311
THE DETERMINATION OF FIVE STEROIDS IN AVIAN PLASMA BY
RADIOIMMUNOASSAYAND COMPETITIVE PROTEIN-BINDING
J.C. Wingfield and Donald S. Farner
Department of Zoology, University of Washington
Seattle, Washington 98195
Received: k/29/75
ABSTRACT
A method has been developed for the simultaneous determination of
testosterone, 5a-dihydrotestosteroneand corticosterone,or of estrone,
estradiol-176 and corticosterone,after separation on a Celite:propylene
glycol:ethylene glycol column (6:1.5:1.5
w/v/v).
The lower quarter of
the column was packed with a Celite:watermixture (3:l w/v) as a stationary phase (glycol) 'trap'. This effectively prevented leaching of the
glycols into the eluate as the concentration of ethyl acetate in the
mobile phase was increased to elute the more polar steroids. In addition, a second system utilizing a Celite:ethyleneglycol column (2~1 w/v)
for the separation of estrone and estradiol-178 is described.
Testosterone, 5a-dihydratestosterone,estrone and estradiol-178
were measured by radioimmunoassayand corticosteroneby a competitive
protein-binding technique. Reliability criteria are presented showing
that the assay systems used are accurate and reproducible.
Plasma-steroid levels of eight avian species are also presented
and compared with those found by other investigators.
INTRODUCTION
Simultaneous measurement of a number of plasma steroids such as
testosterone, 5a-dihydrotestosterone(li'B-hydroxy-5a-androstan-3-one),
estrone, estradiol-17B and corticosteronethroughout a breeding cycle
would greatly increase our knowledge of the endocrine control of reproduction in birds.
rhynchoe)
In large species such as the Mallard (Anas platy-
and domestic fowl, relatively large blood samples (5-10 ml)
can be obtained and subdivided for the assay of each steroid. Bowever,
in small passeriform species (e.g. the White-crowned Sparrow, &no-
trichia
leucophrys
gambelii,
which has been under intensive study in
this laboratory for many years) the maximum plasma volume obtainable
VoZwne 26, Number 3
S
WDDOXDI
September,
2975
312
S
from one bird is 0.5-0.8 ml.
~DEOfDrn
If serial sampling is involved, this volume
is reduced to 100-300 ~1 if the bird is to survive and subdivision of
such a small sample of plasma for separate steroid assays is not feasible.
Therefore it would be an advantage to separate chromatographicallythe
steroids to be measured from one sample. We wish to report the development of such a system using Celite column chromatography as described
by several authors (l-4).
MATERIALS AND METHODS
Collection of plasma samples
Plasma samples were collected from the following species: Mature
rooster (CaZtus domesticus),
Mallard, Lesser Scaup (Aythya affinis)
,
White-crowned Sparrow (Z. 2. pugetensia),
Harris' Sparrow (Z. querula),
House Finch (Carpodacue mexicanti),
and Brown-headed Cowbird (MoZothrus
Blood was collected from the jugular vein with heparinized
ater).
syringes or from a wing vein with heparinized micro-hematocrit capillary
tubes after puncture of the blood vessel with a 26-gauge needle. Plasma
samples were stored frozen until analyzed.
Solvents
Chloroform, benzene, ethyl acetate (all purchased from Mallinckrodt,
St. Louis, Missouri), methanol and ethanol (Matheson, Coleman and Bell,
Los Angeles, California) were redistilled once before use. Iso-octane
(nanograde),
dichloromethsne (nanograde), propylene glycol, ethylene
glycol (all purchased from Matheson, Coleman and Bell) and toluene
(Mallinckrodt,Scintillsr) were used without further purification.
Steroid standards
Corticosterone, testosterone, 5a-dihydrotestosterone,estrone and
estradiol-17B were purchased from Sigma (St. Louis, Missouri). Radioactive steroids, 1,2,6,7-3H-corticosterone 1,2,6,7-3H-5a-dihydrotestosterone, 1,2,6,7-3H-testosterone,2,4,6,7-3H-estroneand 2,4,6,7-3Hestradiol-178 were obtained from New England Nuclear (Los Angeles, California) and stored in benzene:ethanol (9:l v/v, 250 uC!i/lOOml) at 4'C.
