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Radioimmunoassay of estrone sulfate in the serum of normal men after a chromatographic procedure that eliminates dehydroepiandrosterone sulfate interference
RADIOIMMUNOASSAY OF ESTRONE SULFATE IN THE SERUM OF NORMAL MEN AFTER A CHROMATOGRAPHIC PROCEDURE THAT ELIMINATES DEHYDROEPIANDROSTERONE SULFATE INTERFERENCE Joel L. Brinda. b. c, Klavida Norman Orentreicha
Chervinskya,
Joseph
H. Voglemana,
and
aOrentreich Foundation for the Advancement of Science, Inc.. New York, NY 10021; bDepartment of Natural Sciences. Baruch College, CUNY, New York, NY 10010; ‘Department of Medicine, Division of Endocrinology and Metabolism, Beth Israel Medical Center, New York, NY 10003; USA Correspondmg author Joel L Brand, CUNY,
17
Received Rewed
Lexington May
June
Avenue,
New
Department of Natural Sciences, Baruch York,
NY
10010,
College
USA
25.1988 10.
1989
ABSTRACT Fifty fresh-frozen normal male sera containing tritiated estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were extracted with ethanol after ether Washed extracts extraction of unconjugated steroids. were defatted and chromatographed on polyamide-coated plates by reversed phase paired ion TLC. Plates were scanned for radioactivity, and ES peaks were cut, eluted and assayed by direct RIA with a commercially available antiserum. Mean ES values were 445 +/- 209 pg/mL (SD), in agreement with the three lowest of the seven laboratories which had previously reported normal male ES values. No differences were observed in ES values when samples were rechromatographed prior to assay, or when up to 4 pg/mL unlabeled DS was added to serum before extraction. These data confirm the absence of interference by DS in the current study and suggest that previously reported high (716-1194 pg/mL) mean normal male ES values reflect DS interference. The present study also demonstrates the the stability of ES in sera stored frozen at -40 C for an average of 17 years (mean: 406 +/-258 pg/mL; [SD]; n=41).
STEROIDS
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21
22
Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
INTHDDUCTION Since 1971 at least
seven different methods for
measuring estrone sulfate (ES) in normal male serum or plasma have appeared in the literature (l-7).The methods differ substantially and so do the reported normal values. Whereas, for example, Myking et al (1)reported the lowest mean value (282 +/- 106 pg/mL; n=53), Towobola et al (2) reported a more than quadruple mean value (1194 +/-228 pg/mL; n=13). A careful review of previous investigators' methods suggested to us that the wide range of normal male ES values obtained was due to a difficulty which was generally acknowledged but inadequately treated, namely, interference by cross-reacting dehydroepiandrosterone sulfate (DS). For example, Towobola et al (2) pointed out that an antiserum with a cross-reaction even as low as l-2% would be unacceptable, considering the 1,000-fold excess of DS over ES in plasma.
However, if the known
mean normal young male values of plasma DS (8) are compared with the lowest reported normal values of ES (l), the estimate of DS excess is found to be about lO,OOOfold. Thus, even a very slight cross-reaction of the order of 0.01% would be unacceptably high, and none of the previous studies (where antiserum cross-reactivity was
STEROIDS
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July 1989
Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
reported at all) demonstrated
MEN
23
a DS cross-reactivity
value below 0.01%. Moreover, chromatographic separation methods, where employed, could not be counted on to remove all significant traces of cross-reacting DS.
For example,
Sephadex LH-20 purification of estrone from a solvolyzed (3) or enzyme-hydrolyzed (1)extract does not separate unconjugated dehydroepiandrosterone (3). Celite chromatography
(DHEA) from estrone
of estrone from a solvolyzed
(4) or enzyme-hydrolyzed (5) extract is better, but DHEA and estrone elute in adjacent fractions from Celite (9), and significant cross-contamination would be expected. Finally, although Hawkins and Oakey (6) assayed radio-receptor assay
ES by a
in which DHEA (or DS) does not cross-
react (lo),their final step before assay requires the borohydride reduction of estrone to estradiol
This
procedure would convert any DHEA present to androst-5-ene3B,17B-dial, which is weakly estrogenic and gives a 1% cross-reaction in the radio-receptor assay they used (11). Since none of the methods thus far reported for ES in men has been conclusively shown specific enough to rule out interference by DS, it seems possible that at least the higher reported normal values (2, 5-7) are artificiallyhigh because of such interference.
However,
since the three groups reporting the lowest normal ES
STEROIDS
54/l
July 1989
24
Brind et al: RIA OF ESTRONE
values
in men
suhstantial
are
in reasonably
methodological
a reasonable
hypothesis
concentration
would
pg/mL, which they In this paper
good
the true
be in the range
had
we report
for obtaining
serum.
The
method
is designed
extract
which is demonstrably
specificity. method
assayed
report
to the estimation
been
freshly
have
been
in long-term
MATERIALS
AND
despite
mean
normal
of about
male ES
300-400
ES from
simple
human
male
to yield a purified ES free
of significant DS con-
by an ES RIA of appropriately
We further
have
MEN
(1,7,8),it seems
a new, relatively
scheme
when
IN NORMAL
found
purification
tamination
agreement
differences that
SULFATE
on the application
of ES in normal
obtained
and
frozen
of this
male sera
frozen, and
high
which
in those
which
storage
METHODS
Frozen serum specimens from normal men were taken from a collection that has been gathered since lY64 as part of a multiphasic health screening program by the (Kaiser) Permanente Medical Group (Oakland, CA). The 41 long term frozen specimens used had been collected between 1966 and 1971 and kept frozen in screw-cap glass viaIs [under air) at -40 C. The mean age of the specimens at the time of assay was 17.1 +/-lo years, and the mean age of the 41 subjects they represented was 49.5 +/-15.3 years at the time of blood collection. The fifty freshfrozen specimens were assayed within 5 months of their collection, and they represented 50 subjects aged 46.7 +/- 13.6 years. AlI specimens had been frozen immediately after collection and were maintained at -20 C until assay.
