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Determination of 16 alpha-hydroxyestrone by radioimmunoassay in systemic lupus erythematosus.

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1122
DETERMINATION OF 16 ALPHA-HYDROXYESTRONE
BY RADIOIMMUNOASSAY IN
SYSTEMIC LUPUS ERYTHEMATOSUS
ROBERT G. LAHITA, RICHARD BUCALA, H. LEON BRADLOW, and JACK FISHMAN
A radioimmunoassay for the feminizing metabolite 16 alpha-hydroxyestrone was applied to a variety of
sera from healthy volunteers, patients with active or
inactive systemic lupus erythemattlsus (SLE), and patients with other rheumatic diseases. A significant increase in this metabolite was detected in patients with
SLE, especially those with active disease, compared with
normal controls (P< 0.001). SLE patients were categorized as having either active or inactive disease by
clinical and laboratory criteria. Many patients who had
clinically and serologically active disease were found to
have normal levels of this estrogenic metabolite, and
several explanations for these differences are explored in
this report. Despite a poor correlation of hormone levels
with age, antibody levels, or complement levels in
patients with SLE, those patients with the highest levels
of hormone were among those whose disease was clinically most active.
The hydroxylation of estradiol at the C-16 alpha
position results in the production of the agonist metabolites, 16 alpha-hydroxyestrone and estriol. Studies of
patients with systemic lupus erythematosus (SLE)
have shown a significant increase of such 16 alpha
hydroxylation in men as well as women (1). Earlier
studies of patients with SLE revealed that urinary 16
alpha-hydroxyestrone was elevated in both sexes,
-~
From the Rockefeller University, New York, New York.
Supported by a grant from the Kroc Foundation and NIH
grant AM-04761.
Robert G. Lahita, MD, PhD; Richard Bucala, PhD; H.
Leon Bradlow, PhD; Jack Fishman, PhD.
Address reprint requests to Robert G. Lahita, MD, PhD,
Rockefeller University, 1230 York Avenue, New York, NY 10021.
Submitted for publication April 17, 1984; accepted in revised form April 3, 1985.
Arthritis and Rheumatism, Vol. 28, No. 10 (October 1985)
whereas estriol was elevated only in women (2) and in
men who had both Klinefelter’s disease, a feminizing
syndrome, and SLE (3).
Sixteen alpha-hydroxyestrone is a potent estrogen with unusual properties (4).Although the steroid is
quite uterotrophic, it binds only modestly to the cytosol receptor. It also has very little affinity for the
testosterone-estrogen-binding globulin (TEBG). Additionally, it has been reported recently that this steroid
can form stable covalent bonds with amino groups in
proteins (5).
The measurement of 16 alpha-hydroxyestrone
in plasma was anticipated to be a potentially useful
adjunct to the study of a variety of disease states such
as SLE. Because of this, a specific radioimmunoassay
(RIA) for 16 alpha-hydroxyestrone was developed (6).
In this report, this RIA procedure is applied to the
study of SLE patients and patients with a variety of
other immunologic diseases that occur predominately
in females, to determine whether their plasma levels of
16 alpha-hydroxyestrone are elevated, as would be
predicted by the previously described increase of 16
hydroxylation (1).
PATIENTS AND METHODS
SLE patients. All SLE patients fulfilled the SLE
classification criteria of the American Rheumatism Association (7); none had thyroid or liver disease at the time of the
study. Samples were obtained from female patients who
were in the midfollicular phase of their menstrual cycles,
when levels of 16 alpha-hydroxyestrone have been shown to
be 5.8 rfr 3.0 pg/ml(6). Care was taken to select patients who
either were not taking steroids or who were taking only
modest doses of the drug (<15 mg/day).
Plasma samples. Plasma samples were obtained from
normal volunteers, patients with SLE, and patients with
1123
16 ALPHA-HYDROXYESTRONE IN SLE
other rheumatologic conditions who were being studied at the
Rockefeller University Hospital. In addition, several samples were obtained from SLE patients at the Hospital for
Special Surgery, New York. Twenty milliliters of anticoagulated blood was obtained by venipuncture. Serial samples
were obtained from several patients who were inpatients at
the :Rockefeller University Hospital, or were frequent visitors in our clinic.
