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Measurement of serum levels of dehydroepiandrosterone sulfate: A comparison of radioimmunoassay and enzymatic analysis
MEASUREMENT OF SERUM LEVELS OF DEHYDROEPIANDROSTERONE SULFATE: COMPARISON OF RADIOIMMUNOASSAY ENZYMATIC ANALYSIS W. David Holtzclawa
A AND
and Gary B. Gordonb
aDepartment of Pharmacology and Molecular Sciences and bOncology Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Corresponding author: Caw B. Cordon Received May 12,1989 Revised July 27, 1989
ABSTRACT Dehydroepiandrosterone sulfate (DHEAS) and unconjugated dehydroepiandrosterone (DHEA) are secretory products of the adrenal cortex. Measurement of serum levels of these steroids is of increasing epidemiologic interest, since low serum concentrations of DHRAS or DHRA have been associated with an increased risk of dying of cardiovascular disease or of developing cancer. Radioimmunoassays (RIAs) are the most convenient systems for the measurement of serum DHRAS concentrations in multiple samples. However, using sera from four individuals we show that different RIA kits provide quite different estimates of serum DHRAS concentrations. Moreover, these results do not always agree with the serum concentrations determined by an independent chromatographic and enzymatic reference method. The results highlight the need for an independent method of determining DHRAS levels in sera that can provide guidance in selecting an appropriate RIA, and in interpreting the results.
INTRODUCTION Low serum sulfate of
death
(DHRAS) from
have
of
been
dehydroepiandrosterone correlated
arteriosclerotic
presence
and
presence
or risk
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54/4 October
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increased
cardiovascular
of coronary
of developing
with
artery
breast
(DHRA)
disease
disease
cancer
(2),
in women
risk, (l),
and
its
in men, or
the
and with
the
(3,4).
Levels
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of both DHRA and DHRAS decline markedly with age (5-7), unlike those of cortisol, which is also secreted by the adrenal cortex (8).
The
issue of whether the decline in DHRA and DHRAS in old age is related to the development of cardiovascular disease and cancer remains unresolved. prevents
However,
spontaneous
dietary and
administration
chemically-induced
of DHRA
tumors
to mice
(9-11)
and
reduces the severity of atherosclerosis in cholesterol-fed rabbits (12). Meaningful
interpretation
of
such
epidemiologic
findings
depends on reliable methods for the accurate and specific estimation of DHRA and DHRAS in human serum. values
Widely divergent normal serum
for these steroids have been reported in the literature
(1,526). analytical
There
is also a disturbing lack of agreement between
results
obtained
on
the
same
samples
in
different
laboratories in which the same RIA kits were used (13).
Thus the
DHRAS concentrations of four serum samples submitted to more than 200 laboratories by the College of American Pathologists (13) were reported to vary by more than a factor of 4 between the high and low determinations.
These variations highlight
the need
to use an
independent method of analysis for validating the accuracy of RIA kits for measuring DHRAS. We have compared measurements of serum DHRAS levels obtained by means of four commercial radioimmunoassay (RIA) kits and values obtained
on
the
same
sera
by
an
independent
enzymatic
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chromatographic
method.
The
results
of
these
experiments
are
presented in this paper.
MATERIALS
AND METHODS
Serum After obtaining informed consent, blood was collected from four healthy volunteers, ages 22-64 years. Three of the donors, M36, M42, and M64, were male and one, F22, was female. The blood samples were collected into lo-mL red-stoppered Vacutainers (Becton Dickinson, Rutherford, NJ), allowed to clot, the sera prepared by centrifugation, and stored at -20 C until use. Samples were usually collected between 8:00 and 11:00 AM without regard to time since last meal. New batches of sera were collected as needed. Reagents DHBA was obtained from Diosynth (Oss, Holland). HPLC grade hexane and 2-propanol were from Fisher Scientific (Fair Lawn, NJ). Reagent grade methanol and ethyl acetate were products of J.T. Baker (Phillipsburg, NJ). [7-3H]DHEAS (23 Ci/mmol) was obtained from New England Nuclear (Boston, MA). ACS, an aqueous counting scintillant, was purchased from Amersham (Arlington Heights, IL). 3B-hydroxysteroid dehydrogenase (3D-HSD) was purified Pseudomonas SD. TB as described by Shikita and Talalay (14).
from
Purification of DHEA DHEA was recrystallized three times from acetone/hexane and then further purified by sublimation (80-100 C at 5 pm of Hg pressure). Purity was assessed by enzymatic analysis (15,16). Standardization of DHEA Solutions Stock solutions of recrystallized and sublimed DHEA were made in methanol at an approximate concentration of l-2 mM. These solutions were calibrated enzymatically by a spectrophotometric assay (15,16). The assay system contained in a final volume of 1 mL: 100 pmol of 2-amino-2-methyl-1-propanol, pH 9.9, lpmol of NAD+, 0.05% BSA and 50 PL of methanol. Steroid to be assayed was added in 30 PL of methanol. After addition of 48 ImU of 3B-HSD, the oxidation of the steroid was monitored by the increase in absorbance at 340 nm until the reaction was complete. The oxidation is more
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A molar than 99% complete under these assay conditions (15). extinction coefficient of 6270 M-lcm-1 for NADH at 340 runwas used for the calculations (17). The spectrophotometric assay was linear with increasing concentrations (O-60 nmol/mL) of DHFA. All solutions of DHEA were standardized by this assay. Stock solutions were uniformly within + 2.0% of the expected concentration when the enzymatic reactivity and expected concentration (weight to volume) were compared. High Performance Liquid Chromatography (HPLC) Steroids were separated on a 4 x 250 mm Lichrosorb Diol column (EM Reagents, Gibbstown, NJ) (18,19). For unconjugated steroids, the solvent system consisted of a concave hexane/2-propanol gradient from 97:3 to 80:20 (v:v) over 23 min at a flow rate of 2 mL/min. [7-3H]DHRAS was purified utilizing a concave solvent gradient of hexane/2-propanol from 35:65 to lo:90 (v:v) over 30 min at a flow rate of 1 mL/min. The appropriate fractions were pooled and used as tracer in the enzymatic assays. The material was reassayed and repurified as