STEROIDS
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Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
25
Diethyl ether (Nanograde) was obtained from Mallinkrodt (Paris, KY), anhydrous ethanol (95% ethanol, 2.5% methanol, 2.5% isopropanol), methanol, ethyl acetate, chloroform and acetonitrile were all HPLC grade. Concentrated ammonium hydroxide and triethylamine (TEA) were reagent grade and were obtained from Fisher (Fairlawn, NJ). The tritium-labelled steroid sulfates [7-3H]-DS (SA=23 Ci/mmol) and [6,7-3HJ-ES (SA=40 Ci/mmol) were obtained from Dupont-NEN (Boston, MA), and were repurified before use by the TLC method of Longcope (12)on Analtech (Newark, DE) silica gel G plates. Unlabeled ES (sodium salt) was obtained from Sigma (St. Louis, M0) and unlabeled DS (sodium salt) was obtained from Steraloids (Wilton,NH).
Ten microliters each of an aqueous stock solution of [3H]-ES (4,500 dpm) and [3H]-DS (4,500 dpm) were added to clean 12 x 75 mm glass test tubes and allowed to air dry. To each tube was added 0.5 mL of serum (each specimen in duplicate). The tubes were mixed gently and allowed to equilibrate overnight at 4 C. After the addition of 0.5 mL water, each tube was extracted twice with 3 mL diethyl ether to remove neutral lipids and unconjugated steroids. To the remaining aqueous phase was added 3 mL ethanol. The cloudy mixture was centrifuged 5 min at 2,500 rpm and the supernatant extract decanted into a clean tube. The pellet was washed once with 1 mL ethanol and the ethanol extracts combined. The pooled ethanol extract was dried overnight at 45 C in a water bath and reconstituted in 2 mL ethyl acetate. Fifty microliters of 0.1 M aqueous TEA was then added, and each tube vortexed about 5 set and centrifuged as above. The ethyl acetate was decanted and the aqueous phase washed with 2 mL fresh ethyl acetate. The ethyl acetate extracts were pooled and dried under nitrogen at 45 C or overnight in air at room temperature. The dried serum extracts were loaded onto 15 x 15 cm Micropolyamide TLC plates (Schleicher & Schuell, Keene, NH) with 10 PL glass micropipets, using small volumes of chloroform. The chromatographic system is described in detail elsewhere (1.3).Briefly, the samples were defatted by a normal phase development in ethyl acetate, and ES and DS separated by a reverse phase development in
STEROIDS
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26
Brindetal:RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
20% acetonitrile in 0.05 M aqueous TEA. The plates were scanned for radioactivity for 25 min on a Berthold (Nashua, NH) linear analyzer. ES peak areas were cut out with scissors and eluted by immersion in 2 mL of ethanol/l M aqueous ammonium hydroxide (9:l)for 2 min at room temperature with initial gentle shaking Eluates were dried under nitrogen at 45 C or overnight in air at room temperature and reconstituted in 0.5 mL RIA buffer. Aliquots of 70 PL were taken to assess sample recovery by counting for 10 min in a Packard (Downers Grove, IL) Tri-Carb model B2450 liquid scintillationcounter following the addition of 5 mL Budget-Solve HFP universal scintillant (Research Products International, Mount Prospect, IL).
Four hundred microliters of each reconstituted sample were taken for RIA according to the method recommended by the vendor of the antiserum (Radioassay Systems Laboratories, Carson, CA). The antiserum had been raised in ewes v. estrone-3-carboxymethyl ether-BSA conjugate. It was diluted l/20,000 in assay buffer and 100 ~Lof diluted antiserum was added to each sample aliquot. Tritiated ES tracer (12,500 dpm/lO(l PL assay buffer) was added to each tube to bring the total radioactivity per tube to 12,500 dpm. This was done by subtracting the mean recovered radioactivity for all the samples in the assay from the tracer aliquot. Sample volumes were brought up to 700 pl_with assay buffer and tubes were incubated overnight at 4 C. Bound radioactivity was separated from free by dextran-coated charcoal_ ES Values were obtained from a standard curve of authentic ES in the range of lo-1,000 pg per tube, and zero binding was always 60-70%. Along with every ten patient samples were run purified extracts of charcoalstripped, normal male plasma containing 0, 200, and 400 pg/mL of added, authentic ES, and an aliquot of a single normal male serum specimen RESULTS
Recovery
of added tritiated ES and DS tracers from
serum was invariably greater than 95% through the ethanol extraction step, as measured
by liquid scintillation
STEROIDS
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Brind et al: RIA OF ESTRONE
counting.