Both radioactive and non-radioactive steroids were purified by
silica gel thin layer chromatography in chloroform:methsnol:water,
9O:lO:l (corticosterone)or ethyl acetate:benzene (1:l) (all other
steroids).
Buffers
A phosphate saline buffer (PBS) containing phosphate buffer (0.1 M,
pII7.0), sodium chloride (0.9% w/v) and sodium azide (0.1% w/v) was used.
313
In the actual assay procedure PBS containing 0.1% (w/v) gelatin (PBS-g)
was utilized.
Dextrsn-coated charcoal
1.25 g of charcoal (Norit A. Pfanstiehl, Waukegan, Illinois) was
suspended in a solution of 125 mg of Dextran T-70 (Pharmacia, Uppsala,
Sweden) per liter of PBS-g.
Celite
Celite Analytical Filter-Aid (Johns-Manville,Lompoc, California)
was incubated in an oven at 54O'C for at least 24 hours before the columns
were packed.
Antisera
Testosterone antiserum was purchased from Wien Laboratories, Inc.
(Succasunna,New Jersey). This antibody was raised in rabbits sgainst
a testosterone-3-oxime-albuminpreparation (5) and was received as lyophilized plasma in glass smpules. Each empule was reconstituted in
50 ml of PBS-g and stored at k°C. This stock solution remains stable for
at least six weeks as shown by the weight of testosterone required to
give 50% binding on the standard curve (Table 1). Data provided by Wien
Laboratories (5) show that this antiserum is highly specific and of 11
steroids tested only 5a-dihydrotestosteroneand 178-hydroxy-1, k-androstadien-3-one gave significant cross reactions, Of 1000 pg of 5a-dihydrotestosteroneadded to the assay system 545 pg were recovered as a
testosterone equivalent (5) which enabled a radioimmunoassay for this
steroid to be developed (6).
Anti-estradiol-17Bserum was provided courtesy of Dr. V.L. Estergreen (Washington State University, Pullman, Washington), The antibody
was raised in sheep against estradiol-17B-17-hydrogensuccinate-bovine
serum albumin (7) and diluted 1:150 in PBS-g and stored in 0.2 ml aliquota, at -2OOC. As required for assay:, aliquots were thawed and made
up to 1:37,500 in PBS-g and stored at 4 C. This stock solution remains
stable for at least 10 weeks as shown by the weight of estradiol-17B
required to give 50% binding on the standard curve (Table 1). Of the
steroids likely to be found in avian plasma only estrone, 1,3,5(10)estratrien-3, 17a-diol and 1,3,5(10)-estratrien-3,16a, la&trio1 showed
significant cross-reaction (62, 33, and 27% respectively) . The high
degree of cross-reactionof estrone was utilized in this study for the
radioimmunoassayof this steroid.
Corticosteroid-bindingglobulin
Corticosterone is the major plasma corticosteroidof all avian
species so far studied (8,9) and a corticosteroid-bindingcapacity similiar to that found in mammals has been demonstrated in the blood of the
domestic fowl (10). For these reasons chicken plasma was selected as a
source of corticosterone-bindingglobulin,
S
314
TEIEOI131
&Percentsge cross-reaction was defined as pg of estradiol-17f3
exhibited by 100 pg of the steroids listed (7).
equivalent
TABLE1
Stability of antisera stock solutionsa
Steroid
Weight of steroid
required to give
50% binding
Time
Anti-estradiol-17B serum
-p
Estrone
75.5
10 weeks
pf: (12)
C.V. = 9.815
Estradiol-178
10 weeks
108.5 ~~$2'
c.v.
. I
--_-_-.
Anti-testosterone serum
---I_-
-_--
----_--__-
Testosterone
6 weeks
5a-dihydrotestosterone
6 weeks
-C.V.
-----
----
__.._.___---._-_-._--_-__
sta.ndarddeviation
= coefficient of variation calculated as -x 100
mean
Numbers in parentheses represent the number of determinations.
'During the time period stated, the standard curves, 50% binding values
and sensitivity were very constant. After this time period 50% binding
values began to increase resulting in loss of sensitivity and shallow
standard curves.
-~---~-1_-_1_.-.