required. All solvents were evaporated by centrifugation under reduced pressure in a Speed Vat Concentrator (Savant Instruments, Hicksville, NY) at room temperature. Fluorometric Enzymatic Assay for Serum DHEAS &traction of DHRAS from Serum for Enzvmatic Assavs, Trace amounts of purified [7-jH]DHEAS (40,000 dpm) were added to 0.6-mL aliquots of serum as an internal standard to quantitate the recovery during the assay procedure. Serum samples were equilibrated at 4 C for 3 h and, after warming to room temperature, 25-PL aliquots were removed for counting. Steroids were extracted from the serum by means of Sep-Pak cl8 octadecylsilane cartridges (Waters Associates, Milford, MA) (20). Aliquots (0.5-mL) of serum were loaded onto the cartridges and the cartridges were washed with 5 mL of water. Steroids were eluted with 4 mL of 100% methanol. The methanol extracts were evaporated to dryness. Hvdrolvsis of DHRAS to DHEA and Isolation of DHFA, The dried extract was dissolved in 1 mL of 100 mM sodium acetate, pH 4.7, and the DHEAS was hydrolyzed to DHFA by heating at 100 C for 6 h (21). Hydrolyzed steroids were extracted from the hydrolysis mixture by The solvent was evaporated and the the use of ethyl acetate. residue stored at -20 C. This method of hydrolysis is selective for A5-steroid-3D-sulfates (21) and thus limits the number of potential 38-hydroxysteroids that could arise from other 3g-sulfated steroids
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by a more general method of hydrolysis. Immediately prior to the isolation of DHEA by HPLC the residues were dissolved in 250 pL of a mixture of hexane:2-propanol (97:3). DHEA was isolated by the HPLC procedure described above. Fluorometric Enzvmatic Assav of DHEA, After the HPLC separations the dried fractions containing DHFA were dissolved in 100 ML of methanol and diluted with 900 PL of 100 mM 2(cyclohexylamino)-ethane sulfonate (CHES), pH 9.5, containing 200 After addition of 2.4 ImU of 30-HSD and complete oxidation @MAD+. of the steroid, the change in fluorescence was measured in a Farrand Ratio Fluorometer-2 (Farrand Optical Co., [now Optical Technology Devices, Elmsford, NY]. The assay was linear over the range of O2500 pmol of DHEA/mL assay mixture. The sensitivity was 50 pmol/mL. Standards of enzymatically calibrated DHEA were included with each set of serum assays. A quinine sulfate standard was used for calibration of the fluorometer and the range setting was 0.1. At the completion of the enzymatic analysis, 100 PL of each sample was counted for tritium content in order to determine recoveries of [73H]DHEAS. The final DHEAS values actually represent the sum of serum DHEAS plus DHEA; however, the contribution of DHEA to the total is less than 1% for all four serum samples. Radioimmunoassays for DHEAS RIA kits for DHEAS were obtained from four manufacturers: Diagnostic Products (Los Angeles, CA), Immuchem (Carson, CA), Pantex (Santa Monica, CA), and Wien Laboratories (Succasunna, NJ). Kits are designated A,B,C, and D. The pertinent characteristics of these kits are listed in Table 1. Each kit was used according to the manufacturer's instructions or advice. Kits A, B and C used an 1251 tracer. Kit D used a 3H tracer. All kits measured the antibody-bound steroid. Kits A and B employed polypropylene tubes coated with antibodies against DHEAS, and the tubes were counted after the assay mixtures were removed. Disposable borosilicate glass tubes (12 x 75 mm) were used for Kits C and D. Kit C used a second antibody to precipitate the DHEAS-specific antibody and the precipitate was then counted. In Kit D Dextran-coated charcoal was added to remove the unbound fraction of steroids and the supernatant fraction was counted. If 1251 tracers were used, each sample was counted twice for 1 min and the two counts averaged. For Kit D, the supernatant fractions were mixed with 10 mL of scintillation fluid, and each vial was counted once for 5 min after allowing time for dark adaptation.
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Table 1 CHARACTERISTICS OF RIA KITS FOR DHEAS
Kit
Property
A
B
c
D
Immunizing antigen
DHEAS-7conjugate
Radioactive tracer
12s1
125I
1251
50
25
50
0.5b
30
60
37
4
Serum volume (/IL) Incubation time (min)
120
DHEA-3BhemisuccinateBSAa
120
Temp. of incubation (C)
37
37
Separation method
Coated tube
Coated tube
Number of standards
b"
6
6
DHEA-3BhemisuccinateHSAa
DHEA-3EhemisuccinateBSAa 3H
Second antibody
CharcoalDextran 5
8
BSA, bovine serum albumin; HSA, human serum albumin. 20 PL of a 1:39 dilution.
A standard curve was obtained for each assay using a logit transformation (22). The resulting regression lines were used to calculate serum DHEAS concentrations. All values for DHEAS are expressed as nmol/mL of the free acid (molecular weight of 368.5). Extraction of DHF.AS from Serum for use with Kit C, Internal standards of [7-'HIDHEAS were added to 1-mL aliquots of serum and
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the mixtures were applied to Sep-Pak Cl8 cartridges. After a 5-mL water wash, conjugated steroids, including DHEAS, were eluted (free of unconjugated steroids) with 4 mL of 50% (v/v) methanol (20). The 50% methanol fractions were dried and reconstituted in Kit C's DHF.AS "blank" calibrator.
RESULTS Serum Levels of DHEAS in Four Healthy Individuals Enzvmatic Assavs,
The
enzymatic procedure
described was
developed to provide reference values for serum DHEAS for comparison with the results obtained by RIA. from four healthy assayed
individuals
(Table 2).
(three male and one female) were
Serum was
several occasions.
To this end, several lots of sera
obtained from each volunteer
on
The sera and their dates of collection are
identified in Table 2. ranged from 64 to 80%.
The recovery of the
[7-3H]DHEAS tracer
Most assays were performed in duplicate and
a single average value reported.
For some samples the same serum
was assayed on multiple days and the mean value reported.
Each
individual had a characteristic DHEZASconcentration that was similar (mean f 20%) but not identical at each donation of serum.
This
variation is partially attributable to day-to-day changes in serum DHFAS
concentration.
However,
consistency
is observed
in the
ranking of the levels for the four individuals, with F22 (the only female) having the lowest DHEAS concentration and M42 the highest. In addition, assay of a mixture of equal volumes of sera M36-3 (5.27 nmol/mL) and M42-4
(8.85 nmol/mL) gave an average value of 7.10
nmol/mL, in close agreement with the expected value of 7.06 nmol/mL.