SULFATE
IN NORMAL
However, ZO-30%
losses
MEN
27
were
routinely
incurred
during the TEA wash step, due to the high affinity of the relatively polar steroid sulfates for the aqueous Nevertheless, this step provided a necessary
phase.
tenfold
purification, reducing the dry residue in the ethanol extract of 0.5 mL serum from 10.0 +\- 1.7 mg to 0.91 +/- 0.35 mg (SD; n=4). Substantial (lo-20%) losses were also routinely incurred during loading of washed extracts onto the TLC plates; however, elution from the TLC plates was invariably quantitative (>99%). Thus, net post-elution recoveries of ES averaged 56.7 +/- 7.7% (SD; n=220). Figure 1 shows a scan of a typical chromatogram of a
ES
I/
Figure 1. Typical thin-layer chromatogram of a washed ethanol extract of a normal male serum labeled with 4,500 dpm each of tritiated estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS). "SF" indicates solvent front.
STEROIDS
5411
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28
Brind ?t al: RIA OF ESTRONE
serum extract.
SULFATE
IN NORMAL
MEN
Peaks of ES and DS were well resolved from
each other and easily distinguished from background in a 25-30 min scan
the linear analyzer also permits
quantitation without elution; however, the low count level resulted in only a rough correlation with liquid scintillation data (r=U.79 and r=O.SO in two separate experiments with a combined total of 37 extracts]. The apparently lower recovery of tritiated IX (Fig. 1) was a consistent finding and was due to degradation of the DS tracer during sample processing.
The commercially obtained ES antiserum is listed by the supplier as giving a 100% cross-reaction with both unconjugated estrone and estradiol, which are both low in males and removed
by ether extraction.
No other cross-
reactions are significant in male serum except for that of DS, which is listed as 0.05%. In our hands, this antiserum gave a cross-reaction which is plotted along with the ES standard curve in Figure 2.
The cross-reaction curve is
relatively shallow, giving a variable percent crossreaction ranging from 0.01% at B/Bo=.36 to U.U8% at B/Bo=.80, and with a value of 0.02% at B/Bo=.50.
As
was discussed earlier, even this slight cross-reactivity looms unacceptably large in view of the 5,000- lO,OOO-
STEROIDS
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bind
et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
29
fold excess of DS over ES in male serum; hence, chmmatography prior to RIA is needed. 501 0
40-
5
30-
iz !i s 5 a
zo-
lo-
I
I
I
1
10
30
100
PS ESTRONE
SULFATE
(@) OR
“g
DEHYDROEPIANDROSTERONE
t
1 1000
300 SULFATE
(0)
Figure 2. Typical standard curve of estrone sulfate (ES) RIA and curve showing the cross-reaction of dehydroepiandrosterone sulfate (DS) with the same antiserum.
A typical standard curve is shown in Figure 2. The working range of the assay was 10-1,000 pg, with a 511% B/Be at 41 pg.
(AII ES values given in this paper,
including those cited from other reports, are in units of pg or pg/ml of the monosodium salt of ES.) A value of 10 pg was always significantly different from zero.
Values
obtained from a specimen of charcoal-stripped, normal male
STEROIDS
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bind
30
plasma averaged
et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
5-8 pg/mL. The intra-assay variability was
found to be 12.0% (CV) for 23 measurements
of a normal
male serum specimen (mean value: 396 +/- 48 pg/mL; SD). The interassay CV for ten determinations of the same specimen in each of two assays
was 17.9% (mean value:
346 +/- 79 pg/mL; SD). The mean recovery of 200 pg/ml unlabeled ES added to this specimen (determined in duplicate in each of two assays) was 179 +/-30 pg/mL (SD). Data on recovery of added unlabeled ES to aliquots of charcoal-stripped plasma are shown in Table 1.
TABLE 1. Recovery of added estrone sulfate to charcoal-stripped plasma
x/mL
Pg/mL
200 400 600
242 470 661
aMean of 10 measurements
22 45 117
9.0 9.6 17.7
in a single assay.
As a further check on the specificity of our assay system it was necessary
to verify that the TLC procedure
removed sufficient DS to render the cross-reaction of the antiserum with DS insignificant. While the chromatograms appeared to show complete resolution of ES and DS (Fig. l),it should be noted that even a 99% removal of DS from the ES extract would still leave the ES region
STEROIDS
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Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
31
MEN
Therefore, two
with a 50- to loo-fold excess of DS. additional experiments were performed.
In the first,
duplicate aliquots of male serum specimens (from another clinical study) were processed such that one of each duplicate was assayed
for ES as described above, and the
other aliquot was taken through the TLC elution step and rechromatographed
in the same TLC system
All samples were measured
before RIA.
in the same assay, and a good
correlation was obtained between the two procedures, as shown in Figure 3.
I
In the second experiment, unlabeled DS
I
100 ESTRONE
I
200 SULFATE
I
300 RIA (pg/mLl
L
400 AFTER SINGLE
1 500 TLC
Fig. 3. Comparison of male serum estrone sulfate (ES) values obtained by specific RIA following a double v. single TLC step to remove contaminating dehydroepiandrosterone sulfate (DS). The line shown is the calculated regression line.
STEROIDS
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Brind et al. RIA OF ESTRONE
32
at 2 Pg/mL specimen
and 4 pg/mL
known
was
to contain
by a standard
DS produced
value
of ES (320 pg/mL).
to a normal
2 pg/mL
endogenous
(8). Neither
RIA method
added
added
a detectable
SULFATE
DS, assayed
concentration
change
sera
was
mean
445
C/-209
in 41 long-term years).