Blood WRS removed from mature roosters with a heparinized syringe
from the jugular vein and centrifuged at 2000 rpm for 15 min. The plasma
was stored in sealed glass ampules in 1 ml aliquots at -2O'C. For each
corticosterone assay the required amount of rooster plasma was thawed
and passed down a Sephadex G-25 coarse column (Pharmacia) to remove endogenous steroids as described by Bassett and Hinks (11). Briefly, Sephadex G-25 was swelled in distilled water for 24 hours at room temperature.
A glass column (20 nnn in diameter) was packed to a height of 75 cm in
PBS. Rooster plasma (l-2 ml) was passed through the column in PBS
(2 drops/see) and the protein fraction collected and diluted to 2% (v/v)
in PBS-g.
Liquid scintillation counting
All radioactive steroids were counted on a Beckman Liquid Scintillation System (Beckman Instruments, Inc., Fullerton, California), in 5 ml
of counting medium prepared as follows: 4 g Cmnifluor (New Englsnd
Nuclear) per liter of toluene. Samples were counted to an accuracy of 2%.
Purification of plasma steroids
Extraction: plasma samples ranging in volume from 100-2000 ul were
mixed with known amounts (approximately2000 cpm) of respective 3H-steroids for subsequent recovery determinations. Plasma samples with less
than 1 ml volume were made up to 1 ml with distilled water and those
with volumes between 1 and 2 ml were made up to 2 ml. In addition, two
1 ml distilled water blanks were extracted and processed as were the
plasma samples. Each sample was extracted with 4 ml of dichloromethane
and the organic phase aspirated off and dried under a stream of nitrogen
in a 40°C water bath. The dried extract was taken up in a total of 1.5
ml of 2% ethyl acetate in iso-octane and transferred to the top of one
of the columns.
Celite columns
1 g of a Celite-stationaryphase mixture (see Table 2) was packed
in each column (5 ml disposable glass pipette) supported by a glass bead.
In cases in which corticosteronewas also to be eluted, high concentrations of ethyl acetate (Table 2) were required in the mobile phase. ,In
these a Celite-water 'trap' (3:l w/v, 0.3~) was packed beneath the Celitestationary phase mixture. This 'trap' effectively prevented leakage of
the glycols into the eluted extract without drastically changing the
elution pattern of the steroids to be measured. Two 3.5 ml aliquots of
iso-octane were percolated through each column before the extracts were
applied. The flow rate was never allowed to exceed 1 drop/7 seconds
and was maintained by nitrogen pressure.
Chromatography
The elution patterns of the steroids measured are presented in
Table 2, in relation to the respective stationary phase mixture. The
simultaneous radioinnnunoassay
of testosterone and 5a-dihydrotestosterone
in avian plasma using a Celite:propyleneglycol:ethylene glycol column
(6:1.5:
1.5 w/v/v) without a water trap has already been described (6).
Therefore, presentation of the asssy procedure and reliability criteria
in this report will be restricted to the eluates of the other columns
used.
After collection of the steroid fractions, eluates were dried under
a stresm of nitrogen in a 40°C water bath. Dried extracts were taken up
in 1 ml of dichloromethane:methanol(9:l v/v), subdivided as required
and taken to dryness under a stream of nitrogen in a 40°C water bath.
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316
T-EOXDI
TABLE 2
Elution pattern of steroids
from Celite column
% ethyl acetate
in iso-octane
Volume
ml
Eluate
Celite:propyleneglycol:ethylene glycol column with water trap
0
10
20
4.0
4.5
4.5
40
50
4.5
4.0
Discard
5a-dihydrotestosterone
Testosterone plus
estrone
Estradiol-17g
Corticosterone
Celite:ethylene glycol column
0
15
40
Discard
Estrone
Estradiol-17B
Radioimmunoassay and competitive protein-binding assay
For standard curves, duplicate tubes containing standard steroid in
100 ul of PBS-g covering the range of O-1000 pg for testosterone, 5adihydrotestosterone,estrone aa estradiol-178, ana O-10,000 pg for corticosterone were used. To the dried extract tubes, 100 yl PBS-g were
added to keep assay incubation volumes constant. To all tubes 100 ul of
respective 3H-steroid, usually lo4 cpm, were added followed by 100 ul of
antiserum from the respective stock solutions (for the sex steroid assays) or 0.5 ml of fresh corticosterone-bindingglobulin (CBG) for the
corticosterone assay.