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Table 2 SERUM DHEAS AS DETERMINED BY THE FLUOROMETRIC ENZYMATIC METHOD
Serum Sample
Date Collected
Meana (nmol/mL)
Nb
SD=
CV(%)d
F22-1 F22-2 F22-3
06/24/87 07/06/87 09/2o/aa
1.47 1.78 2.22
1 2 2
0.08 0.00
4.2 0.0
M64-1 M64-2 M64-3
02/27/87 07/07/87 09/20/88
3.38 2.86 2.58
3 2 2
0.10 0.30 0.06
2.9 0.9 2.3
M36-1 M36-2 M36-3 M36-4
01/05/87 05/15/87 06/16/87 09/2o/aa
4.17 4.45 5.27 4.46
2 1 4 2
0.10
2.5
0.04 0.06
0.7 1.4
M42-1 M42-2 M42-3 M42-4 M42-6
02/09/87 05/15/87 06/16/87 07/06/87 04/06/88
8.61 7.90 8.82 8.85 8.61
2 1 2 4 2
0.03
0.4
0.02 0.40 0.23
0.2 4.7 2.7
7.12
2
0.08
1.1
M36-3:M42-3 (1:l)
-
-
a Mean of replicate single value. bN = number of assays on each sample. ' SD = standard deviation. d cv - coefficient of variation - (SD/mean) x 100.
Radioimmunoassavs.
RIA Kits A, B, and C were tested using
aliquots from identical serum samples that had been enzymatically assayed for DHEAS.
Kit D, which became available at a later time,
was used to assay new batches of enzymatically assayed serum from the same donors.
In all cases: a) all kits from the same supplier
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OF STEROIDS
had the same lot number; b) 5 assays were done using each kit, four times by one operator, and once by a second operator; and c) within each assay non-specific binding, maximum binding, and each serum sample were assayed in six replicates, whereas each manufacturersupplied standard was measured in four replicates. these assays performed with Kits
The results of
A, B, and C are presented in Table
3, together with the values obtained by enzymatic assays of each serum.
Kits A, B, and C agreed in giving the same ranking for the
four sera: from F22 having the lowest values to M42 the highest. However, the kits usually did not agree with each other in assessing the amount of DHKAS in the serum samples.
In almost every case,
Kits A and B gave lower DHKAS values than Kit C.
A variation of 50%
was seen at the highest concentrations of DHKAS (M42 serum). C
exhibited
the
lowest
intra-assay
and
inter-assay
Kit
percent
coefficients of variation, but all kits exhibited reasonable (about 10%) intra-assay and inter-assay coefficients of variation. The higher values obtained with Kit C in the face of its otherwise excellent performance led us to speculate whether there were other components in the serum that might cause this phenomenon. Therefore, a number of experiments were carried out with this kit to determine if extraction of DHEAS from the serum had any effect on the assayed DHKAS values (see Methods).
Both the 50% methanol
Sep-Pak fractions containing DHKAS, and the original unextracted sera were assayed for DHKAS using radioimmunoassay Kit G; aliquots
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Table 3 MEAN SERUM DHEAS VALUES AS DETE~INED BY RIA KITS A, B, AND C
DHEAS Values (nmol/mL) Serum Sample Method of measurement
Kit
A
Kit B
Kit C
Enzymatic assay
Means and CV (X)
F22-2
M64-2
M36-3
M42-4
2.11
3.11
6.28
11.37
0.26
0.23
0.43
0.90
12.51
7.44
6.82
7.92
9.60
6.40
8.90
6.70
Inter-assay mean Inter-assay SD Inter-assay CV (%> Avg intraassay CV (X)
1.74
3.23
5 45
8.36
0.13
0.29
0 30
0.86
7.36
8.87
5 50
10.26
9.60
8.70
8 10
9.80
Inter-assay mean Inter-assay SD Inter-assay 03 (%> Avg intraassay CV (X)
2.05
4.34
7.04
13.06
0.09
0.17
0.10
0.58
4.52
3.83
1.48
4.42
7.50
6.70
5.40
5.70
1.78
2.86
5.27
8.85
Inter-assay mean Inter-assay SD Inter-assay CV (%I Avg intraassay CV (X)
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were counted to determine recoveries (Table 4).
365
Recoveries of [7-
3H]DHEA were greater than 95% for all four sera.
Table 4 EFFECT OF SERUM EXTRACTION ON DHEAS VALUES AS DETERMINED BY RIA KIT C
DHEA (nmol/mL)
Unextracted Serum
Serum
F22-1 M64-2 M36-3 M42-5
Extracted Seruma
2.21 4.70 7.70 11.66
Recoveries (%)
1.69 3.56 5.33 8.68
Extracted Unextracted (%)
97.2 98.4 95.5 97.5
76.5 75.7 69.2 74.4
aCorrected for recoveries.
However,
when
assayed
by
RIA
only
69
- 76%
of
the
original
immunoreactive material was found in the extracted fractions. Thus, the much lower values obtained by RIA for the extracted sera did not result from loss of DHEAS during the extraction and are much closer to those obtained enzymatically.
The higher values observed with
this Kit could result from other serum components binding to the anti-DHEAS antibody, specific binding of the 1251-tracer by a serum component, or other less specific effects. RIA Kit D.
Kit D was tested with new lots of enzymatically
assayed serum (Table 5).
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It differed from the other kits in that
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Table 5 MEAN SERUM DHEAS VALUES AS DETERMINED BY KIT D
DHEAS Values (nmol/mL) Serum Sample Method of measurement
Kit D
Means and CV (X)
Inter-assay mean Inter-assay SD Inter-assay CV (%) Avg intraassay CV (X)
Enzymatic assay
F22-3
M64-3
M36-4
M42-6
2.42
3.63
5.62
9.58
0.06
0.08
0.06
0.77
2.48
2.32
1.04
8.04
2.40
2.00
2.00
2.70
2.22
2.58
4.46
8.61
it used a 3H-tracer instead of 125I, and required far less serum per assay
(0.5 PL compared to 25 or 50 pL).
Kit D gave the same
relative order of DHEAS concentration for the four sera as the other kits.
Except for serum M64-3, the mean values agree most closely
with those obtained with Kit B and with the enzymatic values.
DISCUSSION The development of an enzymatic method for the measurement of serum DHEAS concentration was prompted by different commercial the same sera.
our observation
that
RIA kits often gave quite different values with
These discrepancies rendered a rational choice of
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a kit for surveying large numbers of samples impossible.