All serial samples of plasma were frozen to -80°C
after collection. At the end of a period of serial collections,
all samples were analyzed at the same time by RIA. All
procedures were approved by the institutional review board
of the Rockefeller University Hospital.
Patients with other diseases. Plasma samples from
patients with a variety of clinical rheumatologic or hematologic syndromes were also assayed. Since most immune
diseases are more common in females, the majority of tested
patients in this group were women. We tested plasma
samples from patients with rheumatoid arthritis (3 patients),
mixed connective tissue disease (1 patient), myositis ( 2
patients), scleroderma (6 patients), Sjogren's syndrome (1
patient), Reiter's disease (3 patients), osteoarthritis (1 patienl), autoimmune hepatitis (2 patients), cryoglobulinemia
( 1 patient), and a variety of other arthropathies (6 patients).
Clinical activity of SLE. The majority of SLE patients
studied were not taking corticosteroids. In some instances,
patients were taking low doses of prednisone (0-30 mg/day).
The usual clinical criteria, along with laboratory measurements of anti-DNA, anti-single-stranded DNA (anti-ssDNA)
(8),total hemolytic complement (9), leukopenia, or other
indices, such as analyses of urinary sediment and erythrocyte sedimentation rates, were used to substantiate the
activity of the illness at the time of the study. Table 1
summarizes the clinical activity and mean prednisone doses
of the patients with SLE.
Karyotyping. All males were karyotyped by Dr.
James German of the New York Blood Center to rule out
Klin efelter' s syndrome.
Plasma RIA. 16 alpha-hydroxyestrone. Plasma levels
of 16 alpha-hydroxyestrone were determined as described
elsewhere (6). Briefly, 5,000 counts per minute of 3H-16
alpha-hydroxyestrone was added to 1 ml of plasma and then
extracted twice with ethyl ether. After evaporation under
nitrogen at 40°C, the residue was dissolved in ethanol, and
an aliquot was saved for determination of the percent
recovery. The ethanol was evaporated under NZ,and the
Table 1. Characteristics of systemic lupus erythematosus (SLE)
patients, by disease activity group*
Active SLE
(n = 28)
Age
Anti-DNA (normal O-l5%)t
Anti-ssDN AS
CH50 (normal 150-250 hu)§
Prednisone dose (mg/day)
* Valiues are mean
33.8 t 11.7
32.8 29.3
60.1 t 25.1
113.0 t 78.7
7.6 t 9.2
*
f SD.
t Antibody binding by radioimmunoassay.
S Anti-single-stranded DNA.
8 Total complement in hemolytic units (hu).
Inactive SLE
(n = 33)
36.4
6.9
27.9
200.1
6.8
2
13.7
t 4.4
t 8.6
t 44.0
t 9.9
final residue was taken up in 0.1 ml of phosphate buffered
saline (pH 7.4) and subjected to RIA. A standard curve of 01,000 pg of 16 alpha-hydroxyestrone was determined during
each assay run.
Dilutions of the standard tracer and the antisera
(prepared in rabbits immunized with a 3,6-alpha,16-alphatrihydroxyestra-1,3,5[trienl-17-one-6-hemisuccinate-bovine
serum albumin adduct) (10) were incubated overnight at 4°C.
treated with 1 ml of a dextran (0.06%)<harcoal (0.6%)
suspension, and then centrifuged at 2,000 revolutions per
minute for 10 minutes. Supernatants thus prepared were
diluted with 10 ml of hydrofluor and counted for 10 minutes
in a Packard scintillation counter.
Sensitivity of assay. The assay for 16 alpha-hydroxyestrone was performed on duplicate samples. When possible, triplicate or quadruplicate samples were tested. In
previous studies (6), we found that normal, healthy women
have the highest 16 alpha-hydroxyestrone levels during the
periovulatory period (14.5 5 5.7 pg/ml). The inter- and intraassay coefficients of variation were 7.6% and 5.7%, respectively. Consistent blank values of 8-10 pg/ml could be
obtained using fresh diethyl ether for extraction.