In addition to the standard curve and samples, three duplicate assay
blank tubes were incubated. Blank (Bl) contained 100 ul PBS-g, 100 1.11
3H-steroid and 100 ul antiserum (0.5 ml CBG for the corticosterone assay). These tubes represent the total counts added to the assay system
(no dextran-coated charcoal is added to these tubes). Blank (B2) contained 200 ul (600 ul in the corticosterone assay) of PBS-g and 100 vl
of 3H-steroid (i.e., no antiserum/CBG blank) and represents the residual
free counts not absorbed by the charcoal. Blank 3 (B3) was the same as
Bl except that charcoal is added (no hormone blank) and represents 100%
binding.
After the appropriate incubation time (12 hours at 4'C for the sex
steroid assays and 10 min. at 45'C followed by 2 hours at 4'C for corticosterone), 1 ml of dextran-coated charcoal (0.5 ml for the corticosterone assay) was added to all tubes except Bl (1 or 0.5 ml of PBS-g added)
at 4'C and whirl-mixed. After a 10 min. (corticosterone,testosterone
and 5a-dihydrotestosterone)or 20 min. (estrone ana estradiol-178) incubation with charcoal, all tubes were centrifuged at 2000 rpm for 10
min. at 4OC. Supernatants were decanted into counting vials and 5 ml of
scintillation fluid added. Vials were allowed to stand overnight before
counting to insure complete extraction of 3H-steroid the counting medium.
Calculations
Mean cpm in B2 was subtracted from the mean cpm in B3 and the mean
cpm of both standard curve and plasma extract duplicates, and then expressed as % bound as follows:
(cpm in standard curve or plasma extract duplicates)-(cpmin B2)
(cpm in B3)-(cpm in B2)
Standard curves were plotted as % bound against log pg of steroid.
The % 3R-steroZd bound in the plasma extract tubes was read off the standard curve as pg of respective steroid present. Recoveries (Table 3)
were calculated as % activity eluted from the columns of total activity
added prior to extraction with dichloromethsne. All plasma samples were
adjusted for recoveries and the results were expressed as ng/ml or pg/ml
(sex steroids) or ng/lOO ml (corticosterone)plasma (Table 8).
TABLE:3
Recovery of radioactive steroids following extraction and chromatography
Steroid
Mean Recovery %
Range
Celite:propyleneglycol:ethyleneglycol column with water trap
Sa-di~~otestosterone
77.6 (33)
59.7-93.0
Testosterone
78.8 (30)
63.5-92.7
Estrone
68.5 (30)
52.2-92.0
Estradiol-176
74.4 (30)
62.0-91.0
Corticosterone
68.5 (33)
54.9-88.8
Celite:ethyleneglycol column
Estrone
69.7 (33)
50.4-93.2
Estradiol-17B
79.7 (26)
60.5-94.6
Numbers of replicates are shown in parentheses.
S
318
=EltlEOIDI
RESULTS
Method blank
Plasma samples (100-2000 ul rooster plasma) treated with dextrancoated charcoal were taken through the entire purification and assay
procedures. Percentage-binding values obtained were indistinguishable
from those of the distilled water blanks. Therefore, Table 4 represents
the combined results of both plasma and water blanks. Occasionally blanks
were encountered with % binding levels greater than 100.
In these cases,
such values were treated as 100%. As the method blanks for each assay
were so low and well off the upper part of the standard curves, they were
not considered an important source of error in the assay systems.
Sensitivity
The smallest smount of steroids detectable by each standard curve is
presented in Table 5.
These amounts were estimated by considering the
highest point on the standard curve whose standard deviation did not
overlap that of the method blank as the least detectable dose.
Intra- aa
inter-ass.5
variation
-----
Intra-assay variation was determined by taking a number of replicates
containing a known amount of steroid throughout the entire assay procedure and expressing results as pg of steroid measured (Table 6).
Inter-
assay variation was estimated from the repeated assay of a rooster plasma
pool (1 ng each of estrone and estradiol-17B were added to the plasma
pool for the estrogen assays) and results are presented in Table 7.
The
coefficients of variation are given in both Tables 6 and 7 and indicate
good precision in all the assay systems.