A key
requirement of any epidemiologic study is to be certain that the desired component is being measured.
Therefore a reference method
for determining serum DHRAS was required for the evaluation of RIA methods.
The RIA methods are clearly superior to any other method
for ease, rapidity and adaptability to large numbers of samples. Nevertheless,
their performance must be judged not against each
other, but against an independent standard. by a fluorometric enzymatic assay.
This was accomplished
The importance of this type of
approach was reinforced by the observations of Chasalow and coworkers (23) who compared the results obtained with two serum RIAs in measuring DHEAS in samples of breast cyst fluid.
Although both
assays gave identical results with sera, they gave statistically different
results
with
breast
cyst
fluid.
A
chromatographic
separation of DHRAS resolved these discrepancies. The following lines of evidence support the validity of the enzymatic assay in providing reliable and accurate values for serum DHEAS:
(a) the measurements depend on the specific and complete
oxidation of the 30-hydroxyl group of DHRA by 3D-hydroxysteroid dehydrogenase.
Conditions for achieving the complete oxidations and
for measuring the stoichiometric changes in NADH have been carefully defined
(15,16). Under
appropriate conditions
the oxidation
of
substrate and reduction of NAD+ are strictly stoichiometric, and the reaction proceeds to greater than 99% completion.
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In contrast, RIAs
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are
based on an equilibrium binding phenomenon which is sensitive
to
assay
conditions
components.
and
subject
to
interference
by
competing
In the enzymatic assay possible interference from other
compounds is reduced by separating the DHRA by HPLC and by the specificity of the hydrolysis method for only A5-steroid-3I3-sulfates (21);
(b)
Replicate measurements on the same serum samples gave
excellent agreement, and mixtures of two different sera gave DHRAS Potential
values that agreed within 2% of the expected values. limitations correcting
of for
the
enzymatic
recoveries,
methods
and
this
are in
that turn
they
depend
depends
on
on the
reasonable assumption that exogenous [7-3H]DH13ASbehaves in the same manner as endogenous DHRAS at all of the analytical steps. It was satisfying that all of the RIAs gave the same rank of DHRAS concentrations in the four sera examined; the same ranking was also given by the enzymatic assays.
However, there did appear
to be real differences in the actual values given by the different kits for the same sera.
The reason for these discrepancies is not
known, but several explanations may be offered: 1) differing crossreactivities with other steroids.
This does not seem to be very
likely because Kits B, C, and D use the same immunizing antigen and the
cross-reactivities
of
manufacturers, are similar.
the
antibodies,
as
The antibody of Kit
reported
by
the
A, as indicated by
the manufacturer, shows little cross-reactivity with other steroids; 2) interference by some other component(s) of the serum,
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protein(s).
ANALYSIS
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OF STEROIDS
Our results with Kit C, giving better agreement with
the chromatographic-enzymatic assay after extraction of the steroids from serum and reconstitution in the kit's blank calibrator, suggest that
there are other
factors
in serum, such as cross-reacting
ligands or compounds that may alter the steroid binding assay.
in the
Notably, the two kits giving the highest DHEAS values, A and
C, both
use
50 pL
of
serum.
These
results confirm
that
the
demonstration of linearity with added amounts of known ligand is not a sufficient condition to establish the validity of an RIA. essential
that
the
concentration
of
the
endogenous
It is
ligand
be
established by an independent technique and the results of the RIA be compared to this value. In conclusion, we have described a chromatographic-enzymatic method for the analysis of DHEAS that uses only a small volume of serum and that can be used to verify the accuracy of an RIA for DHEAS.
We have also demonstrated the need for such validation.
ACKNOWLEDGMENTS This work was supported by a Grant from the National Cancer Institute, National Institutes of Health (1 PO 1 CA 44530). G.B.G. is the recipient of a Clinician-Scientist Award from The Johns Hopkins Medical Institutions. We wish to thank Nancy Edwards for technical assistance and Gale Doremus for secretarial help.
REFERENCES 1.
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Barrett-Connor E, Khaw KT, and Yen SSC (1986). A prospective study of dehydroepiandrosterone sulfate, mortality and cardiovascular disease. N ENGL J MED 315:1519-1524.
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13.
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Herrington DM, Gordon GB, Achuff SC, Kwiterovich PO, Trejo JF, Weisman HF, and Pearson TA (1988). Dehydroepiandrosterone sulfate (DHEAS) and coronary artery disease (CAD). CVD EPIDEMIOLOGY NEWSLETTER 43:ll. Brownsey B, Cameron EHD, Griffiths K, Gleave EN, Forest APM, and Campbell H (1972). Plasma dehydroepiandrosterone sulfate levels in patients with benign and malignant breast disease. EUR J CANCER 13:477-482. Zumoff B, Levin J, Rosenfeld RS, Markham M, Strain GW and Fukushima DK (1981). Abnormal 24-hr meanplasma concentrations of dehydroisoandrosterone sulfate in women with primary operable breast cancer. CANCER RES 41:3360-3363. Zumoff B, Rosenfeld R, Strain GW, Levin J, and Fukushima DK (1980). Sex differences in the twenty-four-hour mean plasma concentrations of dehydroisoandrosterone and (DHA) dehydroisoandrosterone sulfate (DHAS) and the DHA to DHAS ratio in normal adults. J CLIN ENDCRINOL METAB 51:330-333. Orentreich N, Brind JL, Rizer RL, and Vogelman JH (1984). Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J CLIN ENDCRINOL METAB 59:551-555. Migeon CJ, Keller AR, Lawrence B, and Shepard II TJ (1957). Dehydroepiandrosterone and androsterone levels in human plasma. Effect of age and sex; Day-to-day and diurnal variation. J CLIN ENDOCRINOL METAB 17:1051-1062. Parker L, Gral T, Perrigo V, and Skowsky R (1981). Decreased adrenal androgen sensitivity to ACTH during aging. METAB CLIN EKP 30:601-604. Gordon GB, Shantz, LM and Talalay P (1987). Modulation of differentiation and carcinogenesis growth, by dehydroepiandrosterone. ADV ENZYME REGUL 26:355-382. Schwartz AG, Whitcomb JM, Nyce JW, Lewbart ML and Pashko LL (1988). Dehydroepiandrosterone and structural analogs: A new ADV CANCER RES class of cancer chemopreventive agents, 51:391-424. Schwartz AG and Tannen RH (1981). Inhibition of 7,12lung tumor and urethan-induced dimethylbenzanthracenewith long-term treatment formation in A/J mice by dehydroepiandrosterone. CARCINOGENESIS (LOND) 2:1335-1337. Gordon GB, Bush DE, and Weisman HF (1988). Reduction of atherosclerosis by administration of dehydroepiandrosterone: A study in the hypercholesterolemic New Zealand white rabbit with aortic intimal injury. J CLIN INVEST 82:712-720. College of American Ligand Assay - Series 2 1986 Survey. Pathologists, Skokie, IL.