Estradiol. Since the menstrual history of several
patients was unavailable, concurrent estradiol levels were
also obtained. Previous work with normal, healthy subjects
from this laboratory had shown peak 16 alpha-hydroxyestrone levels to be 14.5 2 5.7 pg/ml in the periovulatory
phase. During the other parts of the cycle, levels were found
to be between 4 and 10 pg/ml. Estradiol levels were determined by RIA according to a standard method (1 1).
Statistics. Statistical analysis was carried out by
using the BNDP statistical programs P31) for correlation, on
a PDP 11/70 computer.
RESULTS
The levels of 16 alpha-hydroxyestrone in the
plasma of most SLE patients were significantly greater
than those observed in the plasma of healthy people or
patients with other forms of collagen disease (P <
0.001). Figure 1 shows the distribution of the values of
16 alpha-hydroxyestrone in the 4 groups of patients
studied. The overall range of values for all patients
with SLE was greater than the range for either of the
other 2 groups (Table 2). Patients with SLE had
significantly higher levels of 16 alpha-hydroxyestrone
(25.4 k 20.6, n = 61) than did either the normal
controls (12.1 k 11.6, n = 22) or the patients with
other rheumatic diseases (11.1 5 7.1, n = 26) (P <
0.00 1).
When the SLE patients were categorized into
active and inactive disease groups, based on clinical
and serologic activity, a significant difference (P <
0.02) in the mean level of 16 alpha-hydroxyestrone
was seen between the 2 subgroups. When the patients
with active SLE were compared with normal controls
or with patients who had other rheumatic diseases, the
I124
0
E
m
LAHITA ET AL
-
m
males
females
o=
0
a
-
80-
a=
Table 3. Correlation between laboratory parameters of systemic
lupus erythematosus (SLE) activity and levels of 16 alphahydroxyestrone*
r value
a
E
a
\
aJ
a
C
2 60c
7!?
a
Normals
Other
Inactive
Active
diseases
SLE
SLE
Figure 1. Scatterplot of levels of 16 alpha-hydroxyestrone, determined by radioimmunoassay, in normal volunteers, patients with a
variety of rheumatic diseases, and patients with active or inactive
systemic lupus erythematosus (SLE). Bars indicate the mean levels
in each group.
differences were very significant ( P < 0.001), indicating the exceptionally high levels of hormone found in
this group. When patients with inactive SLE were
compared with the population of normal volunteers,
the P value was <0.1. No numerical correlation with
anti-ssDNA, anti-DNA, or age was found in either the
active or inactive SLE group (Table 3 ) , when each
parameter was individually compared with the level of
hormone.
Several patients and normal controls were studied serially over several days, with multiple bleedings
on the same day. In 1 normal male and 1 normal
female, the levels of 16 alpha-hydroxyestrone fluctuTable 2. Levels of 16 alpha-hydroxyestrone, determined by
radioimmunoassay , in the plasma of systemic lupus erythematosus
(SLE) patients, patients with other rheumatic diseases, and normal
controls*
Age
Anti-DNA
Anti-ssDNA
CHSO
Prednisone dose
~~
Active SLE
Inactive SLE
-0.020
-0.272
-0.093
-0.006
0.202
-0. I47
0.188
0.406
0.545
0.375
~
* Plasma samples were subjected to serologic analysis
at the same
time the radioimmunoassay for 16 alpha-hydroxyestrone was performed. No significant correlation was found for any variable. AntissDNA = anti-single-stranded DNA.
ated but never exceeded 18 pg/ml (Figure 2). In
addition, the values observed in the normal male
seemed to increase gradually with time, whereas no
defined pattern was found in the normal female. There
was no apparent explanation for these findings in the
normal male. Two women with SLE were also studied, and we found that the levels varied from hour to
hour and from day to day (Table 4). No diurnal rhythm
was observed, and in both women, several of the
values were in the range of 20-25 pglml.