319
TABLE 4
Method blanks
Steroid
Amount of steroid
'apparent' from %
bound in blank
Range of
percentage
binding
Celite:propyleneglycol:ethyleneglycol column with water trap
Sa-dihydrotestosterone
'3 pg
Testosterone
'5 Pg
Estrone
'2 Pg
Estradiol-17B
'2 P&
Corticosterone
90.7-100
(20)
97.0-100
(20)
91.0-100
(20)
93.0-100
(20)
88.5-100
(20)
>lOO pg
Celite:ethyleneglycol column
Estrone
<2 pg
Estradiol-17B
<2 PQ
93.4-100
(24)
96.0~loo
(24)
Numbers of replicates are in parentheses.
Plasma steroid levels in 8 species of birds
Results for the passerifonn species are,presented in Table 8.
laying female Mellard, 92.8 pg/ml (2) estrone and 184.4
In
pg/ml (2)
estradiol-17gwere measured. In non-laying female P!allard,levels were
much lower (17.8
+ 4.0 and 21.8 + 7.6 pg/ml respectively). Almost equal
levels of dihydrotestosteroneand testosterone were found in the plasma
of female Aythya
affinis ssmpled in February/March,being 0.63 + 0.15
ana 0.54 + 0.14 r&ml respectively. In male A. affinis dihydrotestosterone (1.01 ng/ml, 2 samples) and testosterone (0.72
r&ml,
2 samples)
levels were slightly higher than in the female sampled at the ssme time.
S
320
TDEOXDI
TABLE 5
Sensitivity"
Steroid
Least detectable dose
Testosterone
5a-dihydrotestosterone
Estrone
Estradiol-176
Corticosterone
15.6
12.0
10.5
14.0
150.0
P&
pg
pg
pg
pg
aThe smallest amount of steroid on the standard curve distinguishable
from the method blank.
TABLE 6
Intra-assay variation'
Amount of
steroid
added
Steroid
Amount otC
steroid
measured
Coefficient
of variation
$
Celite:propyleneglycolzethylene glycol column with water trap
5a-dihydrotestosterone 1000
500 pg
Pe,
250 pg
Testosterone
1000 pg
500 PF:
250 PB
Estrone
1000 pg
500 pg
250 PA
Estradiol-17B
1000 pg
500 PR
250 m
Corticosterone
40 ng
20 nf:
10 ng
Estrone
Celite:ethylene glycol column
1000 pg
985 PFld
Estradiol-176
b
1095
523 pg
PF:
252 pg
1223 pg
553 PR
269 PR
1053 pg
533 PE
272 PQ
1139 PR
5.2’1 w
261 ~6;
36.2 ng
18.6 ng
9.8 np
1000 pg
906 ped
z*;
12:8
14.6
7.6
13.5
13.4
4.6
12.0
5.6
9.6
12.0
9.6
14.5
7.3
9.5
8.1
Steroids were added to distilled water or plasma (treated with charcoal) and t&en through the entire assay procedure.
'Mean of 12 replicates.
4 dean of 10 replicates.
TABLE 7
a
Inter-asssy variation
Coefficient of
variation ($1
Steroid
Amount
measured
5a-dihydrotestosterone
1076 p&ml
17.6
Testosterone
3844 pg/ml
15.7
Estroneb
1352 pg/ml
3.0
Estradiol-17Bb
1338 p&ml
9.3
Corticosterone
20.5 g/100 ml
12.6
gEstimates are based on means of 20 0.5 ml replicates.
b
Includes measurements from Celite:ethyleneglycol columns.
1 ng each of standard estrone and estradiol-17f3
were added to the
plasma pool.
DISCUSSION
The method blanks presented in Table 4 were determined on rooster
plasma and distilled water. Plasma blanks were also measured for Mallard
and White-crowned Sparrow (after dextran-coated charcoal treatment). In
both cases blanks were well within the percentage-bound ranges given in
Table 4.
No plasma blanks were determined for the other five species
listed in Table 8 and thus the plasma steroid levels given could be subject to error. However, their steroid levels compared so well with the
other three species that method blanks were not considered. Over-all
the method blanks in this study compare well with those found by others
using Celite column chromatography and radioimmunoassay for measurement
of steroids in human plasma (3,4,12,13).