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14.
15. 16.
17. 18.
19.
20.
21. 22.
23.
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Shikita M and Talalay P (1979). Preparation of highly purified 3a- and 3B-hydroxysteroid dehydrogenases from Pseudomonas SD. ANAL BIOCHEM 95:286-292. Hurlock B and Talalay P (1957). Principles of the enzymatic measurement of steroids. J BIOL CHEM 227~37-52. Payne DW, Shikita M, and Talalay P (1982). Enzymatic quantities by steroids in subpicomole estimation of hydroxysteroid dehydrogenases and nicotinamide nucleotide cycling. J BIOL CHEM 257:287-300. Lowry OH and Passonneau JV (1972) In: A flexible system of enzymatic analysis, Academic Press, New York, p 4. Payne DW and Talalay P (1986). A one-step enzymatic assay for the measurement of 17B-hydroxy- and 17-oxo-steroid profiles in biological samples. J STEROID BIOCHEM 25:403-410. and Adashi EY (1988). The Payne DW, Holtzclaw WD, steroidogenic characteristics of primary testicular cell cultures from adulthypophysectomized rats: enhanced formation of C21 steroids. BIOL REPROD 39:581-599. Payne DW, Holtzclaw WD, and Adashi EY (1989). A convenient, unified scheme for the differential extraction of conjugated and unconjugated serum Cl steroids on Sep-Pak C18-cartridges. J STEROID BIOCHEM (in pr & ss). Bitman J and Cohen SL (1951). Hydrolysis of urinary conjugated 17-ketosteroids by acetate buffer. J BIOL CHEM 191:351-363. Rodbard D, Rayford PL, Cooper JA, and Ross GT (1968). J CLIN Statistical quality control of radioimmunoassays. ENDOCRINOL METAB 28:1412-1418. Chasalow FI, Blethen SL, and Bradlow HL (1988). Dehydroepiandrosterone sulfate (DHEA-S) and DHEA-S-like compounds in fibrocystic disease of the breast. STEROIDS 52:205-215.
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A AND
and Gary B. Gordonb
aDepartment of Pharmacology and Molecular Sciences and bOncology Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Corresponding author: Caw B. Cordon Received May 12,1989 Revised July 27, 1989
ABSTRACT Dehydroepiandrosterone sulfate (DHEAS) and unconjugated dehydroepiandrosterone (DHEA) are secretory products of the adrenal cortex. Measurement of serum levels of these steroids is of increasing epidemiologic interest, since low serum concentrations of DHRAS or DHRA have been associated with an increased risk of dying of cardiovascular disease or of developing cancer. Radioimmunoassays (RIAs) are the most convenient systems for the measurement of serum DHRAS concentrations in multiple samples. However, using sera from four individuals we show that different RIA kits provide quite different estimates of serum DHRAS concentrations. Moreover, these results do not always agree with the serum concentrations determined by an independent chromatographic and enzymatic reference method. The results highlight the need for an independent method of determining DHRAS levels in sera that can provide guidance in selecting an appropriate RIA, and in interpreting the results.
INTRODUCTION Low serum sulfate of
death
(DHRAS) from
have
of
been
dehydroepiandrosterone correlated
arteriosclerotic
presence
and
presence
or risk
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54/4 October
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increased
cardiovascular
of coronary
of developing
with
artery
breast
(DHRA)
disease
disease
cancer
(2),
in women
risk, (l),
and
its
in men, or
the
and with
the
(3,4).
Levels
355
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of both DHRA and DHRAS decline markedly with age (5-7), unlike those of cortisol, which is also secreted by the adrenal cortex (8).
The
issue of whether the decline in DHRA and DHRAS in old age is related to the development of cardiovascular disease and cancer remains unresolved. prevents
However,
spontaneous
dietary and
administration
chemically-induced
of DHRA
tumors
to mice
(9-11)
and
reduces the severity of atherosclerosis in cholesterol-fed rabbits (12). Meaningful
interpretation
of
such
epidemiologic
findings
depends on reliable methods for the accurate and specific estimation of DHRA and DHRAS in human serum. values
Widely divergent normal serum
for these steroids have been reported in the literature
(1,526). analytical
There
is also a disturbing lack of agreement between
results
obtained
on
the
same
samples
in
different
laboratories in which the same RIA kits were used (13).
Thus the
DHRAS concentrations of four serum samples submitted to more than 200 laboratories by the College of American Pathologists (13) were reported to vary by more than a factor of 4 between the high and low determinations.
These variations highlight
the need
to use an
independent method of analysis for validating the accuracy of RIA kits for measuring DHRAS. We have compared measurements of serum DHRAS levels obtained by means of four commercial radioimmunoassay (RIA) kits and values obtained
on
the
same
sera
by
an
independent
enzymatic
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chromatographic
method.
The
results
of
these
experiments
are
presented in this paper.