The
the fresh
obtained
pg/mL
frozen
two
statistically There
value
sera
406
Patient
of normal
+/-258
A%
frozen
pg/mL
(SD)
age: 17.1 +/-1.0
(mean serum
populations
of
in the measured
for ES in 50 fresh
(SD), and
MEN
male serum
ge and The
IN NORMAL
male sera
were
indistinguishable. was
a sufficient
frozen
shown
in Table
among
the three
serum
2.
There
age
patient
values were
groups,
age
into three
range age
to divide groups,
as
no significant differences in serum
ES values
or their
variability.
Table values Age
2. Comparison of serum estrone m no&men of dlfkent apes
range (yr)
Serum ES (np/rnL)
24-39 40-59 60-70
423 458 470
SD 219 189 201
sulfate
No. of suhlects 17 18 15
DISCUSSI0N The
normal
male
serum
ES values
we have
obtained
STEROIDS
54/l
in
July 1989
Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
33
MEN
the present study confirm the relatively low values reported by three other laboratories (1,3,4), and strengthen the suggestion that those laboratories which have reported much higher values (2,5-7) did not take adequate account of the enormous fold) of DS in serum.
excess (5,000- to lO,OOO-
The high variability approximately
50% CV) we obtained in both fresh and frozen serum is comparable to that reported by others (1,4,7). Of the other laboratories which have reported normal male ES values, only Myking et al (1)and Franz et al (4) reported specifically on age differences. The former group reported a modestly but significantly lower mean value (258 pg/mL) for 26 men aged 50-87 years (mean=70) as opposed to 27 younger men (305 (mean=31).
pg/mL) aged 20-49 years
However, this correlation might be
attributable to interference by DS, which decreases
by
about 80% in men between age 31 and age 70 (8). Thus, DS interference to the extent of 20% in the younger age group would correspond to only a 4% interference in the older group, thereby accounting entirely for the observed decrease in ES.
Although Franz et al(4) reported an
inverse correlation between ES (but not DS) and age in normal men, their population was small (n=19) and the age range of the subjects was very narrow (21-38 years).
STEROIDS
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34
kind
et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
Both Franz et al(4) and Remy-Martin et al(5) reported sharply decreased
ES values in cirrhotic men (29% and 44X,
respectively), but age was also a variable. Thus, half the 74% drop in serum DS reported by Franz et al would be expected on the basis of the age difference alone (8),and this could partially account for the difference observed in serum ES, were DS interference in the ES assay significant
Likewise, the 44% drop in serum ES in cirrhotics
(mean age=51 years) v. normal subjects (mean age=36 years) reported by Remy-Martin et al(5) is about the magnitude of the expected drop in serum DS (which was not measured) between those two age groups in normal men (8). It therefore appears likely that, as the present study shows, serum ES concentration is not age-dependent
in normal men.
The lack of effect of long-term frozen storage on the measurement
of serum ES is consistent with the stability
we have previously reported for DS in similarly aged frozen sera (8). The long-term stability of ES contrasts with the instability of unconjugated estrogens stored under similar conditions, as observed
by us (unpublished
data) and others (14). REFERENCES 1. Myking 0, Thorsen T, and Stoa KF (1980) Conjugated and unconjugated plasma aestrogens--oestrone, oestradiol and oestriol--in normal human males. J STEROID BIOCHEM =1215-1220.
STEROIDS
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Brindetal:RIAOFESTRONESULFATE INNORMAL
2.
MEN
35
Towobola OA, CriLly RC, and Oakey RE (1980) Oestrone sulphate in plasma from postmenopausal women and the effects of oestrogen and androgen therapy. CLIN ENDOCRINOL u461-471. 3. Loriaux DL, Ruder HJ, and Lipsett MB (1971).The measurement of estrone sulfate in plasma. STEROIDS 18: 463-472. 4. Franz C, Watson D, and Longcope C (1979).Estrone sulfate and dehydroepiandrosterone sulfate concentrations in normal subjects and men with cirrhosis. STEROIDS =563-572. 5. Remy-Martin A, Prost 0, Nicollier M, Burnod J, and Adessi GL (1983) Estrone sulfate concentrations in plasma of normal individuals, postmenopausal women with breast cancer, and men with cirrhosis. CLIN CHEM =86-89. 6. Hawkins RA and Oakey RE (1974) Estimation of oestrone sulfate, oestradiol-17B and oestrone in peripheral plasma: concentrations during the menstrual cycle and in men. J ENDOCRINOL Lin_:3-17. 7. Wright K, Collins DC, Musey PI, and Preedy JRK (1978). A specific radioimmunoassay for estrone sulfate in plasma and urine without hydrolysis. J CLIN ENDOCRINOL METAB 4;L:1092-1098. 8. Orentreich N, Brind JL, Rizer RL and Vogelman JH (1984). Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations through=551-555. out adulthood. J CLIN ENDOCRINOL . METAB . 9. Abraham GE (1977).-ok of Radlolmmunoassav. Vol 5, Marcel Dekker, New York, p 591. 10. Korenman SG, Perrin LE, and McCallum TP (1969) A radio-ligand binding assay system for estradiol measurement in human plasma. J CLIN ENDOCRINOL METAB 29:879-883. 11. Korenman SG (1969) Comparative binding affinity of estrogens and its relation to estrogenic potency. STEROIDS Bl63-177. 12. Longcope C (1972) The metabolism of estrone sulfate in normal males. J CLIN ENDOCRINOL METAB =113-122. 13. Brind JL, Kuo S-W, Chervinsb K, and Orentreich N (1988) A new reversed phase paired ion thin-layer chromatographic method for steroid sulfate separations. STEROIDS, %: 561-570. 14. Phillips GB, Yano K, and Stemmermann GN (1984) Decrease in serum estradiol values with'storage. N ENGL J MED m1635.