Six female patients with SLE were studied
serially over 2-month periods. We obtained 6-8 plasma samples from each woman, and levels of 16 alpha-
-E
16 alpha-hydroxyestrone (pglml)
Normal controls
Patients with other
rheumatic diseases
SLE patients
Active SLE
Inactive SLE
n
Mean t SD
Range
SEM
22
26
12.1 2 11.6
11.1 2 7.1
0-35.8
0-28.0
2.46
1.39
61
28
33
25.4 2 20.6t
32.3 f 20.3t
18.6 2 21.0
0-92.0
0-92.0
0-73.9
3.30
4.29
5.26
* See Patients and Methods for description of patients with other
rheumatic diseases.
t P < 0.001 compared with normal controls.
6
2
'0
20
40
60
80
100
120
140
160
DAYS
Figure 2. Levels of 16 alpha-hydroxyestrone, obtained serially, in
2 normal (NL) individuals ( I male, 1 female). Picograms of hormone
are plotted against time in days.
16 ALPHA-HYDROXYESTRONE IN SLE
Table 4. Levels of 16 alpha-hydroxyestrone in 2 women with
systemic lupus erythematosus (SLE), over a period of several days
and with multiple same-day bleedings
16 alpha-hydroxyestrone
SLE patient
Day
Time
IDe/ml)
CK
1
10:10 AM
6.6
22.0
21.4
11.5
19.8
26.4
1:oo PM
1050 PM
2
4:20
AM
9:OO PM
JIJ
3
12:00 PM
1
2
7
11:m PM
4:OO AM
8
12:m PM
16.8
23.1
24.7
3.9
9:OO PM
hydroxyestrone, total hemolytic complement, antiDNA antibodies, and anti-ssDNA antibodies were
determined for each of the samples. Values obtained
from serologic testing were compared with the levels
of 16 alpha-hydroxyestrone measured by RIA, and
Correlation coefficients were determined (Table 5 ) .
With few exceptions, no correlations between these
values and hormone levels were observed. Interestingly, 1 woman who was receiving 19-nortestosterone
showed a negative correlation between her level of 16
alpha-hydroxyestrone and her anti-DNA, antissDNA, and CH50 levels. Another woman, who had
been treated with cyclophosphamide 10 years previously., had been amenorrheic for 10 years and had
good correlations between both anti-DNA and antissDNA values and 16 alpha-hydroxyestrone (0.7 and
0.6, respectively).
When the levels of hormone were measured in
patients who had collagen diseases other than
SLE, all of the values were found to be within the
normal range. There were only 2 men in each group of
SLE patients tested. The values obtained from the 2
1125
men in the active SLE group were above the 95%
confidence limits for other men, for women with
inactive SLE, and for normal women (Figure 1). Both
had normal male karyotypes, and neither was feminized, according to the usual clinical criteria.
When possible, women were studied during the
midfollicular phase of their menstrual cycle. Estradiol
levels from 20 patients in the SLE group were measured by RIA (11) to see if there was a correlation
between estradiol and 16 alpha-hydroxyestrone. An r
value of -0.373, obtained when estradiol was plotted
against 16 alpha-hydroxyestrone (data not shown),
indicated that estradiol levels did not correlate with 16
alpha-hydroxyestrone in those patients tested.
Levels of 16 alpha-hydroxyestrone were also
determined in some of the patients who had been
previously studied and described as having an elevated
rate of hydroxylation of estradiol at C-16 (1). There
was no correlation between the previously determined, elevated extent of 16 hydroxylation and levels
of 16 alpha-hydroxyestrone. However, data on the
extent of hydroxylation had been obtained more than 1
year before the plasma samples had been taken.
13ecause plasma estradiol fluctuates and 16 alpha-hydroxyestrone increases during pregnancy, we
measured the levels in 1 pregnant SLE patient and 8
healthy pregnant women. The difference between the
healthy pregnant women and the 1 pregnant SLE
patient was insignificant: the mean value for the 8
pregnant women was 273.6 ( 5 118.2) p g h l ; the pregnant SLE patient had a value of 276.9 pg/ml.