MATERIALS
AND METHODS
Serum After obtaining informed consent, blood was collected from four healthy volunteers, ages 22-64 years. Three of the donors, M36, M42, and M64, were male and one, F22, was female. The blood samples were collected into lo-mL red-stoppered Vacutainers (Becton Dickinson, Rutherford, NJ), allowed to clot, the sera prepared by centrifugation, and stored at -20 C until use. Samples were usually collected between 8:00 and 11:00 AM without regard to time since last meal. New batches of sera were collected as needed. Reagents DHBA was obtained from Diosynth (Oss, Holland). HPLC grade hexane and 2-propanol were from Fisher Scientific (Fair Lawn, NJ). Reagent grade methanol and ethyl acetate were products of J.T. Baker (Phillipsburg, NJ). [7-3H]DHEAS (23 Ci/mmol) was obtained from New England Nuclear (Boston, MA). ACS, an aqueous counting scintillant, was purchased from Amersham (Arlington Heights, IL). 3B-hydroxysteroid dehydrogenase (3D-HSD) was purified Pseudomonas SD. TB as described by Shikita and Talalay (14).
from
Purification of DHEA DHEA was recrystallized three times from acetone/hexane and then further purified by sublimation (80-100 C at 5 pm of Hg pressure). Purity was assessed by enzymatic analysis (15,16). Standardization of DHEA Solutions Stock solutions of recrystallized and sublimed DHEA were made in methanol at an approximate concentration of l-2 mM. These solutions were calibrated enzymatically by a spectrophotometric assay (15,16). The assay system contained in a final volume of 1 mL: 100 pmol of 2-amino-2-methyl-1-propanol, pH 9.9, lpmol of NAD+, 0.05% BSA and 50 PL of methanol. Steroid to be assayed was added in 30 PL of methanol. After addition of 48 ImU of 3B-HSD, the oxidation of the steroid was monitored by the increase in absorbance at 340 nm until the reaction was complete. The oxidation is more
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A molar than 99% complete under these assay conditions (15). extinction coefficient of 6270 M-lcm-1 for NADH at 340 runwas used for the calculations (17). The spectrophotometric assay was linear with increasing concentrations (O-60 nmol/mL) of DHFA. All solutions of DHEA were standardized by this assay. Stock solutions were uniformly within + 2.0% of the expected concentration when the enzymatic reactivity and expected concentration (weight to volume) were compared. High Performance Liquid Chromatography (HPLC) Steroids were separated on a 4 x 250 mm Lichrosorb Diol column (EM Reagents, Gibbstown, NJ) (18,19). For unconjugated steroids, the solvent system consisted of a concave hexane/2-propanol gradient from 97:3 to 80:20 (v:v) over 23 min at a flow rate of 2 mL/min. [7-3H]DHRAS was purified utilizing a concave solvent gradient of hexane/2-propanol from 35:65 to lo:90 (v:v) over 30 min at a flow rate of 1 mL/min. The appropriate fractions were pooled and used as tracer in the enzymatic assays. The material was reassayed and repurified as required. All solvents were evaporated by centrifugation under reduced pressure in a Speed Vat Concentrator (Savant Instruments, Hicksville, NY) at room temperature. Fluorometric Enzymatic Assay for Serum DHEAS &traction of DHRAS from Serum for Enzvmatic Assavs, Trace amounts of purified [7-jH]DHEAS (40,000 dpm) were added to 0.6-mL aliquots of serum as an internal standard to quantitate the recovery during the assay procedure. Serum samples were equilibrated at 4 C for 3 h and, after warming to room temperature, 25-PL aliquots were removed for counting. Steroids were extracted from the serum by means of Sep-Pak cl8 octadecylsilane cartridges (Waters Associates, Milford, MA) (20). Aliquots (0.5-mL) of serum were loaded onto the cartridges and the cartridges were washed with 5 mL of water. Steroids were eluted with 4 mL of 100% methanol. The methanol extracts were evaporated to dryness. Hvdrolvsis of DHRAS to DHEA and Isolation of DHFA, The dried extract was dissolved in 1 mL of 100 mM sodium acetate, pH 4.7, and the DHEAS was hydrolyzed to DHFA by heating at 100 C for 6 h (21). Hydrolyzed steroids were extracted from the hydrolysis mixture by The solvent was evaporated and the the use of ethyl acetate. residue stored at -20 C. This method of hydrolysis is selective for A5-steroid-3D-sulfates (21) and thus limits the number of potential 38-hydroxysteroids that could arise from other 3g-sulfated steroids
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by a more general method of hydrolysis. Immediately prior to the isolation of DHEA by HPLC the residues were dissolved in 250 pL of a mixture of hexane:2-propanol (97:3). DHEA was isolated by the HPLC procedure described above. Fluorometric Enzvmatic Assav of DHEA, After the HPLC separations the dried fractions containing DHFA were dissolved in 100 ML of methanol and diluted with 900 PL of 100 mM 2(cyclohexylamino)-ethane sulfonate (CHES), pH 9.5, containing 200 After addition of 2.4 ImU of 30-HSD and complete oxidation @MAD+. of the steroid, the change in fluorescence was measured in a Farrand Ratio Fluorometer-2 (Farrand Optical Co., [now Optical Technology Devices, Elmsford, NY]. The assay was linear over the range of O2500 pmol of DHEA/mL assay mixture. The sensitivity was 50 pmol/mL. Standards of enzymatically calibrated DHEA were included with each set of serum assays. A quinine sulfate standard was used for calibration of the fluorometer and the range setting was 0.1. At the completion of the enzymatic analysis, 100 PL of each sample was counted for tritium content in order to determine recoveries of [73H]DHEAS. The final DHEAS values actually represent the sum of serum DHEAS plus DHEA; however, the contribution of DHEA to the total is less than 1% for all four serum samples. Radioimmunoassays for DHEAS RIA kits for DHEAS were obtained from four manufacturers: Diagnostic Products (Los Angeles, CA), Immuchem (Carson, CA), Pantex (Santa Monica, CA), and Wien Laboratories (Succasunna, NJ). Kits are designated A,B,C, and D. The pertinent characteristics of these kits are listed in Table 1. Each kit was used according to the manufacturer's instructions or advice. Kits A, B and C used an 1251 tracer. Kit D used a 3H tracer. All kits measured the antibody-bound steroid. Kits A and B employed polypropylene tubes coated with antibodies against DHEAS, and the tubes were counted after the assay mixtures were removed. Disposable borosilicate glass tubes (12 x 75 mm) were used for Kits C and D. Kit C used a second antibody to precipitate the DHEAS-specific antibody and the precipitate was then counted. In Kit D Dextran-coated charcoal was added to remove the unbound fraction of steroids and the supernatant fraction was counted. If 1251 tracers were used, each sample was counted twice for 1 min and the two counts averaged. For Kit D, the supernatant fractions were mixed with 10 mL of scintillation fluid, and each vial was counted once for 5 min after allowing time for dark adaptation.