STEROIDS
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Chervinskya,
Joseph
H. Voglemana,
and
aOrentreich Foundation for the Advancement of Science, Inc.. New York, NY 10021; bDepartment of Natural Sciences. Baruch College, CUNY, New York, NY 10010; ‘Department of Medicine, Division of Endocrinology and Metabolism, Beth Israel Medical Center, New York, NY 10003; USA Correspondmg author Joel L Brand, CUNY,
17
Received Rewed
Lexington May
June
Avenue,
New
Department of Natural Sciences, Baruch York,
NY
10010,
College
USA
25.1988 10.
1989
ABSTRACT Fifty fresh-frozen normal male sera containing tritiated estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were extracted with ethanol after ether Washed extracts extraction of unconjugated steroids. were defatted and chromatographed on polyamide-coated plates by reversed phase paired ion TLC. Plates were scanned for radioactivity, and ES peaks were cut, eluted and assayed by direct RIA with a commercially available antiserum. Mean ES values were 445 +/- 209 pg/mL (SD), in agreement with the three lowest of the seven laboratories which had previously reported normal male ES values. No differences were observed in ES values when samples were rechromatographed prior to assay, or when up to 4 pg/mL unlabeled DS was added to serum before extraction. These data confirm the absence of interference by DS in the current study and suggest that previously reported high (716-1194 pg/mL) mean normal male ES values reflect DS interference. The present study also demonstrates the the stability of ES in sera stored frozen at -40 C for an average of 17 years (mean: 406 +/-258 pg/mL; [SD]; n=41).
STEROIDS
54/I
July1989(21-35)
21
22
Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
INTHDDUCTION Since 1971 at least
seven different methods for
measuring estrone sulfate (ES) in normal male serum or plasma have appeared in the literature (l-7).The methods differ substantially and so do the reported normal values. Whereas, for example, Myking et al (1)reported the lowest mean value (282 +/- 106 pg/mL; n=53), Towobola et al (2) reported a more than quadruple mean value (1194 +/-228 pg/mL; n=13). A careful review of previous investigators' methods suggested to us that the wide range of normal male ES values obtained was due to a difficulty which was generally acknowledged but inadequately treated, namely, interference by cross-reacting dehydroepiandrosterone sulfate (DS). For example, Towobola et al (2) pointed out that an antiserum with a cross-reaction even as low as l-2% would be unacceptable, considering the 1,000-fold excess of DS over ES in plasma.
However, if the known
mean normal young male values of plasma DS (8) are compared with the lowest reported normal values of ES (l), the estimate of DS excess is found to be about lO,OOOfold. Thus, even a very slight cross-reaction of the order of 0.01% would be unacceptably high, and none of the previous studies (where antiserum cross-reactivity was
STEROIDS
54/l
July 1989
Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
reported at all) demonstrated
MEN
23
a DS cross-reactivity
value below 0.01%. Moreover, chromatographic separation methods, where employed, could not be counted on to remove all significant traces of cross-reacting DS.
For example,
Sephadex LH-20 purification of estrone from a solvolyzed (3) or enzyme-hydrolyzed (1)extract does not separate unconjugated dehydroepiandrosterone (3). Celite chromatography
(DHEA) from estrone
of estrone from a solvolyzed
(4) or enzyme-hydrolyzed (5) extract is better, but DHEA and estrone elute in adjacent fractions from Celite (9), and significant cross-contamination would be expected. Finally, although Hawkins and Oakey (6) assayed radio-receptor assay
ES by a
in which DHEA (or DS) does not cross-
react (lo),their final step before assay requires the borohydride reduction of estrone to estradiol
This
procedure would convert any DHEA present to androst-5-ene3B,17B-dial, which is weakly estrogenic and gives a 1% cross-reaction in the radio-receptor assay they used (11). Since none of the methods thus far reported for ES in men has been conclusively shown specific enough to rule out interference by DS, it seems possible that at least the higher reported normal values (2, 5-7) are artificiallyhigh because of such interference.
However,
since the three groups reporting the lowest normal ES
STEROIDS
54/l
July 1989
24
Brind et al: RIA OF ESTRONE
values
in men
suhstantial
are
in reasonably
methodological
a reasonable
hypothesis
concentration
would
pg/mL, which they In this paper
good
the true
be in the range
had
we report
for obtaining
serum.
The
method
is designed
extract
which is demonstrably
specificity. method
assayed
report
to the estimation
been
freshly
have
been
in long-term
MATERIALS
AND
despite
mean
normal
of about
male ES
300-400
ES from
simple
human
male
to yield a purified ES free
of significant DS con-
by an ES RIA of appropriately
We further
have
MEN
(1,7,8),it seems
a new, relatively
scheme
when
IN NORMAL
found
purification
tamination
agreement
differences that
SULFATE
on the application
of ES in normal
obtained
and
frozen
of this
male sera
frozen, and
high
which
in those
which
storage
METHODS
Frozen serum specimens from normal men were taken from a collection that has been gathered since lY64 as part of a multiphasic health screening program by the (Kaiser) Permanente Medical Group (Oakland, CA). The 41 long term frozen specimens used had been collected between 1966 and 1971 and kept frozen in screw-cap glass viaIs [under air) at -40 C. The mean age of the specimens at the time of assay was 17.1 +/-lo years, and the mean age of the 41 subjects they represented was 49.5 +/-15.3 years at the time of blood collection. The fifty freshfrozen specimens were assayed within 5 months of their collection, and they represented 50 subjects aged 46.7 +/- 13.6 years. AlI specimens had been frozen immediately after collection and were maintained at -20 C until assay.