DISCUSSION
A specific radioimmunoassay for the potent
estrogen 16 alpha-hydroxyestrone has been developed
in our laboratory, and was used to measure absolute
Table 5. Correlations between levels of 16 alpha-hydroxyestrone and CH50, anti-DNA, and antisingle-stranded DNA (anti-ssDNA) antibodies studied serially (over time) in 6 women with systemic
lupus erythernatosus*
r value of 16 alpha-hydroxyestrone
Patient
HB
CT
ML
KM
BG
JH
Time period over
which samples
were obtained
No. of samples
acquired and
assayedt
24 mo.
6
7
18 mo.
35 mo.
15 mo.
23 mo.
36 mo.
-
(pg/ml) compared with:
CH.50
Anti-DNA
Anti-ssDNA
0.4
0.3
-0.6
0.2
-0.09
0.1
-0.5
0.5
0.7
0.1
7
-0. I
0.4
6
0.8
7
8
0.09
0.2
0.5
0.08
0.6
* Patients had various degrees of clinical activity. Patient CT was taking 19-nortestosterone; patient
JH had been amenorrheic for 10 years.
t Samples were obtained at least 30 days apart.
1126
amounts of this substance in the plasma samples from
patients with SLE and a variety of other diseases.
Previous quantitation of urinary estrogenic metabolites revealed increased 16 alpha-hydroxyestrone in
patients with SLE (2), and studies of the extent of
hydroxylation showed an increased rate of formation
of this metabolite in both men and women with SLE.
Despite the previous findings, the absolute plasma
levels of free, unconjugated 16 alpha-hydroxyestrone
were normal in about half of the SLE patients. Interestingly, the 2 men with active SLE had plasma levels
of 44.4 pglml and 38.3 pg/ml, much higher than the
mean levels found in women. Both men were of
normal karyotype and had no feminized characteristics, such as lack of pubic hair or gynecomastia.
Although patients with active SLE had higher
levels of hormone, estrone levels could not be correlated with any serologic parameter. By these criteria,
most patients with very high levels of the hormone
could not be easily differentiated from those with
either low or no detectable levels of hormone.
Overall, >25% of the patients with SLE had
values of the hormone which were above the range
obtained for normal individuals and for patients with a
variety of other immune diseases. Many patients,
however, had either low or no detectable levels of the
hormone, and among those were some of the most
clinically active cases. Since previous studies showed
a statistically significant increase in 16 alpha hydroxylation in both males and females with SLE when
compared with normal subjects (2), the low plasma
level of 16 alpha-hydroxyestrone observed in some
patients was inconsistent, although the overall increase in 16 alpha-hydroxyestrone levels found in the
plasma samples from the SLE patients correlate with
those found previously.
Patients studied serially over several hours had
significant fluctuations of 16 alpha-hydroxyestrone levels. Our inability to detect uniformly elevated levels in
SLE patients might be related to the variation in such
levels over finite periods of time. Such fluctuations
were also observed in normal individuals, but peak
values never reached those observed in patients with
SLE. These fluctuations observed in normal individuals and in patients with SLE escape explanation at
this time. While no apparent circadian rhythm exists
regarding this hormone, more data are needed to reach
firm conclusions. It is possible that there are minute-tominute changes in the level of this hormone in plasma.
Such fluctuations are probably a reflection of overall
estrogen metabolism, and this awaits further investigation.
LAHITA ET AL
There are several possible reasons for the incongruously low levels of hormone in some patients.
One possible reason might be the sequestration of the
hormone via protein binding. Testosterone-estrogenbinding globulin could not account for this sequestration since 16 alpha-hydroxyestrone has poor affinity
for TEBG, and the RIA used in our study eliminates
TEBG binding as a variable. A proportion of 16 alphahydroxyestrone might be sequestered by being covalently bound to protein and hence nonextractable via a
nonenzymatic reaction, which has recently been described (5). Some other mechanism, such as rapid
clearance or instability of the molecule in plasma,
might also be responsible for the low levels in some
patients with active SLE. The possibility that 16
alpha-hydroxyestrone binds as antigen to antibody or
to an acute-phase reactant in these plasmas is also
possible.
