fold doses are shown in tables 4 and 5. There is a significant positive correlation
(r = 0.89 to 0.99) between the treatment-dose
and the residue concentration determined. Qualitative and quantitative differences between tissues are apparent with liver showing the highest residues and conjugated TBOH being the major residue fraction. The lowest residuelevels were found in muscular tissue. TBA could only be quantitated in fat. In figure 5 the appearance of TBOH in plasma after implantation is shown. Peak levels (13.8 ng/ml) were obtained within 6 hours and a rapid drop was observed afterwards. However, TBOH was still present 67 days after implantation (0.17 ng/ml). In general conjugated TBOH amounted up to 20 % of the free TBOH.
S
520
TDEOIDII
Table 4 Residuelevelsof TBA and free TE?OHin varioustissues 69 days after treatmentwith increasingdosagesof R.evalorR (20mg estradiol-1713 + 140 mg TFA) levels
residue treatment
muscle (WH)
n
(rig/g tissue)
kidney liver (TBOH) (TEW
10
fat (TBOH)
fat (TEA)
6
6
6
6
RevalorRG
0.125
0.911
0.434
0.477
0.118
5
0.032
0.097
0.086
0.024
0.024
11
n
8
6
10
6
5
(lofold)G
0.500
2.409
1.153
5.873
0.626
S
0.030
0.310
0.093
0.262
0.032
18
16
II
n
(25fold);; S
8
6
7
1.175
4.770
5.538
14.091
0.765
0.035
0.183
0.287
1.328
0.092
n = mnber of singledeterminations x =meanvalue s = standarddeviation
Table 5 Residueconcentrations(meanvalues of duplicates)of conjugated TaOHinvarioustissues 69 days after treatmentwith increasing dosagesof R.evaorR (20mg estradiol-17fi + 140 mg TBA) conjugated treatment RevalorR
TBOH (ng/g) kidney
muscle
liver
fat
n.d.
1.194
0.452
n.d.
"(IO-fold)
0.013
9.125
3.874
0.119
'(25-fold)
0.048
21.550
5.482
0.332
n.d. = not detectable
Figure 5 Free and conjugated TBOH in peripheral plasma (ng/ml) of a calf after implantation of RevalorR (20 mg estradiol-17B + 140 mg TBA) CI 15 z . z ; E ‘; P s ‘L’ CI
O---Q
free conjugcted
50
60
u
10
5
10
20
30
40
days
ihplcntction ( 20mg
67
0e2 -170 + 140 mg TBA )
Discussion In connection with the use of anabolic agents in animal production the requirement has to be met to qualitative and quantitative estimate possible residues - either of the administered compound or a major metabolite - in edible tissues. As was demonstrated in radio-tracer studies, the concentrations to be expected few days apart from the treatment are in the pg-range
(low ppb-level)
(11,
12). The successful quantification of these low level residues by RIA in the case of steroid-estrogens in various bovine tissues of treated and untreated animals has been reported
(10, 131, similarly as the determination of
testosterone in bovine fatty tissue (5) and of progesterone in milk products
(14).
With the present paper the successful development of a RIA for quantification of tissue residues of an anabolic
S
TDROIDS
steroid not naturally occuring is described. Main emphasis was based on the estimation of free and conjugated TBOH, since they are the main metabolites of TBA, the originally administered compound which - with the exception of the implantation site - can be found as a residue in fat only (3). With a lower limit of sensitivity of 40 pg for TBOH and about 70 pg for TBA the reliability of the method could be established. With the experimental design selected and the results obtained and without tissue-extracts)
(calibration curves with
especially unspecific inter-
ference with the antigen-antibody
reaction could be ruled
out (10, 15). Furtheron the approach to plot calibration curves as "ng-introduced
(y-axis)" versus "counting-time
necessary for a pre-set instrument-error
(x-axis)" and to
express the function as a polynomial regression of 3rd degree has shown to be very easy, precise and satisfactory. With the methods developed residue quantitation even 69 days after treatment with a regular dose of RevalorR
(20 mg estradiol-17B + 140 mg TBA) was possible; depending on the tissue total residues
(TBOH, free and conjugated and
TBA) were in a range of about 0.1 to 2.0 rig/g.. About 100 samples can be handled by one person per week. If it is taken into consideration, that liver could be classified as a "marker" tissue, the method could be used for regulatory purposes in laboratories having the necessary equipment.
ACKNOWLEDGEMENT Labelled and unlabelled TBOH and TBA were kindly donated by Roussel/Uclaf, Paris, France.
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TIIEOIDI
523
REFERENCES 1.
2.
3. 4.
5.
6. 7. 8. 9. 10.
11. 12. 13. 14. 15.
Gropp, J., Boehncke, E., Schulz, V., v.Sandersleben, J., Geisel, 0. and H;inichen, Th.: Z. TIERPHYSIOL., TIERERNAHRG., FUTTERMITTELKUNDE, Beiheft 6, 33 (1976) Heitzman, R.J.: FAO/WHO Symposium on the use of anabolic agents in animal production and its public health aspects. FAO Headquarters, Rome, Italy, March 17 - 19, 1975 ENVIRONMENTAL QUALITY AND SAFETY, in press Poitter, J., Busigny, H. and Grandadam, J.A.: J. ANIM. SCI., 37, 256(1973) Hoffmann, B., Heinritzi, K.H., Kyrein, H.J., Oehrle, K.L., Oettel, G., Rattenberger, E., Vogt, K. and Karg, H.: 2. TIERPHYSIOL., TIERERNAHRG., FUTTERMITTELKUNDE, Beiheft 6, 80 (1976) Hoffmann, B. and Karg, H.: FAO/WHO Symposium on the use of anabolic agents in animal production and its public health aspects. FAO Headquarters, Rome, Italy, March 17 - 19, 1975, ENVIRONMENTAL QUALITY AND SAFETY, in press Karg, H., Kyrein, H.J. and Hoffmann, B.: J. ANIM. SCI. 2, 256 (1973) Erlanger, B.F., Borek, F., Beiser, S.M. and Liebermann, S .: J. BIOL. CHEM. 228, 713 (1957) Hoffmann, B., SchamrD., Gimgnez, T., Ender, M.L., Herrmann, Ch. and Karg, H.: ACTA ENDOCR. (KBH) 2, 385 (1973) Neill, J.D., Johansson, E.D.B., Datta, J.K. and Knobil, E . : J. CLIN. ENDOCRINOL. 27, 1167 (1967) Hoffmann, B., Karg, H., Hanritzi, K.H., Behr, H. and Rattenberger, E.: MITT. GEBIETE LEBENSM. HYG. 66, 20 (1975) Aschbacher, P.W. and Thacker, E.J.: J. ANIM. SCI. 39, 1186 (1974) Sharp, G.D. and Dyer, J.A.: J. ANIM. SCI. 34, 176 (1972) Henricks, D.M.: Proc. of the first annual NCTR Hormone Symposium, November 21 - 22 (1975), p. 139 Hoffmann, B., Hamburger, R. and Karg, H.: Z. LEBENSM. UNTERS,-FORSCH. 158, 257 (1975) KUSS, E.: 21. SymTsium d. Dt. Ges. f. Endokrinologie, Munich, February 26 - March 1, 1975