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SULFATE
IN NORMAL
MEN
25
Diethyl ether (Nanograde) was obtained from Mallinkrodt (Paris, KY), anhydrous ethanol (95% ethanol, 2.5% methanol, 2.5% isopropanol), methanol, ethyl acetate, chloroform and acetonitrile were all HPLC grade. Concentrated ammonium hydroxide and triethylamine (TEA) were reagent grade and were obtained from Fisher (Fairlawn, NJ). The tritium-labelled steroid sulfates [7-3H]-DS (SA=23 Ci/mmol) and [6,7-3HJ-ES (SA=40 Ci/mmol) were obtained from Dupont-NEN (Boston, MA), and were repurified before use by the TLC method of Longcope (12)on Analtech (Newark, DE) silica gel G plates. Unlabeled ES (sodium salt) was obtained from Sigma (St. Louis, M0) and unlabeled DS (sodium salt) was obtained from Steraloids (Wilton,NH).
Ten microliters each of an aqueous stock solution of [3H]-ES (4,500 dpm) and [3H]-DS (4,500 dpm) were added to clean 12 x 75 mm glass test tubes and allowed to air dry. To each tube was added 0.5 mL of serum (each specimen in duplicate). The tubes were mixed gently and allowed to equilibrate overnight at 4 C. After the addition of 0.5 mL water, each tube was extracted twice with 3 mL diethyl ether to remove neutral lipids and unconjugated steroids. To the remaining aqueous phase was added 3 mL ethanol. The cloudy mixture was centrifuged 5 min at 2,500 rpm and the supernatant extract decanted into a clean tube. The pellet was washed once with 1 mL ethanol and the ethanol extracts combined. The pooled ethanol extract was dried overnight at 45 C in a water bath and reconstituted in 2 mL ethyl acetate. Fifty microliters of 0.1 M aqueous TEA was then added, and each tube vortexed about 5 set and centrifuged as above. The ethyl acetate was decanted and the aqueous phase washed with 2 mL fresh ethyl acetate. The ethyl acetate extracts were pooled and dried under nitrogen at 45 C or overnight in air at room temperature. The dried serum extracts were loaded onto 15 x 15 cm Micropolyamide TLC plates (Schleicher & Schuell, Keene, NH) with 10 PL glass micropipets, using small volumes of chloroform. The chromatographic system is described in detail elsewhere (1.3).Briefly, the samples were defatted by a normal phase development in ethyl acetate, and ES and DS separated by a reverse phase development in
STEROIDS
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Brindetal:RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
20% acetonitrile in 0.05 M aqueous TEA. The plates were scanned for radioactivity for 25 min on a Berthold (Nashua, NH) linear analyzer. ES peak areas were cut out with scissors and eluted by immersion in 2 mL of ethanol/l M aqueous ammonium hydroxide (9:l)for 2 min at room temperature with initial gentle shaking Eluates were dried under nitrogen at 45 C or overnight in air at room temperature and reconstituted in 0.5 mL RIA buffer. Aliquots of 70 PL were taken to assess sample recovery by counting for 10 min in a Packard (Downers Grove, IL) Tri-Carb model B2450 liquid scintillationcounter following the addition of 5 mL Budget-Solve HFP universal scintillant (Research Products International, Mount Prospect, IL).
Four hundred microliters of each reconstituted sample were taken for RIA according to the method recommended by the vendor of the antiserum (Radioassay Systems Laboratories, Carson, CA). The antiserum had been raised in ewes v. estrone-3-carboxymethyl ether-BSA conjugate. It was diluted l/20,000 in assay buffer and 100 ~Lof diluted antiserum was added to each sample aliquot. Tritiated ES tracer (12,500 dpm/lO(l PL assay buffer) was added to each tube to bring the total radioactivity per tube to 12,500 dpm. This was done by subtracting the mean recovered radioactivity for all the samples in the assay from the tracer aliquot. Sample volumes were brought up to 700 pl_with assay buffer and tubes were incubated overnight at 4 C. Bound radioactivity was separated from free by dextran-coated charcoal_ ES Values were obtained from a standard curve of authentic ES in the range of lo-1,000 pg per tube, and zero binding was always 60-70%. Along with every ten patient samples were run purified extracts of charcoalstripped, normal male plasma containing 0, 200, and 400 pg/mL of added, authentic ES, and an aliquot of a single normal male serum specimen RESULTS
Recovery
of added tritiated ES and DS tracers from
serum was invariably greater than 95% through the ethanol extraction step, as measured
by liquid scintillation
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Brind et al: RIA OF ESTRONE
counting.