An interesting and plausible explanation, however, is that the metabolite exists in a conjugated form
in plasma, and thus would not be detectable by the
usual RIA methods. A sulfo-conjugate of estrone is
known to be the preferred substrate for 16 alphahydroxylase (12). Conjugated 16 alpha-hydroxyestrone might therefore be expected to be the major
plasma form of the hormone. Such a finding would
explain the previously observed elevated urinary levels of the metabolite and the overall increase in
hydroxylation without a concurrent increase in the
plasma content of the free material. The SLE patients
with high plasma values of the free compound might
have them either because of defective conjugation or
because of excessive deconjugation. An RIA specific
for sulfated 16 alpha-hydroxyestrone is being developed to resolve this issue.
The data presented here, however, confirm the
findings of previous studies in that values of 16 alphahydroxyestrone are elevated in most SLE patients.
This is particularly true of patients who have active
SLE. Despite persistent attempts to correlate values
of steroid with serologic activity, no such correlation
existed in either the overall single-sample plasmas of
patients or of those patients studied serially over 2month periods. There were a few exceptions, but no
uniform pattern was found.
Our radioimmunoassay has also confirmed the
finding that normal pregnant women in the third trimester have elevated plasma 16 alpha-hydroxyestrone
levels. In 1 pregnant patient with active SLE, levels
were reached which were comparablr to those in
normal individuals. Caution is warranted in the interpretation of these data regarding pregnant women.
16 ALPHA-HYDROXYESTRONE IN SLE
Since estriol cross-reacts significantly (0.75%) in the
RIA, used, it is possible that elevated levels of estriol
contribute to the high values of 16 alpha-hydroxyestrone that were observed in these pregnant women.
Studies are now in progress to examine levels of 16
alpha-hydroxyestrone from pregnant SLE patients
before, during, and after gestation.
In summary, most patients with SLE have very
high plasma levels of the estrogenic metabolite 16
alpha-hydroxyestrone. The levels of hormone, however', have no relationship to gender or serologic data.
Elevated levels are common to males and females with
clinically active SLE. Elevated levels in patients with
active SLE might reflect an overall decrease in the
normal mechanisms of conjugation, and it is the conjugated form which may actually be elevated in some
patients with the disease.
ACKNOWLEDGMENTS
The authors wish to acknowledge the expert tecbnical assistance of Gretchen Meyer and Elena Kissin. We
thank Dr. Charles Christian of the Hospital for Special
Surgery for several plasma samples from SLE patients and
Dr. Mortimer Levitz of New York University Medical
Center for the maternal plasma samples.
REFERENCES
Lahita RG, Bradlow HL, Kunkel HG, Fishman J:
Increased 16 alpha hydroxylation of estradiol in SLE. J
Clin Endocrinol Metab 53: 174-178, 1982
Lahita RG, Bradlow HL, Kunkel HG, Fishman J:
Alterations of estrogen metabolism in systemic lupus
erythematosus. Arthritis Rheum 22: 1195-1 198, 1979
Stern R, Fishman J , Brusman H, Kunkel HG: Systemic
1127
lupus erythematosus associated with Klinefelter's syndrome. Arthritis Rheum 20: 18-22, 1977
4. Fishman J, Martucci C: Biological properties of 16 alpha
hydroxyestrone: implications in estrogen physiology
and pathophysiology. J Clin Endocrinol Metab 51 :61 I615, 1980
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6. Ikegawa S, Lahita R, Fishman J: Concentration of 16
alpha hydroxyestrone in human plasma as measured by
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Rothfield N F , Schaller JG, Tala1 N, Winchester RJ: The
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1982
8. Koffler D, Carr R, Agnello V, Thoburn R, Kunkel HG:
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9. Mayer NH: Complement and complement fixation, Experimental Imrnunochemistry. Second edition. Edited
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p 133
10. Ikegawa S, Fishman J: Synthesis of 3,6-alpha,- 16-alphatrihydroxy- 1,3,5(lO)-estratrien- 17-one-6-hemisuccinate
1,3,5(10)-estratrienand (6,7,3H)-3,16-alpha-dihydroxy17-one. Steroids 39557-559, 1982
11. England BG, Niswender GD, Midgely AR Jr: Radioimmunoassay of 17 beta estradiol without chrornatography. J Clin Endocrinol Metab 38:42-50, 1974
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Endocrinology 103: 1227-1233, 1978
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