Abrahsm (3) has described the elution pattern of many steroids from
Celite columns, and of the steroids likely to be encountered in the
eluate fractions collected (Table 2) none gave significant cross-reaction
in the assay systems of the steroids to be measured.
--
ng/ml(14)
---
d = 0.18 + 0.01
ng/ml(19) + 0.06
0 = 0.26
---
---
?a-DHT
1.75 ngfml
+ 0.02
(13)
0.4 ng/ml
(6)
_.____________.__--_-l_l__-
E1
__-~I____
____
---
ng/ml-_~__EL.______---
----.--
-__-~
\,I
.____
-
.___
----_--
202.7p&$il.--+ 123.2
- (5)
‘2’-_-_-2Ti.T pg /ml
2 39.7
(71
- (i3)
<5o--p-gKl--__---~-- (6)
--d = 0.39 j-_ 0.05
ng/ml
(19) + 0.08
0 = 0.46
---
8.03 ng/ml
+ 0.13
(13)
~----~0.55 w/ml
--+ 0.09
- (6)
-----.-------yF&pg77nl
T
B
(7)
I__
_____~_
ug/lO?iml
(14)
l_______--____--75_lglml
5
~~~~__.______-_____~
1.14 ug/lOO ml
+ 0.24
(13)
__0.82 u&100 ml
+ 0.06
(6)
408.5 pg/ml
0.68 r.g/lOOml
f 154.4
+ 0.12
(13)
(13)
CO.5 pg/lOO ml <50 pglml
(6) _---(6)
--d = 0.55 + 0.09 ug/lO?iml
F = 0.81(19)
+ 0.25
B2
--_
“ulumber of samples assayed in duplicate are indicated in parentheses.
b5a-DHT = 5wdihydrotestosterone; T = testosterone; El = estrone; B2 = estradiol-178; B = corticosterone.
_______
_Carpodacus nemk&iT$ April
____________________--ir?otothrus ater
--5 April
August
i. querda
d December
? December
k.$t. pugetensis
1 April
z. 1. pugetenszs
Species
Zonotrichia
leucophrys
pugetensis
d April
i. Z. pugetensis
d"August
TABLE 8
Plasma steroid levels of several passerine avian species f.SE meansa,b
tl
I
n
0
4
u
g
____
----
w/ml(19)
9 = 0.26 + 0.06
n&ml(14)
___
__-
d = 0.39 + 0.05
n&ml(19)
9 = 0.46 + 0.08
w/ml(12)
246.4 pglml
+ 39.7
(7)
282.7 p&ml
+ 123.2
(5)
---
<50JP1
242.5 p&ml
+ 45.6
- (13)
___
___
E1
(7)
<50 pe/ml
(5)
<50 pg/ml
---
<50 p&ml
(6)
408.5 p&ml
+ 154.4
(13)
_--
---
=2
--
d = 0.55 + 0.09
)lg/lOSml
(19)
9 = 0.81 + 0.25
vg/lO?Ym.l
(14)
eo.5 pg/lOO ml
(6)
0.60 )rg/~ooml
+ 0.12
- (13)
0.02 pg/lOO ml
+ 0.00
- (6)
1.14 )rg/lOOml
+ 0.24
(13)
B
(kb
m ers of samples assayed in duplicate are indicated in parentheses.
b 5a-DHT = 5edihydrotestosterone; T = testosterone; El = estrone; E2 = estradiol-176;B = corticosterone.
Molcthm8 ater
9 April
9 April
mezicanu8
quenJ5
d December
9 December
Ccupodacu8
z.
d = 0.10 + 0.01
___
____
9 August
z. 1.
pugeten8i8
_-
____
2. 1. plrgetensicr
9 April
2.
0.55 w/ml
+ 0.09
- (6)
8.03 &ml
i 0.13
(13)
P
0.4 n&n1
(6)
1.75 w/ml
+ 0.02
(13)
5a-DHT
1. plg63t8n8is
d August
jnq&l?l8is
d April
Zcnctrichia
teucophrys
Species
TABLE 8
plasma steroid levels of several passerine aviao species f SE meansa,b
S
TDEOXDI
Intra- and inter-assay variation (Tables 6 and 7) show adequate
precision and coupled with high recoveries and sensitive standard curves
(Table 5) indicate the practical uses of this system for small volumes
of avian plasma. One possible exception is the assay of estrone and
estradiol-178 in the plasma of laying female !bIallards.Rere considerable
quantities of lipid were encountered resulting in a reduction of recoveries to 40-50s.