SULFATE
IN NORMAL
However, ZO-30%
losses
MEN
27
were
routinely
incurred
during the TEA wash step, due to the high affinity of the relatively polar steroid sulfates for the aqueous Nevertheless, this step provided a necessary
phase.
tenfold
purification, reducing the dry residue in the ethanol extract of 0.5 mL serum from 10.0 +\- 1.7 mg to 0.91 +/- 0.35 mg (SD; n=4). Substantial (lo-20%) losses were also routinely incurred during loading of washed extracts onto the TLC plates; however, elution from the TLC plates was invariably quantitative (>99%). Thus, net post-elution recoveries of ES averaged 56.7 +/- 7.7% (SD; n=220). Figure 1 shows a scan of a typical chromatogram of a
ES
I/
Figure 1. Typical thin-layer chromatogram of a washed ethanol extract of a normal male serum labeled with 4,500 dpm each of tritiated estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS). "SF" indicates solvent front.
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Brind ?t al: RIA OF ESTRONE
serum extract.
SULFATE
IN NORMAL
MEN
Peaks of ES and DS were well resolved from
each other and easily distinguished from background in a 25-30 min scan
the linear analyzer also permits
quantitation without elution; however, the low count level resulted in only a rough correlation with liquid scintillation data (r=U.79 and r=O.SO in two separate experiments with a combined total of 37 extracts]. The apparently lower recovery of tritiated IX (Fig. 1) was a consistent finding and was due to degradation of the DS tracer during sample processing.
The commercially obtained ES antiserum is listed by the supplier as giving a 100% cross-reaction with both unconjugated estrone and estradiol, which are both low in males and removed
by ether extraction.
No other cross-
reactions are significant in male serum except for that of DS, which is listed as 0.05%. In our hands, this antiserum gave a cross-reaction which is plotted along with the ES standard curve in Figure 2.
The cross-reaction curve is
relatively shallow, giving a variable percent crossreaction ranging from 0.01% at B/Bo=.36 to U.U8% at B/Bo=.80, and with a value of 0.02% at B/Bo=.50.
As
was discussed earlier, even this slight cross-reactivity looms unacceptably large in view of the 5,000- lO,OOO-
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et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
29
fold excess of DS over ES in male serum; hence, chmmatography prior to RIA is needed. 501 0
40-
5
30-
iz !i s 5 a
zo-
lo-
I
I
I
1
10
30
100
PS ESTRONE
SULFATE
(@) OR
“g
DEHYDROEPIANDROSTERONE
t
1 1000
300 SULFATE
(0)
Figure 2. Typical standard curve of estrone sulfate (ES) RIA and curve showing the cross-reaction of dehydroepiandrosterone sulfate (DS) with the same antiserum.
A typical standard curve is shown in Figure 2. The working range of the assay was 10-1,000 pg, with a 511% B/Be at 41 pg.
(AII ES values given in this paper,
including those cited from other reports, are in units of pg or pg/ml of the monosodium salt of ES.) A value of 10 pg was always significantly different from zero.
Values
obtained from a specimen of charcoal-stripped, normal male
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30
plasma averaged
et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
5-8 pg/mL. The intra-assay variability was
found to be 12.0% (CV) for 23 measurements
of a normal
male serum specimen (mean value: 396 +/- 48 pg/mL; SD). The interassay CV for ten determinations of the same specimen in each of two assays
was 17.9% (mean value:
346 +/- 79 pg/mL; SD). The mean recovery of 200 pg/ml unlabeled ES added to this specimen (determined in duplicate in each of two assays) was 179 +/-30 pg/mL (SD). Data on recovery of added unlabeled ES to aliquots of charcoal-stripped plasma are shown in Table 1.
TABLE 1. Recovery of added estrone sulfate to charcoal-stripped plasma
x/mL
Pg/mL
200 400 600
242 470 661
aMean of 10 measurements
22 45 117
9.0 9.6 17.7
in a single assay.
As a further check on the specificity of our assay system it was necessary
to verify that the TLC procedure
removed sufficient DS to render the cross-reaction of the antiserum with DS insignificant. While the chromatograms appeared to show complete resolution of ES and DS (Fig. l),it should be noted that even a 99% removal of DS from the ES extract would still leave the ES region
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SULFATE
IN NORMAL
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MEN
Therefore, two
with a 50- to loo-fold excess of DS. additional experiments were performed.
In the first,
duplicate aliquots of male serum specimens (from another clinical study) were processed such that one of each duplicate was assayed
for ES as described above, and the
other aliquot was taken through the TLC elution step and rechromatographed
in the same TLC system
All samples were measured
before RIA.
in the same assay, and a good
correlation was obtained between the two procedures, as shown in Figure 3.
I
In the second experiment, unlabeled DS
I
100 ESTRONE
I
200 SULFATE
I
300 RIA (pg/mLl
L
400 AFTER SINGLE
1 500 TLC
Fig. 3. Comparison of male serum estrone sulfate (ES) values obtained by specific RIA following a double v. single TLC step to remove contaminating dehydroepiandrosterone sulfate (DS). The line shown is the calculated regression line.
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32
at 2 Pg/mL specimen
and 4 pg/mL
known
was
to contain
by a standard
DS produced
value
of ES (320 pg/mL).
to a normal
2 pg/mL
endogenous
(8). Neither
RIA method
added
added
a detectable
SULFATE
DS, assayed
concentration
change
sera
was
mean
445
C/-209
in 41 long-term years).
The
the fresh
obtained
pg/mL
frozen
two
statistically There
value
sera
406
Patient
of normal
+/-258
A%
frozen
pg/mL
(SD)
age: 17.1 +/-1.0
(mean serum
populations
of
in the measured
for ES in 50 fresh
(SD), and
MEN
male serum
ge and The
IN NORMAL
male sera
were
indistinguishable. was
a sufficient
frozen
shown
in Table
among
the three
serum
2.