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Table 1 CHARACTERISTICS OF RIA KITS FOR DHEAS
Kit
Property
A
B
c
D
Immunizing antigen
DHEAS-7conjugate
Radioactive tracer
12s1
125I
1251
50
25
50
0.5b
30
60
37
4
Serum volume (/IL) Incubation time (min)
120
DHEA-3BhemisuccinateBSAa
120
Temp. of incubation (C)
37
37
Separation method
Coated tube
Coated tube
Number of standards
b"
6
6
DHEA-3BhemisuccinateHSAa
DHEA-3EhemisuccinateBSAa 3H
Second antibody
CharcoalDextran 5
8
BSA, bovine serum albumin; HSA, human serum albumin. 20 PL of a 1:39 dilution.
A standard curve was obtained for each assay using a logit transformation (22). The resulting regression lines were used to calculate serum DHEAS concentrations. All values for DHEAS are expressed as nmol/mL of the free acid (molecular weight of 368.5). Extraction of DHF.AS from Serum for use with Kit C, Internal standards of [7-'HIDHEAS were added to 1-mL aliquots of serum and
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361
the mixtures were applied to Sep-Pak Cl8 cartridges. After a 5-mL water wash, conjugated steroids, including DHEAS, were eluted (free of unconjugated steroids) with 4 mL of 50% (v/v) methanol (20). The 50% methanol fractions were dried and reconstituted in Kit C's DHF.AS "blank" calibrator.
RESULTS Serum Levels of DHEAS in Four Healthy Individuals Enzvmatic Assavs,
The
enzymatic procedure
described was
developed to provide reference values for serum DHEAS for comparison with the results obtained by RIA. from four healthy assayed
individuals
(Table 2).
(three male and one female) were
Serum was
several occasions.
To this end, several lots of sera
obtained from each volunteer
on
The sera and their dates of collection are
identified in Table 2. ranged from 64 to 80%.
The recovery of the
[7-3H]DHEAS tracer
Most assays were performed in duplicate and
a single average value reported.
For some samples the same serum
was assayed on multiple days and the mean value reported.
Each
individual had a characteristic DHEZASconcentration that was similar (mean f 20%) but not identical at each donation of serum.
This
variation is partially attributable to day-to-day changes in serum DHFAS
concentration.
However,
consistency
is observed
in the
ranking of the levels for the four individuals, with F22 (the only female) having the lowest DHEAS concentration and M42 the highest. In addition, assay of a mixture of equal volumes of sera M36-3 (5.27 nmol/mL) and M42-4
(8.85 nmol/mL) gave an average value of 7.10
nmol/mL, in close agreement with the expected value of 7.06 nmol/mL.
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Table 2 SERUM DHEAS AS DETERMINED BY THE FLUOROMETRIC ENZYMATIC METHOD
Serum Sample
Date Collected
Meana (nmol/mL)
Nb
SD=
CV(%)d
F22-1 F22-2 F22-3
06/24/87 07/06/87 09/2o/aa
1.47 1.78 2.22
1 2 2
0.08 0.00
4.2 0.0
M64-1 M64-2 M64-3
02/27/87 07/07/87 09/20/88
3.38 2.86 2.58
3 2 2
0.10 0.30 0.06
2.9 0.9 2.3
M36-1 M36-2 M36-3 M36-4
01/05/87 05/15/87 06/16/87 09/2o/aa
4.17 4.45 5.27 4.46
2 1 4 2
0.10
2.5
0.04 0.06
0.7 1.4
M42-1 M42-2 M42-3 M42-4 M42-6
02/09/87 05/15/87 06/16/87 07/06/87 04/06/88
8.61 7.90 8.82 8.85 8.61
2 1 2 4 2
0.03
0.4
0.02 0.40 0.23
0.2 4.7 2.7
7.12
2
0.08
1.1
M36-3:M42-3 (1:l)
-
-
a Mean of replicate single value. bN = number of assays on each sample. ' SD = standard deviation. d cv - coefficient of variation - (SD/mean) x 100.
Radioimmunoassavs.
RIA Kits A, B, and C were tested using
aliquots from identical serum samples that had been enzymatically assayed for DHEAS.
Kit D, which became available at a later time,
was used to assay new batches of enzymatically assayed serum from the same donors.
In all cases: a) all kits from the same supplier
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OF STEROIDS
had the same lot number; b) 5 assays were done using each kit, four times by one operator, and once by a second operator; and c) within each assay non-specific binding, maximum binding, and each serum sample were assayed in six replicates, whereas each manufacturersupplied standard was measured in four replicates. these assays performed with Kits
The results of
A, B, and C are presented in Table
3, together with the values obtained by enzymatic assays of each serum.
Kits A, B, and C agreed in giving the same ranking for the
four sera: from F22 having the lowest values to M42 the highest. However, the kits usually did not agree with each other in assessing the amount of DHKAS in the serum samples.
In almost every case,
Kits A and B gave lower DHKAS values than Kit C.
A variation of 50%
was seen at the highest concentrations of DHKAS (M42 serum). C
exhibited
the
lowest
intra-assay
and
inter-assay
Kit
percent
coefficients of variation, but all kits exhibited reasonable (about 10%) intra-assay and inter-assay coefficients of variation. The higher values obtained with Kit C in the face of its otherwise excellent performance led us to speculate whether there were other components in the serum that might cause this phenomenon. Therefore, a number of experiments were carried out with this kit to determine if extraction of DHEAS from the serum had any effect on the assayed DHKAS values (see Methods).
Both the 50% methanol
Sep-Pak fractions containing DHKAS, and the original unextracted sera were assayed for DHKAS using radioimmunoassay Kit G; aliquots
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Table 3 MEAN SERUM DHEAS VALUES AS DETE~INED BY RIA KITS A, B, AND C
DHEAS Values (nmol/mL) Serum Sample Method of measurement
Kit
A
Kit B
Kit C
Enzymatic assay
Means and CV (X)
F22-2
M64-2
M36-3
M42-4
2.11
3.11
6.28
11.37
0.26
0.23
0.43
0.90
12.51
7.44
6.82
7.92
9.60
6.40
8.90
6.70
Inter-assay mean Inter-assay SD Inter-assay CV (%> Avg intraassay CV (X)
1.74
3.23
5 45
8.36
0.13
0.29
0 30
0.86
7.36
8.87
5 50
10.26
9.60
8.70
8 10
9.80
Inter-assay mean Inter-assay SD Inter-assay 03 (%> Avg intraassay CV (X)
2.05
4.34
7.04
13.06
0.09
0.17
0.10
0.58
4.52
3.83
1.48
4.42
7.50
6.70
5.40
5.70
1.78
2.86
5.27
8.85
Inter-assay mean Inter-assay SD Inter-assay CV (%I Avg intraassay CV (X)
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were counted to determine recoveries (Table 4).