Plasma blanks in these samples were within the range
of percent bound given in Table 4 and results for laying Mallard plasma
are considered as being good estima.tesof estrone and estradiol-176.
Table 6 does show some overestimations, especially for 1000 pg of testosterone. This is attributed to the high dose of steroid assayed which
gave a percentage binding on the lower part of the standard curve where
inaccurate results could be obtained.
Of further significance is the comparison of estrone and estradiol178 levels measured in eluates from the Celite:propyleneglycol:ethylene
glycol and Celitezethylene glycol columns (Table 7).
Coefficients of
variation were less than 105 suggestinfrthat measurements from both columns are directly comparable.
Plasma testosterone levels measured in mature rooster (Table 7) and
male Aythya affinis (1.11 n&/ml 5a-dihydrotestosteroneand 0.72 nr/ml
testosterone--2 samples) compare well with the range of values (0.1-2-7
ng/nl) found in the Pekin duck (14), Red-winged Rlackbird (Lpelaitls
@enicezds)
(15), and roosters (16),
but higher than in the pigeon
(CoZwnba Zivia) and Coturnix quail (14). Much higher levels were observed in male 2. 1. pugetensis in April (Table b), but these levels
had fallen considerably by August (8-0.5 ng/ml). It is of interest to
note the considerable smounts of testosterone in female Aptizyaaffinis
(0.63 +
0.15 ng/ml 5a-dihydroteetosterone
and 0.54 + 0.14 testosterone
_-4 ssmples)snd Z. quen.du
(Table
8) sampledin the wintermonths,com-
parableto those found in the layinghen (0.13 - 1.10 W/ml) (17). Testosteronewas not detected(CO.4ng/ml)in the plasmaof femaleZ. 1. pugs&@teia ssmpledfn the summermonths.
In all the speciestested,5a-di~drotestosterone
levelswere generally lower than those of testosterone.Cautionmust be exercisedin the
interpretation
of these data 8s 5a-dibydrotestosterone
has not, to our
knowledge,been definitelyidentifiedin avisn plasma, It has been shown,
however,that the coccygealgland,epididymisand comb of the domestic
fowl haw
the capacityto converttestosterone
to 5a-dihydrotestosterone
(18,19). Thereforeit is possiblethat 5a-dihydrotestosterone
does occur
in avian plasma.
The levelsof estroneand estradiol-178
levels found in lsyingand
non-layingfemaleMallard (17.8+ 92.8 p&ml estroneand 21.8 + 184.4
pg/ml estradioLl7B)were well within the range found for the same steroids in femaledomesticfowl (20,21)and femalering doves (22). Plasma
estroneand estradiol-176
in female2. 1. pu@&?zsis sampledin April
(Table8) were much higherthan those values found in chickensand ring
doves. A similarresult for eatronewas obtafnedwith samplesfrom C.
mexba.?zus
and M. ater taken in April,but estradiol-178
levelswere not
detectable(~50 pg/ml) (Table8). In males and females(sampledin winter) estrogenlevelswere not detectedin the volumesof plasma used
(Cl ml). In futurestudiesit is proposedto pool plasma ssmplesfrcm
several,
individualsto facilitatedetectionof such low concentrations.
Cortieosterone
levelsof Z. 2. puget&ds
(bothmales and females)
sampledin the summermonths appearedslightlylower than those found
S
326
Y#.-BEOXDI
in the White-throated Sparrow (2. ai%icoZlis) (23).
In Z. querula plasma
corticosterone levels of samples taken in December (Table 8) were also
similar (0.1-1.7 ug/lGO ml).
There were no differences between males
and females.
A more intensive study of plasma steroid levels in relation to
season and reproduction is now in progress with Mallard and crowned
sparrows (~!notrichia species), and a more detailed analysis of results
will be published later.
AC~OW~DG~NTS
We are most grateful to Doctor C.W. Dane, Doctor S.A. Rohwer, and
Mr. R.S. Donhsm for samples of plasma from the Lesser Scaup, Harris'
Sparrow and Mallard, respectively. This investigation was supported by
Grant No. R01 HD 06527 from the National Institute of Health.
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