There
age
patient
values were
groups,
age
into three
range age
to divide groups,
as
no significant differences in serum
ES values
or their
variability.
Table values Age
2. Comparison of serum estrone m no&men of dlfkent apes
range (yr)
Serum ES (np/rnL)
24-39 40-59 60-70
423 458 470
SD 219 189 201
sulfate
No. of suhlects 17 18 15
DISCUSSI0N The
normal
male
serum
ES values
we have
obtained
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Brind et al: RIA OF ESTRONE
SULFATE
IN NORMAL
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MEN
the present study confirm the relatively low values reported by three other laboratories (1,3,4), and strengthen the suggestion that those laboratories which have reported much higher values (2,5-7) did not take adequate account of the enormous fold) of DS in serum.
excess (5,000- to lO,OOO-
The high variability approximately
50% CV) we obtained in both fresh and frozen serum is comparable to that reported by others (1,4,7). Of the other laboratories which have reported normal male ES values, only Myking et al (1)and Franz et al (4) reported specifically on age differences. The former group reported a modestly but significantly lower mean value (258 pg/mL) for 26 men aged 50-87 years (mean=70) as opposed to 27 younger men (305 (mean=31).
pg/mL) aged 20-49 years
However, this correlation might be
attributable to interference by DS, which decreases
by
about 80% in men between age 31 and age 70 (8). Thus, DS interference to the extent of 20% in the younger age group would correspond to only a 4% interference in the older group, thereby accounting entirely for the observed decrease in ES.
Although Franz et al(4) reported an
inverse correlation between ES (but not DS) and age in normal men, their population was small (n=19) and the age range of the subjects was very narrow (21-38 years).
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kind
et al: RIA OF ESTRONE
SULFATE
IN NORMAL
MEN
Both Franz et al(4) and Remy-Martin et al(5) reported sharply decreased
ES values in cirrhotic men (29% and 44X,
respectively), but age was also a variable. Thus, half the 74% drop in serum DS reported by Franz et al would be expected on the basis of the age difference alone (8),and this could partially account for the difference observed in serum ES, were DS interference in the ES assay significant
Likewise, the 44% drop in serum ES in cirrhotics
(mean age=51 years) v. normal subjects (mean age=36 years) reported by Remy-Martin et al(5) is about the magnitude of the expected drop in serum DS (which was not measured) between those two age groups in normal men (8). It therefore appears likely that, as the present study shows, serum ES concentration is not age-dependent
in normal men.
The lack of effect of long-term frozen storage on the measurement
of serum ES is consistent with the stability
we have previously reported for DS in similarly aged frozen sera (8). The long-term stability of ES contrasts with the instability of unconjugated estrogens stored under similar conditions, as observed
by us (unpublished
data) and others (14). REFERENCES 1. Myking 0, Thorsen T, and Stoa KF (1980) Conjugated and unconjugated plasma aestrogens--oestrone, oestradiol and oestriol--in normal human males. J STEROID BIOCHEM =1215-1220.
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2.
MEN
35
Towobola OA, CriLly RC, and Oakey RE (1980) Oestrone sulphate in plasma from postmenopausal women and the effects of oestrogen and androgen therapy. CLIN ENDOCRINOL u461-471. 3. Loriaux DL, Ruder HJ, and Lipsett MB (1971).The measurement of estrone sulfate in plasma. STEROIDS 18: 463-472. 4. Franz C, Watson D, and Longcope C (1979).Estrone sulfate and dehydroepiandrosterone sulfate concentrations in normal subjects and men with cirrhosis. STEROIDS =563-572. 5. Remy-Martin A, Prost 0, Nicollier M, Burnod J, and Adessi GL (1983) Estrone sulfate concentrations in plasma of normal individuals, postmenopausal women with breast cancer, and men with cirrhosis. CLIN CHEM =86-89. 6. Hawkins RA and Oakey RE (1974) Estimation of oestrone sulfate, oestradiol-17B and oestrone in peripheral plasma: concentrations during the menstrual cycle and in men. J ENDOCRINOL Lin_:3-17. 7. Wright K, Collins DC, Musey PI, and Preedy JRK (1978). A specific radioimmunoassay for estrone sulfate in plasma and urine without hydrolysis. J CLIN ENDOCRINOL METAB 4;L:1092-1098. 8. Orentreich N, Brind JL, Rizer RL and Vogelman JH (1984). Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations through=551-555. out adulthood. J CLIN ENDOCRINOL . METAB . 9. Abraham GE (1977).-ok of Radlolmmunoassav. Vol 5, Marcel Dekker, New York, p 591. 10. Korenman SG, Perrin LE, and McCallum TP (1969) A radio-ligand binding assay system for estradiol measurement in human plasma. J CLIN ENDOCRINOL METAB 29:879-883. 11. Korenman SG (1969) Comparative binding affinity of estrogens and its relation to estrogenic potency. STEROIDS Bl63-177. 12. Longcope C (1972) The metabolism of estrone sulfate in normal males. J CLIN ENDOCRINOL METAB =113-122. 13. Brind JL, Kuo S-W, Chervinsb K, and Orentreich N (1988) A new reversed phase paired ion thin-layer chromatographic method for steroid sulfate separations. STEROIDS, %: 561-570. 14. Phillips GB, Yano K, and Stemmermann GN (1984) Decrease in serum estradiol values with'storage. N ENGL J MED m1635.
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