365
Recoveries of [7-
3H]DHEA were greater than 95% for all four sera.
Table 4 EFFECT OF SERUM EXTRACTION ON DHEAS VALUES AS DETERMINED BY RIA KIT C
DHEA (nmol/mL)
Unextracted Serum
Serum
F22-1 M64-2 M36-3 M42-5
Extracted Seruma
2.21 4.70 7.70 11.66
Recoveries (%)
1.69 3.56 5.33 8.68
Extracted Unextracted (%)
97.2 98.4 95.5 97.5
76.5 75.7 69.2 74.4
aCorrected for recoveries.
However,
when
assayed
by
RIA
only
69
- 76%
of
the
original
immunoreactive material was found in the extracted fractions. Thus, the much lower values obtained by RIA for the extracted sera did not result from loss of DHEAS during the extraction and are much closer to those obtained enzymatically.
The higher values observed with
this Kit could result from other serum components binding to the anti-DHEAS antibody, specific binding of the 1251-tracer by a serum component, or other less specific effects. RIA Kit D.
Kit D was tested with new lots of enzymatically
assayed serum (Table 5).
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It differed from the other kits in that
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Table 5 MEAN SERUM DHEAS VALUES AS DETERMINED BY KIT D
DHEAS Values (nmol/mL) Serum Sample Method of measurement
Kit D
Means and CV (X)
Inter-assay mean Inter-assay SD Inter-assay CV (%) Avg intraassay CV (X)
Enzymatic assay
F22-3
M64-3
M36-4
M42-6
2.42
3.63
5.62
9.58
0.06
0.08
0.06
0.77
2.48
2.32
1.04
8.04
2.40
2.00
2.00
2.70
2.22
2.58
4.46
8.61
it used a 3H-tracer instead of 125I, and required far less serum per assay
(0.5 PL compared to 25 or 50 pL).
Kit D gave the same
relative order of DHEAS concentration for the four sera as the other kits.
Except for serum M64-3, the mean values agree most closely
with those obtained with Kit B and with the enzymatic values.
DISCUSSION The development of an enzymatic method for the measurement of serum DHEAS concentration was prompted by different commercial the same sera.
our observation
that
RIA kits often gave quite different values with
These discrepancies rendered a rational choice of
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367
a kit for surveying large numbers of samples impossible.
A key
requirement of any epidemiologic study is to be certain that the desired component is being measured.
Therefore a reference method
for determining serum DHRAS was required for the evaluation of RIA methods.
The RIA methods are clearly superior to any other method
for ease, rapidity and adaptability to large numbers of samples. Nevertheless,
their performance must be judged not against each
other, but against an independent standard. by a fluorometric enzymatic assay.
This was accomplished
The importance of this type of
approach was reinforced by the observations of Chasalow and coworkers (23) who compared the results obtained with two serum RIAs in measuring DHEAS in samples of breast cyst fluid.
Although both
assays gave identical results with sera, they gave statistically different
results
with
breast
cyst
fluid.
A
chromatographic
separation of DHRAS resolved these discrepancies. The following lines of evidence support the validity of the enzymatic assay in providing reliable and accurate values for serum DHEAS:
(a) the measurements depend on the specific and complete
oxidation of the 30-hydroxyl group of DHRA by 3D-hydroxysteroid dehydrogenase.
Conditions for achieving the complete oxidations and
for measuring the stoichiometric changes in NADH have been carefully defined
(15,16). Under
appropriate conditions
the oxidation
of
substrate and reduction of NAD+ are strictly stoichiometric, and the reaction proceeds to greater than 99% completion.
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In contrast, RIAs
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are
based on an equilibrium binding phenomenon which is sensitive
to
assay
conditions
components.
and
subject
to
interference
by
competing
In the enzymatic assay possible interference from other
compounds is reduced by separating the DHRA by HPLC and by the specificity of the hydrolysis method for only A5-steroid-3I3-sulfates (21);
(b)
Replicate measurements on the same serum samples gave
excellent agreement, and mixtures of two different sera gave DHRAS Potential
values that agreed within 2% of the expected values. limitations correcting
of for
the
enzymatic
recoveries,
methods
and
this
are in
that turn
they
depend
depends
on
on the
reasonable assumption that exogenous [7-3H]DH13ASbehaves in the same manner as endogenous DHRAS at all of the analytical steps. It was satisfying that all of the RIAs gave the same rank of DHRAS concentrations in the four sera examined; the same ranking was also given by the enzymatic assays.
However, there did appear
to be real differences in the actual values given by the different kits for the same sera.
The reason for these discrepancies is not
known, but several explanations may be offered: 1) differing crossreactivities with other steroids.
This does not seem to be very
likely because Kits B, C, and D use the same immunizing antigen and the
cross-reactivities
of
manufacturers, are similar.
the
antibodies,
as
The antibody of Kit
reported
by
the
A, as indicated by
the manufacturer, shows little cross-reactivity with other steroids; 2) interference by some other component(s) of the serum,
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protein(s).
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369
OF STEROIDS
Our results with Kit C, giving better agreement with
the chromatographic-enzymatic assay after extraction of the steroids from serum and reconstitution in the kit's blank calibrator, suggest that
there are other
factors
in serum, such as cross-reacting
ligands or compounds that may alter the steroid binding assay.
in the
Notably, the two kits giving the highest DHEAS values, A and
C, both
use
50 pL
of
serum.
These
results confirm
that
the
demonstration of linearity with added amounts of known ligand is not a sufficient condition to establish the validity of an RIA. essential
that
the
concentration
of
the
endogenous
It is
ligand
be
established by an independent technique and the results of the RIA be compared to this value. In conclusion, we have described a chromatographic-enzymatic method for the analysis of DHEAS that uses only a small volume of serum and that can be used to verify the accuracy of an RIA for DHEAS.
We have also demonstrated the need for such validation.
ACKNOWLEDGMENTS This work was supported by a Grant from the National Cancer Institute, National Institutes of Health (1 PO 1 CA 44530). G.B.G. is the recipient of a Clinician-Scientist Award from The Johns Hopkins Medical Institutions. We wish to thank Nancy Edwards for technical assistance and Gale Doremus for secretarial help.
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RIA AND ENZYMATIC
ANALYSIS
OF STEROIDS
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